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The Drosophila egg chamber is an organ composed of a somatic epithelium that covers a germline cyst. After egg-chamber formation, the germline cells grow rapidly without dividing while the surface of the epithelium expands by cell proliferation. The mechanisms that coordinate growth and morphogenesis of the two tissues are not known. This study identifies a role for the actomyosin cytoskeleton in this process. Myosin activity is restricted to the epithelium's apical surface, which is facing the growing cyst. The epithelium collapses in the absence of myosin activity; the force that deforms the epithelium originates from the growing cyst. Thus, myosin activity maintains epithelial shape by balancing the force emanating from cyst growth. Further, these data indicate that cyst growth induces cell division in the epithelium. In addition, apical restriction of myosin activity is controlled. Myosin is activated at the apical cortex by localized Rho kinase and inhibited at the basolateral cortex by PP1β9C. In addition, these data indicate that active myosin is apically anchored by the Bazooka/Par-6/aPKC complex (Wang, 2007).
To analyze the correlation between cyst growth and follicle cell division, dividing cells in the follicular epithelium were counted. Within the first 56 hr that are required to form a stage 3 egg chamber, cell-division rates are very low. In the 14 hr period between stage 4 and stage 6, however, cell-division rates continuously increase. During this time, the volume of the cyst increases approximately 11-fold. This parallel increase in mitosis and cyst growth reflects how the growth of the inner cyst is compensated by cell division in the outer follicular epithelium. After stage 7, the follicle cells stop dividing and undergo diverse morphological changes (Wang, 2007).
Newly formed egg chambers are round and change their shape to ellipsoid during early oogenesis. After stage 7, the process of egg-chamber elongation, which is mediated by a polarized actin cytoskeleton within the follicular epithelium, starts. Actin fibers at the basal cortex of the follicle cells run perpendicular to the anterior-posterior axis of the egg chamber, and their contraction leads to an axis expansion. The mechanisms that shape the egg chamber before elongation takes place are unknown. The simultaneous and rapid growth of cyst and epithelium after stage 3 indicates that the development of the two tissues is precisely coordinated. It is, however, unclear how epithelial morphogenesis and proliferation are coupled to the growth of the cyst (Wang, 2007).
The actin cytoskeleton is central for the cell shape, and is thus a possible candidate involved in a controlled epithelial response to the cyst growth. The activity in the actomyosin cytoskeleton was examined by using an antibody specific for the activated form of the regulatory light chain of nonmuscle myosin II (RMLC; Spaghetti squash). The phospho-specific RMLC antibody binds to phosphoSerine21 and reveals myosin in its active state. Around stage 3 of oogenesis, myosin activity restricts to the apical cortex of the follicle cells, where it is maintained until late oogenesis. After stage 7, myosin activity is also present at the basal cortex of the follicle cells in the actin bundles required for egg chamber elongation. Optical confocal sections reveal a pattern of myosin activity in these long parallel bundles that is reminiscent of stress fibers. In contrast, at the apical cortex, myosin is active in short fibers with random orientation reminiscent of a web (Wang, 2007).
The membrane domains of the follicle cells are established before myosin activity restricts to the apical cortex at stage 3. To examine how apical myosin activation relates to follicle cell polarity, mutants affecting epithelial polarity were examined. To avoid perdurance of the wild-type protein after clone induction, focus was placed on large clones, or clones spanning the whole epithelium. The adherence junctions are central for the organization of the apical actin cytoskeleton, and the domain of myosin activity extends into the region where they localize. Therefore null mutants of armadillo (arm), which encodes Drosophila β-catenin, were examined. It has been shown that the adherence junctions are disrupted in arm follicle cell clones since neither DE- or DN-cadherin are detectable. As a result, arm mutant cells exhibit strong cell-shape defects. Surprisingly, it was found that myosin activity is clearly restricted to the apical membrane in arm follicle cell clones. Thus, myosin activity restricts apically in the absence of adherence junctions (Wang, 2007).
The apical membrane domain is established by the Crumbs (Crb)/Stardust (Sdt)/Patj complex and the Bazooka (Baz)/Par-6/aPKC complex. All these proteins localize, like pRMLC, to the apical membrane of the follicular epithelium. In epithelia lacking crb, myosin restriction is affected as revealed by the interrupted apical pRMLC pattern and by ectopic activity at the basal membrane. However, apical myosin activity is not completely disrupted as broad regions of the epithelium still concentrate higher levels of pRMLC at the apical compared to the basal cortex. In contrast, par-6, aPKC and baz mutants abolish the formation of the apical myosin activity. In these mutants, apical pRMLC restriction is lost, and ectopic myosin activity is detectable in the cytoplasm and at the basal cortex. To test whether the two apical complexes cooperate in apical myosin restriction, baz sdt double mutants were examined. The phenotype of the double mutants is, however, very similar to that of the baz single mutants, suggesting that apical myosin activity is controlled by the Baz/Par-6/aPKC complex (Wang, 2007).
To further analyze this interaction, the Baz/Par-6/aPKC complex was immunoprecipitated from ovaries using an antibody against Baz. Western-blot analysis of the precipitated protein complex reveals a strong enrichment of Baz and aPKC. Notably, pRMLC is also present in the precipitated protein complex, indicating an association of Baz and active myosin. Taken together, these genetic data show that baz, par-6, and aPKC are required for apical myosin restriction, and biochemical data show that Baz associates with pRMLC. This suggests the Baz/Par-6/aPKC complex anchors active myosin at the apical cortex. To further analyze the role of the complex in the apical restriction of myosin activity, its localization was examined in mutants that affect pRMLC localization. Consistent with a function in the anchoring of active myosin, it was found that apical aPKC localization is not affected in arm mutants, in which pRMLC is apically restricted. Further, apical aPKC localization is interrupted in crb mutants, in which pRMLC localization is also interrupted. In summary, the data suggest that the Baz/Par-6/aPKC complex anchors active myosin at the apical cortex independently of the adherence junctions (Wang, 2007).
To examine how myosin activity is inhibited during early oogenesis at the basal and lateral cortex, the localization and function of PP1β9C, the phosphatase that deactivates phosphorylated RMLC, was examined. In follicle cells, PP1β9C is ubiquitously distributed as revealed by a hemagglutinin (HA) fusion protein. PP1β9C is encoded by flap wing (flw). Western-blot analysis of the viable flw1 allele showed that the total pRMLC levels in ovaries are increased 2.8-fold compared to those of the wild-type. The total increase is the result of ectopic myosin activity in the follicular epithelium. This is revealed by flw6 follicle cell clones and homozygous flw1 mutant egg chambers, which show pRMLC staining at the basal and lateral cortex. Interestingly, the ectopic Myosin activity is accompanied by an irregular and wavy appearance of the apical surface of the epithelium. In addition, flw mutant egg chambers are not round or ellipsoid like the wild-type but are either stretched or develop bulges. The coincidence of the altered shape with the ectopic pRMLC staining in the follicular epithelium suggests that the abnormal shape is the result of ectopic myosin activity. This is confirmed by the finding that the expression of constitutively active RMLC results in a very similar phenotype. The defects in flw mutants are not secondary effects of mislocalization of the Baz/Par-6/aPKC complex as the localization of aPKC is indistinguishable from that of the wild-type. In summary, these results show that PP1β9C activity is required to prevent myosin activity at the basal and lateral cortex. They further suggest that during early oogenesis, myosin activity has to be restricted to the apical cortex to ensure the development of normally shaped egg chambers (Wang, 2007).
To investigate how myosin is activated at the apical cortex, the function of Rok, which has been shown to regulate myosin phosphorylation, was analyzed. Myosin phosphorylation is greatly reduced but still detectable in rok mutant follicle cell clones. This confirms that Rok phosphorylates myosin in the follicular epithelium, but also indicates that Rok is not the only kinase involved in myosin activation. A HA-Rok fusion protein accumulates in particles at the apical cortex of the follicle cells, which are in close proximity to the web-like myosin fibers. Thus, localized Rok activates myosin in the follicular epithelium (Wang, 2007).
Because RMLC phosphorylation is strongly reduced in rok mutant cells, rok clones were used to examine the function of apical myosin activity. rok mutant follicle cells divide normally, form a monolayered follicular epithelium, and retain polarity. However, rok mutant cells fail to adopt a normal shape. As a consequence, the epithelium is flatter in these regions than it is in regions with rok activity. Optical sections at the level of the zonula adherens show that rok mutant cells are also stretched compared to neighboring wild-type cells. Furthermore, egg chambers with large follicle cell clones develop abnormal shapes as the cyst bulges outwards in the area of the clones. These results show that rok is required for follicle cell and egg-chamber shape, and indicate that the function of the apical myosin activity is to prevent flattening and stretching of epithelial cells (Wang, 2007).
To test the function of the apical myosin activity directly, follicle cell clones were generated using a null mutation for spaghetti squash (sqh). sqh encodes RMLC and was previously shown to be required for other aspects of egg-chamber morphogenesis, like cyst separation and follicle cell migration (Karess, 1991; Edwards, 1996). Follicle cells lacking RMLC activity are extremely flat and appear stretched. In many egg chambers with sqh clones, gaps were found in the follicular epithelium, suggesting that stretching of the follicle cells eventually disrupts the monolayer. The flat sqh mutant cells retain polarity, as revealed by the localization of Discs large (Dlg), a marker for the region where the septate junctions are formed, and the localization of the apical marker aPKC. The change in the shape of the follicle cells is accompanied by a change in the morphology of the egg chamber. Although those regions of the egg chamber covered by wild-type follicle cells retain a normal shape, the germline cyst bulges out in regions covered by sqh mutant cells. In summary, the morphological defects in the sqh clones are very similar to the defects in the rok mutant clones, although the sqh phenotype is much stronger. The stronger morphological defects in sqh mutants are consistent with the finding that RMLC activity is only reduced in rok, whereas it is abolished in sqh mutant cells (Wang, 2007).
sqh function is also required for cytokinesis (Jordan, 1997), and, consistent with this, epithelia with sqh mutant clones show a reduced number of phospho-Histone H3-positive cells, huge nuclei, and abnormally large cells. To examine whether these defects contribute to the morphological defects, epithelia with clones mutant for diaphanous (dia), another gene required for cytokinesis, were examined. Using the weak allele dia5, follicle cell clones were identifed showing cytokinesis defects in the presence of a normal actin cortex. During early oogenesis, these clones retain a rectangular shape, do not flatten, and the underlying cyst bulges out only very mildly. Late clones show no outward bulging over the growing oocyte and maintain a normal cell shape, with the exception that the cells are bigger because of the absence of cytokinesis. Thus, cytokinesis defects alone do not affect the rectangular shape of the follicle cells, and they affect the shape of the egg chamber only mildly and only during early oogenesis. Importantly, the morphological defects are fully penetrant in sqh mutant follicle cell clones. It is therefore concluded that the morphological defects in egg chambers with sqh clones are the result of the loss of apical myosin activity (Wang, 2007).
The epithelial deformations in sqh clones suggest a stress that is acting on the epithelium. The outward bulging of the cyst further suggests that the origin of this stress is the volume increase of the growing cyst. Wild-type cells might resist this stress because of the myosin activity at the apical cortex that is facing the cyst, whereas sqh mutant cells collapse. To test this hypothesis, cyst growth was blocked by using a chromosome carrying an ovoD1 mutation. ovoD1 is a dominant female-sterile mutation that is normally applied in germline mosaics. Importantly, the ovoD1 phenotype is restricted to the germline and does not affect the somatic epithelium. The ovoD1 harbouring chromosome that was used in this experiment leads to a growth arrest after stage 4 resulting in small stage 6 egg chambers, which later degenerate (Wang, 2007).
sqh follicle cell clones were genrated in parallel in wild-type and in ovoD1 mutant backgrounds and cyst and epithelial shape was analyzed. Strikingly, sqh mutant follicle cells maintain their rectangular shape when cyst growth is blocked, whereas sqh cells are deformed when the cyst grows. Moreover, the cyst bulges out underneath the sqh clones only in the wild-type background, but not in the ovoD1 mutant cysts. Thus, myosin activity is required for epithelial and egg-chamber shape only if the cyst is growing. It is therefore concluded that epithelial myosin activity counteracts the force from the growing cyst (Wang, 2007).
How could myosin activity counteract stress from the growing cyst mechanistically? In Dictyostelium, it has been shown that the cell membrane is able to resist deformations induced by a cell poker, revealing stiffness of the cortex. In myosin mutants, the cortical stiffness is greatly reduced, indicating that stiffness is generated by myosin-mediated contractions within the actin cortex. Consistent with this, in vitro studies demonstrated that myosin activity increases the stiffness of crosslinked actin filaments by a factor of 100. Stiffness is generated by diminishing thermal fluctuations within a crosslinked actin network. Myosin is able to suppress these fluctuations by mediating contractions of actin filaments between crosslink points. It is proposed that stiffness is a crucial feature of the apical epithelial cortex in response to the stress emanating from the growing cyst, and that myosin regulates the stiffness by generating tension between actin crosslink points (Wang, 2007).
The pattern of myosin activity reflects the organization of the actin cytoskeleton. The stress fiber-like pattern at the basal cortex reveals activity in the parallel actin arrays, and this activity leads to egg-chamber elongation. In contrast to this polarized pattern of myosin, the apical pattern shows no uniform direction, indicating that actin filaments of all orientations contract. This suggests that the actin filaments at the apical cortex are crosslinked like a net. Thermal fluctuations are higher in actin networks compared to bundled actin filaments. A netlike organization of the actin filaments is therefore consistent with the model, in which myosin-mediated contractions increase cortical stiffness by suppressing thermal fluctuations within the net (Wang, 2007).
The follicular epithelium responds to the cyst growth by increasing the epithelial surface by cell proliferation. The signal that induces mitosis is unknown. These data raise the possibility that the actomyosin cytoskeleton is involved in the coordination of cyst growth and epithelial proliferation. It is likely that the apical cortex, which is stiffened by myosin, perceives the volume increase of the growing cyst as a further tension increase in the crosslinked actin filaments. It is speculated that tension increase above a certain threshold triggers mitosis in the epithelium. The resulting cell divisions lead to an enlarged epithelial surface and thereby to a tension decrease at the apical cortex. The coupling of tension increase and cell proliferation adapts the growth of the epithelium to the volume increase of the cyst and prevents epithelial rupture. The role of tension in regulating cell growth was proposed in the past and has been demonstrated recently in cell culture experiments (Wang, 2007).
If cyst growth and epithelial proliferation are coupled, follicle cell division should be reduced when the cyst volume does not increase. Notably, a dramatic reduction was found in cell division in ovoD1 mutant ovarioles, in which growth is blocked. Consistent with this, it has been reported that block of cyst growth induced by germline clones mutant for the Drosophila Insulin receptor and dMyc does not result in excess follicle cells. These results show that cyst growth and epithelial growth are coupled. However, they allow no conclusion about the coupling mechanism (Wang, 2007).
The restriction of myosin activity to the apical cortex of the epithelium is mediated by at least three different mechanisms. First, myosin phosphorylation at the apical cortex is achieved by apical localization of Rok. Rok is also regulated by the small GTPase Rho1. rho1 mutant follicle cell clones show reduced apical myosin phosphorylation and cell flattening, suggesting that Rho1 binding enables Rok to phosphorylate myosin. In contrast to rok mutant clones, rho1 mutant cells have large nuclei and an increased cell size, indicating that Rho1 is also required for cytokinesis. The second mechanism that restricts myosin activity to the apical cortex is the anchoring of active myosin by the Baz/aPKC/Par-6 complex. The third mechanism is the inhibition of myosin at the lateral and basal cortex via PP1β9C-mediated dephosphorylation. In the future, it will be important to find additional components regulating apical myosin activity, and to find out whether myosin activity is also in other epithelia restricted to certain domains (Wang, 2007).
Although the regulation of epithelial morphogenesis is essential for the formation of tissues and organs in multicellular organisms, little is known about how signalling pathways control cell shape changes in space and time. In the Drosophila ovarian epithelium, the transition from a cuboidal to a squamous shape is accompanied by a wave of cell flattening and by the ordered remodelling of E-cadherin-based adherens junctions. This study shows that activation of the TGFβ pathway is crucial to determine the timing, the degree and the dynamic of cell flattening. Within these cells, TGFβ signalling controlled cell-autonomously the formation of Actin filament and the localisation of activated Myosin II, indicating that internal forces were generated and used to remodel AJ and to promote cytoskeleton rearrangement. TGFβ signalling controlled Notch activity and its functions were partly executed through Notch. Thus, the cells that underwent the cuboidal-to-squamous transition produced active cell-shaping mechanisms, rather than passively flattening in response to a global force generated by the growth of the underlying cells. Thus, this work on TGFβ signalling provides new insights into the mechanisms through which signal transduction cascades orchestrate cell shape changes to generate proper organ structure (Brigaud, 2015: PubMed).
How actomyosin generates forces at epithelial adherens junctions has been extensively studied. However, less is known about how a balance between internal and external forces establishes epithelial cell, tissue and organ shape. This study used the Drosophila egg chamber to investigate how contractility at adherens junctions in the follicle epithelium is modulated to accommodate and resist forces arising from the growing germ line. Between stages 6 and 9, adherens junction tension in the post-mitotic epithelium decreases, suggesting that the junctional network relaxes to accommodate germline growth. At that time, a prominent medial Myosin II network coupled to corrugating adherens junctions develops. Local enrichment of medial Myosin II in main body follicle cells resists germline-derived forces, thus constraining apical areas and, consequently, cuboidal cell shapes at stage 9. At the tissue and organ level, local reinforcement of medial junction architecture ensures the timely contact of main body cells with the expanding oocyte and imposes circumferential constraints on the germ line guiding egg elongation. This study provides insight into how adherens junction tension promotes cell and tissue shape transitions while integrating the growth and shape of an internally enclosed structure in vivo (Balaji, 2019).
Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While nonmuscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. This study used genetics, Airyscan microscopy, live imaging, optogenetics, and Forster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. Myosin was found to accumulate transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin colocalize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo (Mishra, 2019).
During collective cell migration, front cells tend to extend a predominant leading protrusion, which is rarely present in cells at the side or rear positions. Using Drosophila border cells (BCs) as a model system of collective migration, this study revealed the presence of a supracellular actomyosin network at the peripheral surface of BC clusters. The Myosin II-mediated mechanical tension was demonstrated as exerted by this peripheral supracellular network not only mediated cell-cell communication between leading BC and non-leading BCs but also restrained formation of prominent protrusions at non-leading BCs. Further analysis revealed that a cytoplasmic dendritic actin network that depends on the function of Arp2/3 complex interacted with the actomyosin network. Together, these data suggest that the outward pushing or protrusive force as generated from Arp2/3-dependent actin polymerization and the inward restraining force as produced from the supracellular actomyosin network together determine the collective and polarized morphology of migratory BCs (Wang, 2020).
Dramatic changes in the localization of conventional non-muscle myosin characterize early
embryogenesis in Drosophila. During cellularization, myosin is concentrated around
the furrow canals that form the leading margin of the plasma membrane as the membrane plunges inward to
package each somatic nucleus into a columnar epithelial cell. During gastrulation, there is specific
anti-myosin staining at the apical ends of those cells that change shape in regions of invagination.
Both of these localizations appear to result from a redistribution of a cortical store of maternal
myosin. In the preblastoderm embryo, myosin is localized to the egg cortex (the part of the egg immediately underneath the surface membrane), sub-cortical arrays of inclusions, and, diffusely, to the yolk-free periplasm. At the syncytial blastoderm stage, myosin is found within cytoskeletal caps associated with the somatic nuclei at the embryonic surface. Following the final syncytial division, these myosin caps give rise to the myosin rings observed during cellularization. These myosin localizations and the coincident changes in cell morphology are consistent with a key role for non-muscle myosin in powering cellularization and gastrulation during embryogenesis (Young, 1991).
Peanut and Sep1, a second Drosophila septin identified based on its homolog to yeast septins, colocalize to the leading edge in cellularizing embryos. During the interphase between nuclear divisions 13 and 14, actin first reorganizes from the set of caps over the nuclei to form a hexagonal actin-myosin network at the embryo cortex. During the process of cellularization, the cleavage furrows move into the embryo around each nucleus, with actin and myosin concentrate at their leading edges, and spectrin concentrates slightly behind the leading edges. At the beginning of cellularization, Sep1 also assumes a hexagonal pattern similar to (but apparently less uniform than) that of actin and myosin. The nonuniformity of the Sep1 staining is maintained until the end of cellularization. As cellularization proceeds, Sep1 is concentrated at the leading edges of the advancing cleavage furrows, although some diffuse staining is still observed at the embryo cortex and in the underlying cytoplasm. Both Sep1 and Pnut co-localize at least at the resolution of the light microscope. Examination of double-stained embryos reveal that the septins co-localize with actin and myosin at the very leading edge of the cleavage furrows, in a position distinctly ahead of spectrin (Fares, 1995).
Regulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis. Drosophila embryos offer a well-defined system for observing alterations in the cytoskeleton during
the process of cellularization, a specialized form of cytokinesis. During cellularization, the actomyosin cytoskeleton forms a hexagonal array and drives invagination of the plasma membrane between the nuclei located at the cortex of the syncytial blastoderm. Rho, Rac, and Cdc42 proteins are members of
the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenance of the actin cytoskeleton throughout phylogeny and in Drosophila. To investigate how Rho subfamily activity affects the cytoskeleton during cellularization stages, embryos were microinjected with C3 exoenzyme from Clostridium botulinum or with wild-type, constitutively active, or dominant negative versions of Rho, Rac, and Cdc42 proteins. C3 exoenzyme ADP-ribosylates and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound
state to activate downstream effectors. Dominant negative mutations likely inhibit endogenous small G protein activity by sequestering exchange factors. Of the 10 agents microinjected, C3 exoenzyme,
constitutively active Cdc42, and dominant negative Rho have a specific and indistinguishable effect: the actomyosin cytoskeleton is disrupted, cellularization halts, and embryogenesis arrests. Time-lapse video
records of embryos show that nuclei in injected regions move away from the cortex of the embryo, thereby phenocopying injections of cytochalasin or antimyosin. Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is disrupted in a
dose-dependent fashion. Additionally, DNA stain reveals that nuclei in the microinjected embryos aggregate in regions that correspond to actin disruption. These embryos halt in cellularization and do
not proceed to gastrulation. It is concluded that Rho activity and Cdc42 regulation are required for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning (Crawford, 1998).
In mammalian cells, Rho and Cdc42 effectors function antagonistically. In competition are two distinct small GTPase protein-driven processes: the formation of stress fibers drive by Rho and the formation of filopodia driven by Cdc42. In Drosophila, Rho and Cdc42 effectors function antagonistically, but in contrast to the mammalian case the two phenotypes are indistinguishable. By this hypothesis, Rho and Cdc42 effectors function in independent pathways: Rho effector function is required for cellularization and maintenance of the actinomysin hexagonal array, whereas Cdc42 effector function antagonizes this process. Cdc42 effectors may inhibit Rho effector function directly, thereby phenocopying the disruption of Rho function generated by the microinjection of C3 exoenzyme or N19Rho. An alternative hypothesis that explains these data requires a mechanism of Rho subfamily regulation of the actomyosin cytoskeleton in a single pathway that depends only on Rho effector function during Drosophila cellularization. By this hypothesis, Rho and Cdc42 share a common factor that is required for Rho function, but is sequestered by GTP-bound Cdc42 (Crawford, 1998).
What is the role of myosin in cellularization? Since myosin function is necessary for cytokinesis and cellularization, a mechanism whereby myosin is regulated through phosphorylation by a Rho effector provides an attractive model. Because it is likely that force production for cytokinesis continuously requires activated myosin, it would be predicted that inhibition of Rho activity would block further progression of the cellularization front. Thus, agents that interfere with Rho activity would be expected to prevent the activation of myosin function and the formation of myosin bipolar filaments. It is possible that Rho effects on myosin can also explain the observed changes in the organization of actin. Indeed, Rho activity in the bundling of preexisting actin filaments may be directly or indirectly dependent on myosin function. Bipolar myosin filament formation can stimulate the formation and organization of filamentous actin; therefore, inhibition of myosin function may also explain the observed disruption of actin distribution (Crawford, 1998).
Cyclic reorganizations of filamentous actin,
myosin II and microtubules in syncytial Drosophila
blastoderms have been studied using drug treatments, time-lapse movies
and laser scanning confocal microscopy of fixed stained
embryos (including multiprobe three-dimensional
reconstructions). These observations imply interactions
between microtubules and the actomyosin cytoskeleton.
They provide evidence that filamentous actin and
cytoplasmic myosin II are transported along microtubules
towards microtubule plus ends, with actin and myosin
exhibiting different affinities for the cell's cortex. These
studies further reveal that cell cycle phase modulates
the amounts of both polymerized actin and myosin II
associated with the cortex. Pseudocleavage
furrow formation in the Drosophila blastoderm is analogous to how
the mitotic apparatus positions the cleavage furrow for
standard cytokinesis, and these findings are related to polar
relaxation/global contraction mechanisms for furrow formation (Foe, 2000).
Laser scanning confocal microscope (hereafter LSCM) sectional views are provided of anterior ends of Drosophila embryos showing anaphase and interphase of cycle 9 (before centrosomes, microtubules and nuclei reach the cortex) and
cycle 10 (the first round of bud formation and breakdown). In
cycle 9, myosin II staining concentrates in a cortical rim during
interphase but leaves the cortex during anaphase. Likewise, F-actin concentrates during interphase 9 in a cortical rim; the concentration attenuates greatly during anaphase 9. Throughout interphase 9, with no nuclei/asters
near the cortex, cortical myosin II and F-actin co-localize. Similar waxing and waning of cortical F-actin and
myosin II, co-localized and synchronized with globally
synchronous mitotic cycles, is observed in cycle 8. Migrating
nuclei with microtubule arrays reach the cortex 1 minute after
interphase 10 begins. As telophase 9
ends and interphase 10 begins, F-actin and myosin II re-accumulate
co-localized to high levels in a spatially uniform
cortical rim. Two minutes after nuclei reach the cortex, cortical F-actin and
myosin II are no longer co-localized but occur in the
complementary patterns. Myosin II
occurs at high levels between buds, but vacates the cortex
where buds now protrude, while F-actin attains high
levels precisely on the domes of the buds that myosin II has
vacated. During anaphase 10, cortical levels of F-actin and
myosin II are globally low. Cortical F-actin re-accumulation
begins first near centrosomes at anaphase/telophase. Regardless of cortical fluctuations, high levels of
myosin II staining occur throughout the embryo interior. When myosin dissociates from the
cortex, it transiently boosts the concentration of myosin
immediately beneath the cortex, but does not significantly
boost the concentration of internal myosin globally,
presumably because it is dispersing into an ocean of
cytoplasmic myosin filling these large cells. Throughout the
interior cytoplasm, F-actin occurs diffusely, and additionally in
particles, but at lower levels than cortically (Foe, 2000).
When migrating nuclei with associated microtubule
arrays first reach the periphery early in interphase 10, myosin
II staining disappears from a small region immediately above
the nuclei and, during the next 3 minutes of interphase
and prophase, the holes vacated by myosin II staining enlarge
into oblong holes. During metaphase and
anaphase, the cortical myosin staining dims, then,
during telophase, myosin staining returns brightly to
the cortex except near centrosomes where it remains dim. Holes in the cortical myosin pattern, appearing in the
cortex precisely when/where microtubule arrays first contact
the cortex, are interpreted as implying an interaction between myosin II and
microtubules (Foe, 2000).
Four hypotheses have been proposed about consecutive and simultaneous mitotic-cycle-modulated
interactions between the cell cortex (the
approximately 3 mm deep zone immediately
underlying the plasma membrane), F-actin,
myosin II, centrosomes and microtubules. When
working together, they can explain these
experimental results (Foe, 2000).
The same four mechanical hypotheses that are proposed to
explain pseudocleavage furrow and bud formation in the fly
syncytial blastoderm, if operative in dividing mononucleate
cells, could time and initiate localization of the actomyosin
components of the contractile ring for cytokinesis. H1, by melting down prior
interphase F-actin structures during metaphase, and H2 by
causing F-actin to re-polymerize near centrosomes beginning
in anaphase, in effect force a redeployment of the cell's actin
just prior to beginning the specialized task of cytokinesis. By
H1, the cortical concentrations of F-actin and myosin II, having
fallen to low concentrations in metaphase-anaphase, rebuild in
telophase. But the expanding telophase microtubule asters
approaching the cortex at opposite poles of the cell would
trigger, via H4, depletion of cortical myosin II filaments near
the spindle poles where microtubules impinge, while
simultaneously concentrating myosin II filaments by moving
them through the cytoplasm towards the cell mid-zone. H4 will
thus eventually concentrate myosin II in a three-dimensional
disk whose perimeter will become the contractile ring. By H2,
actin polymer will form coincidentally with microtubule
outgrowth initially most concentrated near centrosomes,
followed later in telophase by a migration of F-actin along
astral microtubules away from centrosomes (by H3) and
toward concentration in a three-dimensional disc-shaped
volume centered at the equator where it will co-localize with
myosin II. The 'rings' of cortical F-actin seen in late interphase, correspond to where these equatorial disc
volumes intersect the cortex in the dividing mononucleate cell. The regions
between buds where both F-actin and myosin
II are present together at the cortex during interphase (though
concentrations of F-actin may be higher elsewhere) constitute
the so-called pseudocleavage furrows in the fly syncytial
blastoderm. This region would be homologous to
the cortex of the cleavage furrow, which constricts during
cytokinesis (Foe, 2000 and references therein).
H1-H4 imply a bipolar 'global contraction-polar
relaxation' mechanism for positioning the
contractile apparatus for cytokinesis. Transient
'polar relaxation' would convert a global cortical contraction
into a self-amplifying equatorial contraction. Computer
simulations have shown how contracting
an initially isotropic actomyosin meshwork into an equatorial
belt aligns the filaments parallel to the equator, as in a
contractile ring, positioning them to cleave a cell in two by a
purse-string contraction. Plus-end transport along astral
microtubules of cortical F-actin (H3) and of myosin II
filaments (H4) could provide a mechanistic explanation for this
oft-hypothesized polar relaxation, while simultaneously
causing an increase in equatorial tension (assuming that
cortical contractile strength is proportional to co-localized F-actin
and myosin II concentrations). Either polar relaxation, or
equatorial strengthening, or both together, can set the stage for
the kind of actomyosin contraction-based cytokinesis that has been proposed (Foe, 2000 and references therein).
The establishment of the
contractile furrow is a mechanism requiring two
independent steps: the release in tension at the poles, preceded by (or coincident with) a
global increase in cortical tensioning. The global loss of actin
and myosin from the cortex during metaphase/anaphase and
their return beginning in telophase, which is observed in
Drosophila embryos (H1), if phylogenetically general, could
underlie the cyclic changes in cortical contractility. Operating together, H1-H4 are potentially
capable of implementing a 'global contraction-polar
relaxation' mechanism of furrow initiation for cytokinesis. If
cortical contractile strength is proportional to co-localized F-actin
and myosin II concentrations, then H3 and H4 would
bring about equatorial strengthening of the cortical actomyosin
meshwork, in principal also implementing 'equatorial
stimulation'. Note that, in flattened cells
with small asters, the microtubules in the mid-zone between
spindle poles are positioned to execute the same actomyosin
rearrangements as astral microtubules in spherical cells. Note also that H3 plus H4 can
cause equatorial concentration of whatever cytoplasmic
actomyosin network a cell contains, focusing internal forces on
the furrow cortex with the potential to aid furrow invagination
in non-spherical cells. Homologies between cleavage and
pseudocleavage, while attractive, come with the caveat that
cortical tensioning and microtubule
outgrowth both occur
earlier in the mitotic cycle during cytokinesis in echinoderms
than during pseudocleavage furrow formation in Drosophila
syncytia (Foe, 2000 and references therein).
In summary, this study has aimed to deduce, from
descriptions of wild-type and drug-perturbed cytoskeletal
kinematics of microtubules, F-actin and myosin II in syncytial
Drosophila embryos, the specific ways that these filament
systems must be interacting. The speculative mechanistic
hypotheses that were deduced, H1-H4, are consistent with a large body
of circumstantial and partial evidence reviewed above. This
machinery, if phylogenetically general, could unify old ideas
about cytokinesis with new molecular findings, reconcile polar
relaxation with equatorial stimulation models of furrow
formation, and homologize cytoskeletal pseudocleavage
furrow formation in syncytia with cleavage furrow formation
in mononucleate cells. Future revelations can be expected
of the molecular details by which the products of an ensemble
of key genes (e.g. anillin, centrosomin, diaphanous, KLP-3A,
pavarotti, polo kinase, Rac 1, septins, etc.) collaborate to bring
about and regulate the interactions between F-actin, myosin II,
centrosomes and microtubules (Foe, 2000).
This study addressed the role of tissue-scale physical forces during axis extension, using Drosophila germband extension (GBE) as a model. Previous studies have shown that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. This study further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, gastrulation movements were mapped temporally using light sheet microscopy to image whole Drosophila embryos. Both mesoderm and endoderm invaginations were found to be synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. Evidence of this force was sought in a simplified system without polarized cell intercalation, in acellular embryos. Posteriorwards Myosin II flows were identified towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. Tension was shown to be increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. It is proposeed that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis (Lye, 2015).
The mechanisms that allow or prevent tissue reorganization, especially in the presence of strongly anisotropic forces, remain unclear. This question was studied in the converging and extending Drosophila germband epithelium, which displays planar-polarized myosin II and experiences anisotropic forces from neighboring tissues. In contrast to isotropic tissues, cell shape alone is not sufficient to predict the onset of rapid cell rearrangement. From theoretical considerations and vertex model simulations, it is predicted that in anisotropic tissues, two experimentally accessible metrics of cell patterns-the cell shape index and a cell alignment index-are required to determine whether an anisotropic tissue is in a solid-like or fluid-like state. Changes in cell shape and alignment over time in the Drosophila germband were shown to predict the onset of rapid cell rearrangement in both wild-type and snail twist mutant embryos, where theoretical prediction is further improved when cell packing disorder is also accounted for. These findings suggest that convergent extension is associated with a transition to more fluid-like tissue behavior, which may help accommodate tissue-shape changes during rapid developmental events (Wang, 2020).
Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. This study developed optogenetic tools to activate (optoGEF) or deactivate (optoGAP) Rho1 signaling. These tools were used to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation, and the effects on contractile cell behaviors were analyzed. Uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar polarized pattern of myosin at cell junctions on the timescale of 3-5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar polarized myosin pattern in the germband during axis elongation. Moreover, these results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles, not only in organizing the cell rearrangements that are known to directly contribute to axis elongation, but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow (Herrera-Perez, 2021).
During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur simultaneously in different regions of a tissue. Using quantitative imaging in Drosophila melanogaster, this study investigate how patterned cell shape changes promote tissue bending during early embryogenesis. The transcription factors Twist and Snail combinatorially regulate a multicellular pattern of lateral F-actin density that differs from the previously described Myosin-2 gradient. This F-actin pattern correlates with whether cells apically constrict, stretch or maintain their shape. The Myosin-2 gradient and F-actin depletion do not depend on force transmission, suggesting that transcriptional activity is required to create these patterns. The Myosin-2 gradient width results from a gradient in RhoA activation that is refined through the balance between RhoGEF2 and the RhoGAP C-GAP. These experimental results and simulations of a 3D elastic shell model show that tuning gradient width regulates tissue curvature (Denk-Lobnig, 2021).
Compartment boundaries prevent cell mixing during animal development. In the Drosophila embryo, the mesectoderm is a group of glial precursors that separate ectoderm and mesoderm, forming the ventral midline. Mesectoderm cells undergo one round of oriented divisions during axis elongation and are eventually internalized approximately 6 h later. Using spinning disk confocal microscopy and image analysis, this study found that after dividing, mesectoderm cells reversed their planar polarity. The polarity factor Bazooka was redistributed to mesectoderm-mesectoderm cell interfaces, and the molecular motor non-muscle Myosin II and its upstream activator Rho-kinase (Rok) accumulated at mesectoderm-ectoderm (ME) interfaces, forming supracellular cables flanking the mesectoderm on either side of the tissue. Laser ablation revealed the presence of increased tension at ME cables, where Myosin was stabilized, as shown by fluorescence recovery after photobleaching. Laser nanosurgery was used to reduce tension at the ME boundary, and Myosin fluorescence decreased rapidly, suggesting a role for tension in ME boundary maintenance. Mathematical modelling predicted that increased tension at the ME boundary was necessary to prevent the premature establishment of contacts between the two ectodermal sheets on opposite sides of the mesectoderm, thus controlling the timing of mesectoderm internalization. The model was validated in vivo: Myosin inhibition disrupted the linearity of the ME boundary and resulted in early internalization of the mesectoderm. These results suggest that the redistribution of Rok polarizes Myosin and Bazooka within the mesectoderm to establish tissue boundaries, and that ME boundaries control the timely internalization of the mesectoderm as embryos develop (Yu, 2021).
An essential question of morphogenesis is how patterns arise without preexisting positional information. Cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Studies have suggested that biological polymers such as actomyosin gels have the property to self-organize. This study reports that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. A simple biophysical model is proposed where pattern formation arises from the interplay of myosin contractility and actin turnover. The hypotheses of the model was validated using photobleaching experiments, and it is reported that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, the model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring the model prediction of a transition from steady to chaotic actin patterns at low cortical friction. These results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting (Hannezo, 2015).
Convergence and extension movements elongate tissues during development. Drosophila germ-band extension (GBE) is one example, which requires active cell rearrangements driven by Myosin II planar polarisation. A combinatorial code of Toll receptors downstream of pair-rule genes contributes to this polarization via local cell-cell interactions. Novel computational methods have been developed to analyse the spatiotemporal dynamics of Myosin II. Initial Myosin II bipolar cell polarization gives way to unipolar enrichment at parasegmental boundaries and two further boundaries within each parasegment, concomitant with a doubling of cell number as the tissue elongates. These boundaries are the primary sites of cell intercalation, behaving as mechanical barriers and providing a mechanism for how cells remain ordered during GBE. Enrichment at parasegment boundaries during GBE is independent of Wingless signaling, suggesting pair-rule gene control. An updated cell-cell interaction model is proposed for Myosin II polarization that was tested in a vertex-based simulation (Tetley, 2016).
Tissue morphogenesis relies on the production of active cellular forces. Understanding how such forces are mechanically converted into cell shape changes is essential to understanding of morphogenesis. This study used myosin II pulsatile activity during Drosophila embryogenesis to study how transient forces generate irreversible cell shape changes. Analyzing the dynamics of junction shortening and elongation resulting from myosin II pulses, this study found that long pulses yield less reversible deformations, typically a signature of dissipative mechanics. This is consistent with a simple viscoelastic description, which was used to model individual shortening and elongation events. The model predicts that dissipation typically occurs on the minute timescale, a timescale commensurate with that of force generation by myosin II pulses. This estimate was tested by applying time-controlled forces on junctions with optical tweezers. Finally, it was shown that actin turnover participates in dissipation, as reducing it pharmacologically increases the reversibility of contractile events. These results argue that active junctional deformation is stabilized by actin-dependent dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation (Clement, 2017).
During embryogenesis tissue layers undergo morphogenetic flow rearranging and folding into specific shapes. While developmental biology has identified key genes and local cellular processes, global coordination of tissue remodeling at the organ scale remains unclear. This study combined in toto light-sheet microscopy of the Drosophila embryo with quantitative analysis and physical modeling to relate cellular flow with the patterns of force generation during the gastrulation process. The complex spatio-temporal flow pattern can be predicted from the measured meso-scale myosin density and anisotropy using a simple, effective viscous model of the tissue, achieving close to 90% accuracy with one time dependent and two constant parameters. This analysis uncovers the importance of a) spatial modulation of myosin distribution on the scale of the embryo and b) the non-locality of its effect due to mechanical interaction of cells, demonstrating the need for the global perspective in the study of morphogenetic flow (Streichan, 2018).
The actomyosin cytoskeleton, a key stress-producing unit in epithelial cells, oscillates spontaneously in a wide variety of systems. Although much of the signal cascade regulating myosin activity has been characterized, the origin of such oscillatory behavior is still unclear. This study shows that basal myosin II oscillation in Drosophila ovarian epithelium is not controlled by actomyosin cortical tension, but instead relies on a biochemical oscillator involving ROCK and myosin phosphatase. Key to this oscillation is a diffusive ROCK flow, linking junctional Rho1 to medial actomyosin cortex, and dynamically maintained by a self-activation loop reliant on ROCK kinase activity. In response to the resulting myosin II recruitment, myosin phosphatase is locally enriched and shuts off ROCK and myosin II signals. Coupling Drosophila genetics, live imaging, modeling, and optogenetics, this study uncovered an intrinsic biochemical oscillator at the core of myosin II regulatory network, shedding light on the spatio-temporal dynamics of force generation (Qin, 2018).
Epithelial folding shapes embryos and tissues during development. This study investigated the coupling between epithelial folding and actomyosin-enriched compartmental boundaries. The mechanistic relationship between the two is unclear, because actomyosin-enriched boundaries are not necessarily associated with folds. Also, some cases of epithelial folding occur independently of actomyosin contractility. Shallow folds called parasegment grooves that form at boundaries between anterior and posterior compartments in the early Drosophila embryo were investigated. Formation of these folds requires the presence of an actomyosin enrichment along the boundary cell-cell contacts. These enrichments, which require Wingless signalling, increase interfacial tension not only at the level of the adherens junctions but also along the lateral surfaces. Epithelial folding is normally under inhibitory control because different genetic manipulations, including depletion of the Myosin II phosphatase Flapwing, increase the depth of folds at boundaries. Fold depth correlates with the levels of Bazooka (Baz), the Par-3 homologue, along the boundary cell-cell contacts. Moreover, Wingless and Hedgehog signalling have opposite effects on fold depth at the boundary that correlate with changes in Baz planar polarity (Urbano, 2020).
Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previous studies have shown that cell-cell fusion is an asymmetric process in which an 'attacking' cell drills finger-like protrusions into the 'receiving' cell to promote cell fusion. This study shows that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by chemical signaling initiated by cell adhesion molecules. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. It is proposed that the protrusive and resisting forces from fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion (Kim, 2015). A preview of this article is available: Myoblast Fusion: Playing Hard to Get
During Drosophila germ-band extension (GBE), cell intercalation is the key mechanism for tissue extension, and the associated apical junction remodelling is driven by polarized myosin-II-dependent contraction. However, the contribution of the basolateral cellular mechanics to GBE remains poorly understood. This study characterized how cells coordinate their shape from the apical to the basal side during rosette formation, a hallmark of cell intercalation. Basolateral rosette formation is driven by cells mostly located at the dorsal/ventral part of the rosette (D/V cells). These cells exhibit actin-rich wedge-shaped basolateral protrusions and migrate towards each other. Surprisingly, the formation of basolateral rosettes precedes that of the apical rosettes. Basolateral rosette formation is independent of apical contractility, but requires Rac1-dependent protrusive motility. Furthermore, Src42A was identified as a regulator of basolateral rosette formation. The data show that in addition to apical contraction, active cell migration driven by basolateral protrusions plays a pivotal role in rosette formation and contributes to GBE (Sun, 2017).
The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. This study found that Death-associated protein kinase related (Drak) is necessary for most of the phosphorylation of myosin regulatory light chain during cellularization. Drak was shown to be required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak's regulation of myosin activity. Drak also is involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, these observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization (Chougule, 2016).
Tight regulation of actomyosin is likely critical for many cellular processes, but how this is accomplished is as yet poorly understood. A key input to the regulation of myosin II is through phosphorylation of the Serine-19, or the Serine-19 and Threonine-18 residues of MRLC (Spaghetti squash). The variety of MRLC kinases might allow different specific aspects of actomyosin dynamics, such as localization, organization and contraction to be regulated independently. Such a system would provide greater flexibility and control than either a single kinase, or multiple kinases acting in concert, regulating all of these functions. drak was found to be required for the organization of myosin II into contractile rings, but is not required for localization of myosin to the cellularization front. Since the majority of Sqh phosphorylation during cellularization is dependent on drak activity, Drak either regulates most aspects of myosin II dynamics during cellularization, or Drak-regulated myosin II organization is required for further function of myosin II, such as contraction (Chougule, 2016).
Myosin II is somewhat less disorganized and Sqh phosphorylation is slightly increased during late cellularization in drak mutants, suggesting that phosphorylation of myosin II by other kinases occurs during late cellularization. Thus other kinases might act synergistically with Drak to regulate actomyosin organization during late cellularization. For example, Drak function has been shown to be partially redundant with Rok function during later development. An alternative possibility is that other kinases that do not normally function in myosin II organization in the microfilament rings might phosphorylate Sqh to some degree and lead to some organization of myosin II in the absence of Drak activity (Chougule, 2016).
Myosin II has been implicated in actin bundling and F-actin organization in some contexts. Since F-actin appears to
be organized normally within drak mutant microfilament rings during early cellularization, it is concluded that myosin II does not play a role in initially organizing F-actin within the microfilament rings during cellularization. F-actin is somewhat disorganized during late cellularization in drakdel mutant embryos, but not as severely as myosin II, nor does the pattern of F-actin distribution fit the pattern of myosin II distribution in drakdel mutant embryos. These observations suggest that F-actin disorganization is an indirect consequence of Drak regulation of myosin II activity, and that F-actin disorganization might be due to actomyosin contraction defects or furrow canal structural defects (Chougule, 2016).
Anillin is required for the organization of actomyosin contractile rings during cellularization and cytokinesis. scraps (scra, anillin) mutant embryos have a myosin II organization defect somewhat similar to that of drak mutant embryos: myosin II is found in discrete bars in the actomyosin network. Despite this similarity, myosin II defects differ between scra and drak mutant embryos. Myosin II becomes more disorganized during late cellularization in scra mutant embryos. Myosin II becomes slightly better organized during late cellularization in drak mutant embryos. This organizational difference is likely caused by actomyosin contraction during microfilament ring constriction occurring in a highly disorganized cytoskeleton in scra mutant embryos, and occurring in a disorganized cytoskeleton that has slightly improved during constriction in drak mutant embryos. Anillin only interacts with myosin II when MRLC is phosphorylated. Together with these results, this suggests that Drak phosphorylation of Sqh might be necessary for Anillin-mediated myosin II organization within the contractile ring (Chougule, 2016).
Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 leads to the
unfolding of inactive myosin II hexamers into an open conformation that allows assembly of bipolar myosin II filaments and their association with F-actin to form actomyosin filaments. This is likely how Drak organizes myosin II. Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 also leads to the activation of the Mg2+-ATPase activity of myosin II that slides actin filaments past each other, causing actomyosin contraction. Three aspects of the drak mutant phenotype support the requirement for Drak in actomyosin contraction: wavy cellularization fronts caused by non-uniform furrow canal depths, abnormal microfilament ring shapes, and failure of microfilament rings to constrict during late cellularization. These are the same defects that suggest an actomyosin contraction defect in src64 mutant embryos. However, src64 mutant embryos do not show myosin II organization defects. Because effective actomyosin contraction likely requires properly organized actomyosin filaments within the contractile ring apparatus, it is unclear whether Drak directly regulates actomyosin contraction or whether Drak only enables actomyosin contraction through proper organization of myosin II within the microfilament rings. One possibility is that phosphorylation of Sqh by Drak both organizes actomyosin filaments into a contractile ring apparatus and directs actomyosin contraction. An alternative possibility is that Drak is directly responsible for organizing actomyosin filaments into a contractile ring by phosphorylating Sqh, but Drak is not directly involved in its contraction and different kinases that phosphorylate Sqh regulate actomyosin contraction. Thus, Drak could be an early regulator of myosin II activity
during cellularization, such that further phosphorylation of Sqh and myosin II-driven contraction is dependent on Drak-mediated organization of myosin II. At some level the regulation of actomyosin contraction diverges from the regulation of actomyosin filament organization: Src64 is required for contraction, but has no role in myosin II organization (Chougule, 2016).
Rescue of myosin II organization, actomyosin contraction and F-actin distribution defects in drak mutant embryos by the mono-phosphorylated SqhE21 phosphomimetic suggests that Drak-mediated mono-phosphorylation of Sqh at Serine-21 is sufficient for regulation of actomyosin dynamics during cellularization. Although the diphosphorylated SqhE20E21 phosphomimetic also rescues myosin II organization and actomyosin contraction defects, it does not rescue F-actin distribution defects in drak mutant embryos. These results are consistent with Drak primarily phosphorylating Sqh at Serine-21, and are consistent with reports that DAPK family members phosphorylate MRLC mainly at Serine-19 (Chougule, 2016).
The normal teardrop shape of the furrow canals in early cellularization is likely caused by actomyosin contraction in the microfilament rings. In drak mutant embryos, unexpanded early cellularization furrow canals and failure of many late cellularization furrow canals to expand further suggest that Drak is required for proper furrow canal structure. Some of the furrow canal structural defects in drak mutant embryos are similar to those of nullo mutant embryos: collapsed furrow canals and blebbing. However, nullo mutant embryos, as well as RhoGEF2 or dia mutant embryos, have other, more severe furrow canal defects: missing or regressing furrow canals and compromised lateral membrane-furrow canal compartment boundaries. Furthermore, cytochalasin treatment causes similar defects, suggesting that reduced F-actin levels in the furrow canals are responsible for these defects. Thus Nullo, RhoGEF2 and Dia regulate F-actin and its levels in furrow canals. These observations suggest that Drak regulates myosin II and thereby regulates actomyosin organization and contraction, and that these are necessary for structural integrity and expansion of the furrow canals, but not for their continued existence (Chougule, 2016).
The furrow canals of drak mutant embryos during late cellularization show extensive blebbing into the lumens. This is consistent with a defect in furrow canal membrane or cortex integrity. Blebs can be formed by local rupture of the cortical cytoskeleton or detachment of the plasma membrane from the cortical actomyosin cytoskeleton. Actomyosin contraction has been implicated in bleb formation. Therefore, it is proposed that blebbing in furrow canals is caused by aberrant localized actomyosin contraction during late cellularization in the disorganized actomyosin cytoskeleton of drak mutant embryos. Contraction is presumably driven by phosphorylation of Sqh by kinases other than Drak. Since actomyosin contraction occurs in a disorganized actomyosin cytoskeleton, it does not lead to uniform constriction of the microfilament rings, but instead leads to localized contraction that produces cytoplasmic blebs. However, other causes for furrow canal defects are possible. Plasma membrane attachment sites might not form or function properly in the disorganized furrow canal cytoskeleton in drak mutant embryos. The disorganized cytoskeleton might inhibit vesicle trafficking. Vesicle trafficking itself might be
defective: mammalian DAPKs have been shown to be involved in membrane trafficking and in phosphorylation of syntaxin A1.
Vesiculated lateral plasma membrane in drak mutant embryos during late cellularization suggests that the plasma membrane breaks down. Intriguingly, scra mutant embryos have lines of vesicles where the closely apposed lateral plasma membranes would have been. However in scra mutant embryos, vesiculation is observed during early cellularization, but to a lesser extent than during late cellularization. drak mutant embryos do not show lateral plasma membrane vesiculation defects until late cellularization. drak mutant defects in both the furrow canal membrane and the lateral plasma membrane might reflect a general defect in membrane integrity. It will be interesting to investigate the potential role of myosin II organization in furrow canal structure and plasma membrane integrity (Chougule, 2016).
Pulsatile actomyosin contractility, important in tissue morphogenesis, has been studied mainly in apical but less in basal domains. Basal myosin oscillation underlying egg chamber elongation is regulated by both cell-matrix and cell-cell adhesions. However, the mechanism by which these two adhesions govern basal myosin oscillation and tissue elongation is unknown. This study demonstrates that cell-matrix adhesion positively regulates basal junctional Rho1 activity and medio-basal ROCK and myosin activities, thus strongly controlling tissue elongation. Differently, cell-cell adhesion governs basal myosin oscillation through controlling medio-basal distributions of both ROCK and myosin signals, which are related to the spatial limitations of cell-matrix adhesion and stress fibres. Contrary to cell-matrix adhesion, cell-cell adhesion weakly affects tissue elongation. In vivo optogenetic protein inhibition spatiotemporally confirms the different effects of these two adhesions on basal myosin oscillation. This study highlights the activity and distribution controls of basal myosin contractility mediated by cell-matrix and cell-cell adhesions, respectively, during tissue morphogenesis (Qin, 2017).
During epithelial cytokinesis, the remodelling of adhesive cell-cell contacts between the dividing cell and its neighbours has profound roles in the integrity, arrangement and morphogenesis of proliferative tissues. In both vertebrates and invertebrates, this remodelling requires the activity of non-muscle myosin II (MyoII) in the interphasic cells neighbouring the dividing cell. However, the mechanisms coordinating cytokinesis and MyoII activity in the neighbours are unknown. This study found that in the Drosophila notum epithelium, each cell division is associated with a mechano-sensing and transmission event controlling MyoII dynamics in the neighbours. The ring pulling forces promote local junction elongation, resulting in local E-cadherin (E-Cad) dilution at the ingressing adherens junction (AJ). In turn, the reduction of E-Cad concentration and the contractility of the neighbouring cells promote self-organized actomyosin flows, ultimately leading to MyoII accumulation at the base of the ingressing AJ. While force transduction has been extensively studied in the context of AJ reinforcement to stabilize adhesive cell-cell contacts, an alternative mechano-sensing mechanism able to coordinate actomyosin dynamics between epithelial cells and to sustain AJ remodelling in response to mechanical forces is proposed (Pinheiro, 2017).
Cell delamination is a conserved morphogenetic process important for generation of cell diversity and maintenance of tissue homeostasis. This study used Drosophila embryonic neuroblasts as a model to study the apical constriction process during cell delamination. Dynamic myosin signals both around the cell adherens junctions and underneath the cell apical surface in the neuroectoderm. On the cell apical cortex the non-junctional myosin forms flows and pulses, which are termed as medial myosin pulses. Quantitative differences in medial myosin pulse intensity and frequency are critical to distinguish delaminating neuroblasts from their neighbors. Inhibition of medial myosin pulses blocks delamination. The fate of neuroblasts is set apart from their neighbors by Notch signaling-mediated lateral inhibition. When Notch signaling activity was inhibited in the embryo, it was observed that small clusters of cells undergo apical constriction and display an abnormal apical myosin pattern. Together, this study demonstrates that a contractile actomyosin network across the apical cell surface is organized to drive apical constriction in delaminating neuroblasts (An, 2017).
Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of myosin II. For instance, during Drosophila gastrulation, apical constriction and cell intercalation are mediated by medial-apical myosin II pulses that power deformations, and polarized accumulation of myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of myosin II activation and the ratchet-like myosin II dynamics. This study reports the function of a common pathway comprising the heterotrimeric G proteins Gα12/13 (Concertina), Gβ13F and Gγ1 in activating and polarizing myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog (Poor gastrulation, Pog & CG31660) was identified as being required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of myosin II and irreversible cell shape changes. It is proposed that GPCR and G proteins constitute a general pathway for controlling actomyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators (Kerridge, 2016).
During tissue morphogenesis, cells rearrange their contacts to invaginate, intercalate, delaminate or divide. During Drosophila gastrulation, invagination of the presumptive mesoderm in the ventral region of the embryo and of the posterior midgut requires apical cell constriction, a geometric deformation that occurs in different organisms. Elongation of the ventral–lateral ectoderm requires cell intercalation, a general topological deformation associated with junction remodelling. In the ectoderm, the so-called ‘vertical junctions’, oriented along the dorsal–ventral axis, shrink, followed by extension of new ‘horizontal’ junctions along the anterior–posterior axis. Despite differences in the cell deformations associated with intercalation and apical constriction, recent studies revealed that both processes require myosin II (MyoII) contractility. Cell shape changes rely on the pulsatile activity of MyoII in the apical–medial cortex, whereby MyoII undergoes cycles of assembly and disassembly allowing stepwise deformation1. Moreover, each step of deformation is stabilized and thereby retained, contributing to the irreversibility of tissue morphogenesis. In the mesoderm, each phase of apical area constriction mediated by MyoII pulses is followed by a phase of shape stabilization involving persistence of medial MyoII. In the ectoderm, medial–apical MyoII pulses flow anisotropically towards vertical junctions resulting in steps of shrinkage that are stabilized by a planar-polarized pool of junctional MyoII. This ratchet-like behaviour of MyoII is regulated by the Rho1–Rok pathway and requires quantitative control over MyoII activation. Low Rho1/Rok activity fails to form actomyosin networks, intermediate activation establishes MyoII pulsatility and high activation confers stability. The signalling mechanisms that cause stepwise activation of MyoII by Rho1 remain unknown. It is also unclear whether different pathways for Rho1 activation operate in the mesoderm and in the ectoderm as indeed Rho1 can be activated by numerous signalling mechanisms or whether a common pathway might exist (Kerridge, 2016).
Tissue-specific factors can result in polarized shape changes by signalling through cell surface receptors. For instance, in Drosophila ectoderm, pair rule genes encoding transcription factors control planar-polarized enrichment of MyoII through the combinatorial expression of the surface proteins Toll2, Toll6 and Toll8 in stripes. Likewise, in the mesoderm, Twist and Snail induce expression of Fog, a secreted ligand, and a G-protein-coupled receptor (GPCR) Mist (methuselah-like 1), which is reported to transduce Fog. The downstream G protein Gα12/13 (known as Concertina (Cta) in Drosophila) is required for RhoGEF2 and thereby MyoII apical recruitment. As RhoGEF2 is a known GEF for Rho1, the requirement of Gα12/13 for RhoGEF2 apical recruitment suggests that GPCRs and G-protein signalling mediate MyoII activation through the Rho1 pathway. These considerations prompted asking whether G-protein signalling directly controls the different regimes of MyoII dynamics (pulsatility and/or stability) in the mesoderm and planar polarized activation of Rho1 and MyoII in the ectoderm (Kerridge, 2016).
This study reports the function of the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and regulating MyoII dynamics both in the mesoderm and in the ectoderm. Receptor activation, through the GEF activity of the GPCR, converts Gα from an inactive GDP-bound state, in a complex with Gβγ, to an active GTP-bound state. This results in dissociation of Gβγ, enabling binding of both Gα–GTP and Gβγ to their respective effectors for signalling. This study found that Gα12/13 and the Gβ13F/Gγ1 complex constitute distinct signalling modules, which regulate MyoII dynamics medial–apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog, was found to be required for cell shape changes associated with both mesoderm invagination and ectoderm elongation. During these morphogenetic events, Smog functions with other GPCRs, Mist in the mesoderm and an as yet unknown GPCR in the ectoderm, to activate the Rho1–Rok pathway. This results in stepwise activation of Rho1 and MyoII, ensuring irreversible cell shape changes (Kerridge, 2016).
First, this study reports that Gα12/13 and Gβ13F/Gγ1 function as distinct signalling modules that control Rho1 and MyoII in different domains. Gα12/13 activates medial–apical MyoII through its effector RhoGEF2 both in the ectoderm and the mesoderm. In mammals, p115–RhoGEF interacts directly with Gα12 suggesting that this may be a conserved signalling module. In contrast, Gβ13F/Gγ1 activates MyoII both at cell junctions and in the medial–apical domain. This modularity may provide distinct regulatory mechanisms for the activation of MyoII in different subcellular compartments owing to the existence of different molecular effectors of Gα–GTP and Gβγ. Second, stepwise activation of Rho1 by multiple GPCRs and their ligands determines the emergence of a pulsatile regime medial–apically, or stable activation. In the mesoderm, Smog and Mist GPCRs, together with high expression of their ligand Fog, ensure stabilization and rapid (<5 min) accumulation of MyoII ensuring apical constriction. In the ectoderm, low Fog expression and thus lower activation of Gα12/13 and RhoGEF2 is responsible for intermediate medial–apical activation of MyoII and pulsatility. Indeed, Fog, constitutively active Gα12/13QL and RhoGEF2 overexpression all lead to stable accumulation of MyoII instead of pulsation, similar to constitutively active RhoV14 (Kerridge, 2016).
Interestingly, the same receptor Smog controls MyoII activation in different subcellular domains during intercalation and apical constriction begging the question of how activation of Gα12/13 and Gβγ is differentially achieved in the ectoderm and the mesoderm. The polarization of Smog activation is to some extent imparted by the ligand. Fog/Smog regulates medial–apical accumulation of MyoII in the two tissues: Fog induces medial Rho1 and Rok activation in the mesoderm and ectoderm and, when ectopically expressed in the ectoderm, it can increase Rho1 and Rok in the medial cortex. This argues that another mechanism results in junction-specific activation of Smog, Gβ13F/Gγ1, Rho1 and Rok in the ectoderm (Kerridge, 2016).
It is possible that an unknown ectoderm-specific ligand activates Smog specifically at junctions. Junctional localization of the Rho1 pathway by Smog may also be imparted by subcellular processing of Smog signalling, such as localization/activation of downstream effectors of Gα12/13 and Gβγ. The recently identified Toll receptors required for MyoII planar-polarized activation may bias Smog signalling although the molecular mechanisms remain unclear. This could be through localization of RhoGEFs. In the mesoderm, the transmembrane protein T48 localizes RhoGEF2 apically through binding to its PDZ domain, and is required for apical MyoII activation in parallel with Smog, Gα12/13 and Gβγ. Similarly, other GEFs may be required for junctional Rho1 activation by Smog (Kerridge, 2016).
What might be the advantage of having multiple GPCRs? Gastrulation sets the foundation for all other future processes in development and hence requires robustness. GPCRs with similar functions yet subtle differences such as ligand specificity may offer advantages compared with single ligand–receptor pairs. For instance, high cortical tension associated with mesoderm invagination may require multiple GPCRs activating parallel pathways to attain efficiency of the process. Moreover, multiple GPCRs may concede tissue-specific regulation of the common G-protein subcellular pathways. Finally, multiple GPCRs can allow stepwise activation of MyoII. Although activation by one GPCR is sufficient to induce pulsatility, more GPCRs are required to shift the actomyosin networks to more stable regimes (Kerridge, 2016).
The discovery that Smog and heterotrimeric G protein activate Rho1 and MyoII in two different morphogenetic processes provides a potentially general molecular framework for tissue mechanics. It is proposed that different developmental inputs tune a common GPCR/G-protein signalling pathway to direct specific patterns and levels of Rho1 activation. Quantitative control specifies the regime of MyoII activation through Rho1, namely pulsatility or stability of MyoII. Modular control defines the subcellular domains where MyoII accumulates (medial–apical or junctions) depending on molecular effectors. How developmental signals tune GPCR signalling will be important to decipher (Kerridge, 2016).
Actomyosin networks provide the major contractile machinery for regulating cell and tissue morphogenesis during development. These networks undergo dynamic rearrangements, enabling cells to have a broad range of mechanical actions. How cells integrate different mechanical stimuli to accomplish complicated tasks in vivo remains unclear. This study explored this problem in the context of cell matching, where individual cells form precise inter-cellular connections between partner cells. To study the dynamic roles of actomyosin networks in regulating precise cell matching, focus was placed on the process of heart formation during Drosophila embryogenesis, where selective filopodia-binding adhesions ensure precise cell alignment. Non-muscle Myosin II clusters periodically oscillate within cardioblasts with ~4-min intervals. Filopodia dynamics-including protrusions, retraction, binding stabilization, and binding separation-are correlated with the periodic localization of Myosin II clusters at the cell leading edge. Perturbing the Myosin II activity and oscillatory pattern alters the filopodia properties and binding dynamics and results in mismatched cardioblasts. By simultaneously changing the activity of Myosin II and filopodia adhesion levels, it was further demonstrated that levels of Myosin II and adhesion are balanced to ensure precise connectivity between cardioblasts. Combined, a mechanical proofreading machinery of robust cell matching is proposed, whereby oscillations of Myosin II within cardioblasts periodically probe filopodia adhesion strength and ensure correct cell-cell connection formation (Zhang, 2020).
Although Snail is essential for disassembly of adherens junctions during epithelial-mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, the behavior of E-cadherin-rich junction clusters were tracked, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. These findings provide in vivo evidence for mechanosensitivity of cell-cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT (Weng, 2016).
This study shows that during Drosophila gastrulation, subapical junctions are repositioned toward the apical surface and are strengthened as the cortical tension increases. Both these phenomena follow apical myosin activation and thus may reflect a mechanosensitive response of junctional complexes to the tension generated by this activation of myosin. The junctional responses occur on the time scale of individual myosin pulses and are temporally correlated with those pulses. Such junctional changes depend on myosin activity but do not require Sna, given that ectopic myosin activation recapitulates similar junctional responses in Sna-negative tissues. This phenomenon may not be restricted to Drosophila embryos. The increased contractile actomyosin on the apical cortex of human cell lines deficient for the cortex actin regulator Merlin is associated with a condensation of adherens junctions toward the apical surface, suggesting that the response of adherens junctions to cortical tension can be of general significance (Weng, 2016).
The changes in junction mass and density suggest that, rather than being simple passive anchors for contractile actomyosin filaments, adherens junctions respond to the contractile actomyosin by restructuring and repositioning themselves, potentially involving aggregation and rearrangement of E-Cad molecules within the plasma membrane or vesicle-based redistribution of E-Cad. Indeed, actomyosin organization has been shown to be critical in the lateral clustering of E-Cad molecules. The change in E-Cad clustering is considered an active mechanosensitive mechanism to strengthen the adhesion. Alternatively, the adhesion can also be remodeled through the vesicle-based mechanisms, and endocytosis of E-Cad has been shown to be up-regulated when junctions are under actomyosin-generated stress. The repositioning could also arise through restructuring rather than passive dragging, if for example recycling and turnover rates in the basal regions of the junctions differ from apical regions. Overall, regardless of the underlying mechanism, this mechanosensitivity may be advantageous, providing a direct self-corrective mechanism that allows junctions to adjust their localization and intensity to match the mechanical force they experience (Weng, 2016).
Although the molecular mechanism for the junction strengthening requires further investigation, the data suggest that it is resistant to the posttranscriptional disassembly of adherens junctions downstream of Sna. The phenotype of myosin knockdown in this study resembles that previously described for cta; T48 double mutants, in which apical actomyosin cannot be activated and junctions are lost only in the ventral mesodermal cells. In all scenarios in which Sna expression is associated with junction loss (ventral cells in cta; T48 mutants, ventral cells in myosin knockdown mutants, and ectodermal cells with ectopic Sna expression), Sna is expressed in cells in the absence of myosin contractility. Maintenance of adherens junctions ultimately relies on the balance between assembly and disassembly rates of junctional components. Thus mechanical force likely modulates the assembly/disassembly balance and therefore remains in a homeostatic relationship with the junctions bearing the force (Weng, 2016).
In the early stages of embryogenesis analyzed in this study, E-Cad is maternally provided and thus not subject to direct transcriptional repression. The disassembly of junctions in the absence of myosin contraction must therefore reflect a posttranscriptional regulation on junctions, likely performed by one or several of Sna’s transcriptional targets. Much effort has been invested in identifying transcription targets of Sna, but it is not known which, if any, of its known targets might play such a role. One mesodermally expressed gene, Traf4, is required for fine-tuning junction morphology, but its expression appears to depend on the other mesodermal determinant, Twist, rather than Sna. One gene repressed by Sna in Drosophila mesoderm, bearded, is required for the subapical positioning of adherens junctions in cells not expressing Snail. It is not clear, however, whether Bearded plays a direct role in junction disassembly or a more general role in apical polarity or the apical myosin contractility that drives repositioning. The posttranscriptional regulation of adherens junction disassembly may allow more rapid and effective EMT than a disassembly relying on transcriptional down-regulation of junctional components such as E-Cad. Identifying and characterizing the relevant Sna targets in Drosophila may provide insights into the underlying mechanism for this disassembly, especially with respect to its apparent sensitivity to externally exerted tension. The force-dependent resistance to this Sna function may help in dissecting the underlying molecular functions. Further exploration of Sna’s posttranscriptional effect on junctions and how myosin contraction antagonizes Sna will shed light on understanding of EMT processes (Weng, 2016).
Tissue morphogenesis arises from controlled cell deformations in
response to cellular contractility. During Drosophila gastrulation, apical activation
of the actomyosin networks drives apical constriction in the
invaginating mesoderm and cell-cell intercalation in the extending
ectoderm. Myosin II (MyoII; Zipper) is
activated by cell-surface G protein-coupled receptors (GPCRs), such as
Smog and Mist, that activate G
proteins, the small GTPase Rho1, and
the kinase Rok. Quantitative
control over GPCR and Rho1 activation underlies differences in
deformation of mesoderm and ectoderm cells. The GPCR Smog activity is
concentrated on two different apical plasma membrane compartments, i.e.,
the surface and plasma membrane invaginations. Using fluorescence
correlation spectroscopy, the surface of the plasma membrane was
probed, and it was shown that Smog homo-clusters in response to its
activating ligand Fog. Endocytosis
of Smog is regulated by the kinase Gprk2 and beta-arrestin-2 that clears
active Smog from the plasma membrane. When Fog concentration is high or
endocytosis is low, Smog rearranges in homo-clusters and accumulates in
plasma membrane invaginations that are hubs for Rho1 activation. Lastly,
this study found higher Smog homo-cluster concentration and numerous
apical plasma membrane invaginations in the mesoderm compared to the
ectoderm, indicative of reduced endocytosis. Dynamic partitioning of
active Smog at the surface of the plasma membrane or plasma membrane
invaginations has a direct impact on Rho1 signaling. Plasma membrane
invaginations accumulate high Rho1-guanosine triphosphate (GTP)
suggesting they form signaling centers. Thus, Fog concentration and Smog
endocytosis form coupled regulatory processes that regulate differential
Rho1 and MyoII activation in the Drosophila embryo (Jha, 2018).
Tissue morphogenesis requires control over changes in cell shape and cell-cell contacts, which depend on the spatiotemporal regulation of actomyosin contractility. In Drosophila embryos, mesoderm invagination is driven by apical constriction, a geometric cell shape change facilitated by medial-apical Myosin II activation. In the ectoderm, tissue extension arises from cell-cell intercalation, whereby cells undergo neighbor exchange through the polarized remodeling of cell junctions. Junction remodeling is driven by medial-apical MyoII contractile pulses and MyoII planar polarized accumulation (Jha, 2018).
Actomyosin contractility is regulated by conserved signaling pathways. MyoII regulatory light chain is activated by Rho-kinase (Rok) downstream of the small GTPase Rho1, which in turn is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). This conserved pathway was shown to be under the direct control of signaling at the cell surface, such as Celsr in vertebrate neural tube formation and G protein-coupled receptors (GPCRs) in early Drosophila embryos. The GPCRs Mist (Manning, 2013) and Smog (Kerridge, 2016) transduce signals from the secreted ligand Fog in the Drosophila presumptive mesoderm (Mist and Smog) and ectoderm (Smog). Medial-apical MyoII activation progresses downstream of hetero-trimeric G proteins Gα12/13, Gβ13F, and Gγ1 in both mesoderm and ectoderm. In the mesoderm, high medial-apical MyoII activation is under a stable regime that ensures persistent apical constriction, while in the ectoderm, intermediate medial-apical MyoII activation is under a pulsatile regime that enables cell-cell intercalation. Therefore, to understand how quantitative activation of MyoII is generated and its temporal dynamics encoded, it is necessary to decipher the regulation of GPCR signaling (Jha, 2018).
Differential MyoII activation in the mesoderm and ectoderm is partly imparted by the ligand Fog, co-expression of Mist and Smog in the mesoderm, as well as by the mesoderm-specific transmembrane protein T48, which enhances apical recruitment of RhoGEF2 and, thereby, is proposed to potentiate Rho1 and MyoII activation. High Fog expression in mesoderm activates high MyoII, while in the ectoderm low Fog expression leads to low activation of MyoII. However, in general, ligand availability is one of several mechanisms impacting GPCR activation and signaling. Various cell culture studies have focused on the other modalities that regulate GPCR signaling. The major regulators of GPCR signaling are G protein-coupled receptor kinases (GRKs) that phosphorylate GPCRs and trigger signal termination, by allowing β-arrestin binding and recruitment of other adaptor proteins. In turn, β-arrestins direct activated receptors to clathrin-coated pits and remove them from the plasma membrane by endocytosis. While removal of activated GPCRs from the plasma membrane via endocytosis terminates GPCR signaling, it also reduces the number of receptors present on the surface for ligand stimulation. This effectively sets a quantitative control over GPCR signaling via endocytosis. Drosophila has only one non-visual GRK (Gprk2) and one non-visual β-arrestin-2 (kurtz). Gprk2 mutant mothers show aberrant contractility in the mesoderm lateral cells, and it was suggested that Gprk2 attenuates Fog-dependent MyoII activation in these cells. Eggs lacking Kurtz display cuticle phenotypes and suggest gastrulation defects. These data indicate that Kurtz plays a role with Gprk2 to terminate Fog signaling and could control Rho1 and MyoII via GPCR endocytosis. Its function in the mesoderm and ectoderm has not been addressed (Jha, 2018).
Conventionally, GPCR signaling from the plasma membrane is thought to occur via ligand binding and subsequent signal transduction via G proteins that relay the information to the interior of the cell. Apart from GPCR endocytosis, the localization of GPCR within the cell membrane will influence GPCR signaling. Lateral movement of GPCRs within the plasma membrane is often restricted to specific nano-domains, suggesting that selective compartmentalization is necessary for efficient signaling as it can increase GPCR localization and clustering. GPCR clustering in the form of homo- and hetero-oligomers has been reported to control both signal amplification as well as receptor recycling. Whether the main role of GPCR clustering is for chaperoning active receptors for transport or to control GPCR signaling specificity remains unclear, especially during development. To understand GPCR signaling during tissue morphogenesis, it is important to elucidate both the clustering of GPCRs at the plasma membrane and the role of endocytosis (Jha, 2018).
This study investigated the quantitative regulation of the GPCR Smog signaling by endocytosis in both the ectoderm and the mesoderm. Fog was shown to promote homo-clusters of Smog, while endocytosis rapidly removes Smog homo-clusters from the surface of the plasma membrane in the ectoderm. Dynamic partitioning of active Smog homo-clusters in two plasma membrane compartments, the surface or the plasma invaginations, was shown to directly impact Rho1 and MyoII activation. In the mesoderm, numerous apical plasma membrane invaginations and high Smog homo-clusters correlate with high Rho1 and MyoII activation compared to the ectoderm (Jha, 2018).
Epithelial cells exhibit different types of cell deformations owing to quantitative control over cell contractility that arises from contraction of the actomyosin cytoskeleton. GPCR signaling relays information conveyed by tissue-specific factors in the mesoderm and ectoderm to control this quantitative regulation during tissue morphogenesis. Rho1-dependent activation of MyoII during both apical constriction in the mesoderm and cell-cell intercalation in the ectoderm is controlled by GPCR signaling. Activation of the GPCR Smog underlies Rho1 activation in both mesoderm and ectoderm. It is believed that differential regulation of the GPCR Smog and other GPCRs underlies these tissue-specific differences in MyoII activation. This partly relies on the fact that Fog, the activating ligand, is present at higher levels in the mesoderm than in the ectoderm. This work sheds new light on this process by probing the plasma membrane organization and distribution of Smog in conditions that affect both endocytosis and production of the ligand Fog (Jha, 2018).
Probing the ectodermal cells with FCS, Smog homo-clusters on the surface of apical plasma membrane is reported and this process depends on Fog. When Fog is absent, such as in a fog-dsRNA, the brightness per Smog::GFP unit is lower, suggesting that Fog induces the formation of Smog homo-clusters. Dynamic exchange of homo-clustered Smog occurs sbetween the surface and plasma membrane invaginations. This dynamic distribution of Smog between the two plasma membrane compartments is strongly dependent upon both the rate of Smog endocytosis and Fog concentration. Increasing Fog concentration or reducing Smog endocytosis enhances the presence of Smog homo-clusters in apical plasma membrane invaginations, which results in an apparent decrease in Smog homo-clusters at the cell surface. When Fog concentration is high under conditions where Smog endocytosis is reduced, for example, when β-arrestin-2 is knocked down, Smog homo-clusters accumulate at the surface as well as in the plasma membrane invaginations. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that control the Smog cluster formation and influence the distribution of active Smog in different plasma membrane compartments. Importantly, this controls the quantitative activation of Rho1 and MyoII. Under low-endocytosis regimes (Gprk2 or β-arrestin-2 knockdowns in the ectoderm), high levels of active Rho1 accumulate in the apical plasma membrane invaginations. It is proposed that the apical plasma membrane invaginations are signaling hubs, where signaling components could concentrate, give rise to high G protein signaling (e.g., Gα12/13), and sustain high MyoII activation. The size and stability of these signaling invaginations is tuned by endocytosis, and they may provide a means to control the strength and persistence of signaling. Pulsatile active Rho1 in the ectodermal cells requires intermediate Rho1 activation. In the ectoderm, low Fog expression and rapid Smog endocytosis by Gprk2 and β-arrestin-2 lead to intermediate activation of Rho1. In turn, intermediate Rho1 activation at the apical plasma membrane creates the conditions required for self-organized actomyosin dynamics associated with pulsation (Jha, 2018).
This study also points to the possibility of tissue level regulation of endocytosis and plasma membrane compartmentalization of GPCRs. Large apical plasma membrane invaginations are observed in the mesoderm compared to the ectoderm. In the mesoderm, Smog accumulates in larger, more numerous, apical plasma membrane invaginations, and it displays larger Smog homo-clusters compared to in the ectoderm. In the mesoderm, Rho1 and MyoII activation is higher. Another GPCR, Mist produced in the mesoderm, works synergistically with Smog to boost Rho1 and MyoII activation (Manning, 2013). This is also due to the expression of another GPCR Mist in the mesoderm and to Fog being present at higher levels in the mesoderm. Ectodermal cells have similar properties of high Smog homo-clusters when Fog is overexpressed and GPCR endocytosis is slowed down. An intriguing possibility is that Smog and potentially Mist endocytosis is downregulated in the mesoderm compared to the ectoderm. Interestingly, the E3 ubiquitin ligase Neuralized (Neur), which is uniformly expressed in the embryo, is inhibited in the ectoderm by the small proteins of the Bearded (Brd) family. Brd genes are repressed by the mesoderm transcription factor Snail, so that Neur is only active in the mesoderm. In a Brd mutant, where Neur becomes active in the ectoderm, MyoII activation is increased and Neur degradation or repression in the mesoderm following Brd overexpression both reduce MyoII activation. Previous studies have shown that the E3 ubiquitin ligase targets β-arrestin-2 for ubiquitination and degradation, and, thereby, it affects endocytosis and signaling by GPCRs. It is possible that GPCR endocytosis could be reduced in the mesoderm due to increased Neur activity in this tissue. This may depend on the downregulation of several target proteins, such as β-arrestin-2 (Jha, 2018).
Selective compartmentalization of GPCR on the plasma membrane as in the case of large apical plasma membrane invaginations can increase the concentration and the probability of GPCR clustering and oligomerization. The current data suggest that the dynamic modulation of GPCR signaling can be achieved by a change in their cluster/oligomer formation. Receptor oligomerization may enlarge the signaling capacities by the recruitment of more downstream signaling components during GPCR signaling. G proteins are reported to be expressed at low concentration, and selective compartmentalization of GPCRs on the plasma membrane further increase the probability of GPCR clustering and oligomerization for efficient signaling. Investigation of G protein activation by different GPCRs in vivo will be needed to test if a similar mechanism is in place during epithelial morphogenesis (Jha, 2018).
The effects of the absence of puckered (pucE69) and its overexpression were compared
on the levels and organization of the actin cytoskeleton and nonmuscle myosin. In pucE69 mutants, the expression of myosin
and actin does not change dramatically in the periphery of the cells in lateral regions of
the embryo, but these proteins fail to accumulate along the leading edge of the epidermis. Cell shape
changes proceed almost normally. In contrast, epidermal cells of embryos overexpressing puc fail to
change their shapes and accumulate low levels of spatially disorganized myosin at the leading edge. In these embryos, actin fails to be expressed in the amnioserosa and its levels are reduced
in the epidermis. Actin and myosin tend to form clumps in these epidermal cells.
Thus puc is an essential component in the control of the different steps of dorsal
closure progression and acts by modulating the apical accumulation of actin and myosin at the leading edge. These results correlate with those of the effects of overexpression of
DN-Drac1 and Djun mutants, and further suggest a role for Puc in the control of JNK activity over the
cytoskeleton (Martin-Blanco, 1998).
To explore the nature of the defects seen in the absence of diaphanous
function, wild-type and dia mutant embryos were stained at
nuclear cycles 11-13 with the DNA dye DAPI and with an
antibody directed against F-actin. In the wild type, nuclei are
positioned at the embryo cortex at interphase of nuclear cycles
11-13; a structure referred to as the actin cap is situated
between each nucleus and the plasma membrane.
During the transition to prophase, filament reorganization
results in a concentration of actin at the edge of the caps. At
metaphase, the resulting rings of cortical actin, together with
associated plasma membrane, invaginate to form metaphase
furrows. As viewed from above, actin
staining at these furrows appears as a hexagonal array over the
embryonic surface. In the sagittal view, actin staining
at the metaphase furrow appears as a line between the
metaphase nuclei. In dia-deficient embryos, severe structural changes in the
actin cytoskeleton are manifested after nuclear cycle 11.
Formation of the hexagonal actin arrays is disrupted during
prophase and metaphase and there is an absence of
actin staining between the metaphase nuclei. Similar
patterns of staining are obtained when dia
embryos are stained with antibodies directed against anillin (Drosophila gene: Scraps) and Peanut,
other components of the metaphase furrow. There
is thus a failure in the formation of the metaphase furrow.
Consistent with the known role of metaphase furrows in
maintaining nuclear organization, the nuclei in dia mutant
embryos frequently exhibit abnormal spacing and, in some
cases, fuse in subsequent nuclear cycles. These irregularities
are readily apparent in contrast to the uniform pattern observed
in the wild type. In regions in which
cortical actin staining is weak or absent, nuclei are frequently
found displaced into the interior of the embryo,
although the centrosomes remain at the surface (Afshar, 2000).
To investigate whether the absence of metaphase furrows
results from a failure in membrane invagination, dia
embryos were stained with antibodies directed against myosin. In wild-type
embryos, myosin localizes to the embryonic cortex between
the actin caps at each interphase, appears at the tip
of the invaginating membrane at prophase and
disappears at metaphase. In dia embryos, myosin
staining, albeit very weak and irregular, is detected between the actin caps
at the cortex during interphase. At prophase,
myosin, where detectable, remains at the cortex, with no
detectable membrane pinching or invagination.
Therefore, despite the presence of myosin at the cortex
between actin caps, the membrane invagination that precedes
metaphase furrowing is absent in dia embryos (Afshar, 2000).
Immunolocalization was used to determine whether
Diaphanous plays a role in the recruitment of anillin and Peanut, a Drosophila septin. In wild-type
embryos both anillin and Peanut localize to the embryonic
cortex, between the actin caps at interphase. During prophase and
metaphase, they localize to the metaphase furrow and their
pattern of staining is similar to that of actin. In
dia embryos, the staining patterns of both anillin and Peanut
are very weak during interphase. Similarly, in dia
embryos the localization of both of these proteins is disrupted
during prophase and metaphase, when the metaphase furrow is
being formed in wild-type embryos. Diaphanous
is thus required for recruitment and proper localization of
anillin and Peanut as well as myosin to the regions of
membrane invagination (Afshar, 2000).
During convergent extension in Drosophila, polarized cell movements cause the germband to narrow along the dorsal-ventral (D-V) axis and more than double in length along the anterior-posterior (A-P) axis. This tissue remodeling requires the correct patterning of gene expression along the A-P axis, perpendicular to the direction of cell movement. A-P patterning information results in the polarized localization of cortical proteins in intercalating cells. In particular, cell fate differences conferred by striped expression of the even-skipped and runt pair-rule genes are both necessary and sufficient to orient planar polarity. This polarity consists of an enrichment of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 protein at the reciprocal D-V cell borders. Moreover, bazooka mutants are defective for germband extension. These results indicate that spatial patterns of gene expression coordinate planar polarity across a multicellular population through the localized distribution of proteins required for cell movement (Zallen, 2004).
Polarized cell movement during convergent extension ultimately derives from the asymmetric localization of proteins that direct cell motility. Interestingly, intercalating cells in the Drosophila germband display a polarized localization of the ectopically expressed Slam protein (Lecuit, 2002). Slam is present in a bipolar distribution that correlates spatially and temporally with intercalary behavior. These observations indicate that Slam can serve as a molecular marker for polarized cell behavior. Pair-rule patterning genes expressed in stripes along the A-P axis are necessary for Slam localization and, conversely, altering the geometry of their expression is sufficient to reorient Slam polarity. An endogenous planar polarity in intercalating cells has been shown to be manifested by the accumulation of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 at D-V cell borders. Moreover, germband extension is defective in bazooka mutant embryos, supporting a model where molecular polarization of the cell surface is a prerequisite for polarized cell movement. Therefore, differences in gene expression along the A-P axis may direct planar polarity in intercalating cells through the creation of molecularly distinct cell-cell interfaces that differ in migratory potential (Zallen, 2004).
Cell movement during germband extension is oriented along the D-V axis, suggesting a mechanism that restricts the productive generation of motility to dorsal and ventral cell surfaces. Molecules that are asymmetrically localized during convergent extension may therefore contribute to the spatial regulation of cell motility. Interestingly, intercalating cells in the Drosophila germband display a polarized localization of the ectopically expressed Slam protein, a novel cytoplasmic factor required for cellularization in the early embryo (Lecuit, 2002). While proteins such as Armadillo/β-catenin are uniformly distributed at the cell surface, ectopic Slam is enriched in borders between neighboring cells along the A-P axis. This polarized Slam population is present in a punctate apical distribution, coincident with the adherens junction component Armadillo/β-catenin. Therefore, intercalating cells have distinct apical junctional domains that differ in their capacity for Slam association (Zallen, 2004).
Interestingly, the polarized distribution of ectopic Slam protein is spatially and temporally correlated with intercalary behavior. Slam polarity is not observed in Stage 6 embryos prior to the onset of intercalation. Slam accumulation at A-P cell borders first appears in late Stage 7, when cells of the germband initiate intercalation, and reaches its full extent during the period of sustained intercalation in Stage 8. In contrast, Slam is uniformly distributed in cells of the head region and the dorsal ectoderm, tissues which do not undergo intercalary movements. These results indicate that the polarized distribution of ectopic Slam protein is specific to intercalating cells and that Slam can therefore serve as a molecular marker for the visualization of polarized cell behavior (Zallen, 2004).
The enrichment of Slam at borders between neighboring cells along the A-P axis is consistent with two modes of localization: Slam could mark one side of each cell in a unipolar distribution, or Slam could localize to both anterior and posterior surfaces in a bipolar pattern. To distinguish between these possibilities, mosaic embryos were generated where Slam-expressing cells were juxtaposed with unlabeled cells, using the Horka mutation to induce sporadic chromosome loss in early embryos. Slam protein accumulates at anterior and posterior boundaries of mosaic clone, indicating that ectopic Slam protein is targeted to both anterior and posterior surfaces of intercalating cells in a symmetric, bipolar distribution. The bipolar localization of ectopic Slam corresponds well with the bidirectionality of cell movement during germband extension, where cells are equally likely to migrate dorsally or ventrally during intercalation. Bipolar motility is also observed during convergent extension in the presumptive Xenopus and Ciona notochords and in Xenopus neural plate cells in the absence of midline structures (Zallen, 2004).
To extend the spatial and temporal correlation between Slam polarity and cell movement, it was asked if this polarized Slam localization is achieved in mutants that are defective for intercalation. Cell intercalation is dependent on the transcriptional cascade that generates cell fates along the A-P axis, in the direction of tissue elongation and perpendicular to the migrations of individual cells. A-P patterning reflects the hierarchical action of maternal, gap, and pair-rule genes. Cell fate differences along the A-P axis are abolished in embryos maternally deficient for the bicoid, nanos, and torso-like genes (referred to as bicoid nanos torso-like mutants), and these mutant embryos do not exhibit intercalary behavior. Ectopic Slam is correctly targeted to the apical cell surface in bicoid nanos torso-like mutants, but fails to adopt a polarized distribution in the plane of the epithelium (Zallen, 2004).
Downstream of the maternal patterning genes, gap genes establish overlapping subdomains along the A-P axis. A quadruple mutant for the gap genes knirps, hunchback, forkhead, and tailless lacks A-P pattern within the germband while retaining terminal structures. This quadruple mutant exhibits severely reduced cell intercalation, and mutant embryos also display a loss of Slam polarity. The absence of planar polarity in A-P patterning mutants correlates with a more hexagonal appearance of germband cells, in contrast to the irregular morphology of wild-type intercalating cells (Zallen, 2004).
In response to maternal and gap genes, pair-rule patterning genes expressed in narrow stripes act in combination to assign each cell a distinct fate along the A-P axis. In particular, the even-skipped (eve) and runt pair-rule genes are essential for germband extension. This strong requirement for eve and runt during germband extension contrasts with the more subtle effects in mutants for other pair-rule genes such as hairy and ftz. Consistent with these defects in intercalation, eve and runt mutants also display aberrant Slam localization. These results establish a correlation between intercalary behavior and the polarized localization of the ectopic Slam marker (Zallen, 2004).
The Eve and Runt transcription factors ultimately direct Slam polarity and cell intercalation through the transcriptional regulation of target genes. To identify downstream effectors involved in this process, components of the noncanonical planar cell polarity (PCP) pathway, which is required for convergent extension in vertebrates, were examined. Germband extension occurs normally in the majority of embryos lacking the Frizzled and Frizzled2 receptors. Similarly, germband extension is unaffected in the absence of Dishevelled. Moreover, dishevelled mutants exhibit a normal polarization of the Slam marker. These results demonstrate that molecular and behavioral properties of planar polarity in the Drosophila germband do not require Frizzled or Dishevelled function (Zallen, 2004).
The polarized distribution of ectopic Slam in intercalating cells provides the first clue to a molecular distinction between D-V cell interfaces that generate productive cell motility and A-P interfaces that do not. However, endogenous Slam mRNA and protein are not detected during germband extension, indicating that Slam may not play a functional role in cell intercalation. Slam colocalizes with the Zipper nonmuscle myosin II heavy chain subunit during cellularization and when Slam is ectopically expressed at germband extension (Lecuit, 2002). Therefore, the endogenous distribution of myosin II was examined during germband extension in wild-type embryos. During cell intercalation, myosin II is present in a punctate distribution at the apical cell surface, colocalizing with the adherens junction component Armadillo/β-catenin. In Stage 8 embryos, apical myosin II protein accumulates at interfaces between cells along the A-P axis. Slam can enhance this polarized localization when ectopically expressed (Lecuit, 2002), suggesting that Slam and myosin II may associate with a common localization machinery. Myosin II polarity is not apparent in Stage 6 or early Stage 7 embryos that have not begun intercalation, indicating that the enrichment of myosin II at A-P interfaces is specific to intercalating cells (Zallen, 2004).
The localized distribution of myosin II is not as pronounced as that of ectopic Slam, suggesting that additional asymmetries contribute to the polarization of intercalating cells. To identify such proteins, the localization was examined of components implicated in cell polarity in other cell types. In particular, the PDZ domain protein Bazooka/PAR-3 participates in both apical-basal and planar polarity. Bazooka/PAR-3 also exhibits a polarized distribution in intercalating cells. Bazooka, like myosin II, is present in a punctate apical distribution, coincident with the adherens junction component Armadillo/β-catenin. However, in contrast to the accumulation of myosin II at A-P cell interfaces, Bazooka is enriched in the reciprocal D-V interfaces. Bazooka polarity is specific to intercalating cells, where it first appears at the onset of intercalary movements in late Stage 7. Bazooka polarity is not observed in cells of the head region, which do not undergo intercalation, nor is it observed in germband cells following the completion of germband extension at Stage 9 (Zallen, 2004).
To characterize the relationship between cell shape and the polarized localization of cortical proteins, the orientation of cell borders was measured as an angle relative to the A-P axis (with A-P interfaces closer to 90° and D-V interfaces closer to 0° and 180°). Interfaces from embryos stained for Bazooka and myosin II were ranked according to mean fluorescence intensity as a relative measure of protein distribution. These results illustrate that Bazooka and myosin II are enriched in distinct sets of cell-cell interfaces that adopt largely nonoverlapping orientations relative to the A-P axis. This quantitation confirms the visual impression from confocal images and demonstrates that the molecular composition of a cell surface domain is a reliable predictor of its orientation within the epithelial cell sheet (Zallen, 2004).
The polarized localization of Bazooka is abolished in the absence of A-P patterning information in bicoid nanos torso-like mutant embryos. A similar disruption of myosin II polarity is observed in A-P patterning mutants. The A-P patterning system may therefore mediate cell intercalation through the polarized accumulation of cell surface-associated proteins. Bazooka participates in a conserved protein complex containing the atypical PKC (DaPKC), and DaPKC is also enriched in D-V cell interfaces during germband extension (Zallen, 2004).
Tissue morphogenesis requires stereotyped cell shape changes, such as apical cell constriction in the mesoderm and cell intercalation in the ventrolateral ectoderm of Drosophila. Both processes require force generation by an actomyosin network. The subcellular localization of Myosin-II (Myo-II) dictates these different morphogenetic processes. In the intercalating ectoderm Myo-II is mostly cortical, but in the mesoderm Myo-II is concentrated in a medial meshwork. Spacial constriction is repressed by JAK/STAT signalling in the lateral ectoderm independently of Twist. Inactivation of the JAK/STAT pathway causes germband extension defects because of apical constriction ventrolaterally. This is associated with ectopic recruitment of Myo-II in a medial web, which causes apical cell constriction as shown by laser nanosurgery. Reducing Myo-II levels rescues the JAK/STAT mutant phenotype, whereas overexpression of the Myo-II heavy chain (also known as Zipper), or constitutive activation of its regulatory light chain, does not cause medial accumulation of Myo-II nor apical constriction. Thus, JAK/STAT controls Myo-II localization by additional mechanisms. Regulation of actin polymerization by Wasp, but not by Dia, is important in this process. Constitutive activation of Wasp, a branched actin regulator, causes apical cell constriction and promotes medial 'web' formation. Wasp is inactivated at the cell cortex in the germband by JAK/STAT signalling. Lastly, wasp mutants rescue the normal cortical enrichment of Myo-II and inhibit apical constriction in JAK/STAT mutants, indicating that Wasp is an effector of JAK/STAT signalling in the germband. Possible models are discussed for the role of Wasp activity in the regulation of Myo-II distribution (Bertet, 2009).
Myo-II subcellular localization controls different cell shape changes such as cell constriction or intercalation. The data shed new light on the mechanisms of the subcellular localization of actomyosin networks in the early Drosophila ectoderm. VL ectodermal cells intercalate via the cortical recruitment of Myo-II at AJs, which drives polarized junction remodelling. This contrasts with the behaviour of immediately adjacent cells in the mesoderm, which undergo apical constriction and recruit Myo-II into a medial apical web. The data indicate that the cortical enrichment of Myo-II in ectodermal intercalating cells is not a 'default pathway', and requires at least activity of the JAK/STAT pathway. Indeed, in JAK/STAT pathway mutants, Myo-II is aberrantly recruited in a medial apical meshwork and cells consequently undergo apical constriction. This is surprising, as apical constriction is normally only observed in mesodermal ventral cells and is considered to be a unique attribute owing to their selective expression of Twist and Snail. Twist and Snail induce expression of the ligand Fog in the ventral cells only, which activates RhoGEF2, Rok and Myo-II. It also regulates expression of the transmembrane protein T48, which participates in the apical recruitment of RhoGEF2 and contributes to apical constriction. However it is not clear whether activation of the RhoGEF2 pathway is sufficient to drive the apical medial recruitment of Myo-II. This study shows that apical constriction is not simply induced in mesodermal cells by Fog, but is also prevented in ectodermal cells by activity of the JAK/STAT pathway and that this is essential for germ-band extension (GBE). In JAK/STAT pathway mutants, ectodermal cells undergo apical constriction despite the absence of ectopic Twist expression. Note, however, that apical constriction is not as rapid in these mutants as in mesodermal cells, so Twist and Snail accelerate or render more efficient the capacity to apically constrict. Moreover, the fact that Wasp mediates JAK/STAT function in the ectoderm but is not required in the mesoderm indicates that the mechanisms promoting medial Myo-II in mesoderm cells are likely to be different (Bertet, 2009).
These findings provide a novel opportunity to investigate the regulation of cortical or medial Myo-II localization in the ectoderm. The data document two novel features of this regulation (Bertet, 2009).
MRLC (Sqh) phosphorylation by the RhoGEF2 and the Rok pathway are both necessary for apical constriction; lowering the dose of RhoGEF2, Rho or Rok suppress the apical constriction observed in upd mutants. However, neither constitutive activation of this pathway by expression of a phosphomimetic form of Sqh, ShqE20E21, which rescues Rok inhibition, nor overexpression of MHC (Zip) is sufficient to promote medial accumulation of Myo-II. The medial accumulation of Myo-II requires additional regulation apart from the activation of Myo-II. Since RhoGEF2 and Rok are key regulators of Myo-II, this suggests that activation of the RhoGEF2/Rok pathway is necessary but not sufficient to explain medial Myo-II accumulation and apical constriction (Bertet, 2009).
This analysis of the JAK/STAT mutant phenotypes indicates a key role of Wasp in this process. Wasp is shown to be necessary for medial Myo-II accumulation, at least in ectodermal cells, and very strong activation of Wasp at the cortex (myrWasp) also causes medial Myo-II accumulation. Moreover, although Wasp is normally downregulated in VL ectodermal cells, in JAK/STAT pathway mutants Wasp is strongly recruited and hence activated at the plasma membrane, which suggests that JAK/STAT signalling represses the membrane activation of Wasp. Importantly, lowering the dose of Wasp maternally suppresses medial accumulation of Myo-II in upd mutants, and restores prominent accumulation at the cortex, as in wild-type embryos. Consistent with this, ectopic apical constriction is completely rescued in these double mutant embryos (Bertet, 2009).
Dia and Wasp play different roles in the regulation of Myo-II localization. Consistent with previous data, Dia controls the amount of apical Myo-II, but the specific localization of Myo-II at the cortex or in the medial network is not affected by loss of Dia. Dia promotes polymerization of non-branched filaments, and might control the formation of a good substrate for the stable association of Myo-II minifilaments. The fact that in dia heterozygotes the amount of apical Myo-II is reduced indicates that the amount of actin filaments might be limiting and controlled. Indeed, more F-actin is detected at the cortex of intercalating cells, preceding by a few minutes the enrichment of Myo-II. The role of Wasp is more surprising and unique; it is shown to mediate specifically repression of medial Myo-II accumulation and, hence, cell constriction in the germband. Because activation of Wasp leads to activation of medial web formation and reduction of Wasp dosage rescues cortical Myo-II in JAK/STAT mutants, it is concluded that Wasp controls an essential feature of Myo-II subcellular localization that is essential for the regulation of apical constriction. How does Wasp control Myo-II localization? Two non-exclusive models. In the first model, Wasp controls actin branching through activation of the Arp2/3 complex. Because Wasp has been implicated in endocytosis via Arp2/3 in Drosophila, Wasp could promote Myo-II web formation indirectly by regulating endocytosis of a surface protein required to anchor the medial actomyosin network at the membrane, such as E-cadherin. Consistent with this, downregulation of E-cadherin by RNAi disrupts the faint medial Myo-II pool. In mesodermal cells, E-cadherin appears to anchor the strong medial Myo-II pool. In the second model, Wasp might act more directly via the regulation of actin network architecture and its impact on the dynamic interactions between the medial web and the cortex, and thereby might affect the steady-state distribution of Myo-II exchanging between these two pools. Although Wasp uniquely mediates Myo-II regulation via JAK/STAT in the ectoderm and not in the mesoderm, regulation of Arp2/3 might be more generally implicated in the control of Myo-II regulation (Bertet, 2009).
Although wasp is an important mediator of JAK/STAT function in the ectoderm, it is unlikely to be the only one. Indeed wasp mutants rescue the cortical accumulation of Myo-II and apical constriction in upd mutants, but GBE is still strongly affected; it was noticed that cortical Myo-II distribution was not properly polarized in the plane of the epithelium. This suggests that other subcellular processes are also perturbed in the mutant. The fact that a reduction of Myo-II levels suppresses the upd defects indicates that the overall dosage of Myo-II is important as well. Identifying the transcriptional targets of JAK/STAT might shed light on its complex regulatory role during embryonic morphogenesis (Bertet, 2009).
Finally, although this work identifies an important regulator of Myo-II network subcellular distribution in epithelial cells, it is still not clear what regulates the polarized distribution of Myo-II at the cortex (Bertet, 2009).
JAK/STAT signalling controls a number of developmental processes. Importantly, this pathway has been implicated in diverse morphogenetic processes, such as convergent extension movements in the zebrafish embryo, hindgut elongation in Drosophila embryos, which probably involves intercalation movements as well, and posterior spiracle morphogenesis in Drosophila embryos. JAK/STAT signalling also controls border cell migration. The data indicate that JAK/STAT signalling plays an important and hitherto unappreciated morphogenetic function in gastrulating embryos. These data document evidence that JAK/STAT controls, via Wasp, a morphogenetic switch based on the regulation of medial or cortical Myo-II distribution. Interestingly, dorsal cells do not undergo apical constriction in JAK/STAT mutants. Indeed, dorsal cells exhibit neither cortical nor medial web Myo-II and are thus unable to participate in profound tissue remodelling. It appears that DV patterning provides a first general subdivision within the embryonic epithelium whereby Myo-II is globally repressed dorsally, and activated laterally and ventrally. Cortical or medial web distribution then results from the combinatorial input of Fog and JAK/STAT (Bertet, 2009).
Cell rearrangements shape the Drosophila embryo via spatially regulated changes in cell shape and adhesion. This study of axis elongation (germband extension) shows that Bazooka/Par-3 (Baz) is required for the planar polarized distribution of myosin II and adherens junction proteins and polarized intercalary behavior is disrupted in baz mutants. The myosin II activator Rho-kinase is asymmetrically enriched at the anterior and posterior borders of intercalating cells in a pattern complementary to Baz. Loss of Rho-kinase results in expansion of the Baz domain, and activated Rho-kinase is sufficient to exclude Baz from the cortex. The planar polarized distribution of Baz requires its C-terminal domain. Rho-kinase can phosphorylate this domain and inhibit its interaction with phosphoinositide membrane lipids, suggesting a mechanism by which Rho-kinase could regulate Baz association with the cell cortex. These results demonstrate that Rho-kinase plays an instructive role in planar polarity by targeting Baz/Par-3 and myosin II to complementary cortical domains (de Matos Simões, 2010).
The spatially regulated activity of protein kinases with multiple substrates provides an efficient strategy for the control of cell polarity in different contexts. This study shows that Rho-kinase is an asymmetrically localized protein that plays an instructive role in planar polarity in the Drosophila embryo by excluding its substrate Baz/Par-3 from the cell cortex. Rho-kinase prevents expansion of the Baz domain and Baz in turn directs the localization of contractile and adherens junction proteins that are required for axis elongation, converting a localized source of kinase activity into a robust bias in polarized cell behavior. The effect of Rho-kinase on Baz planar polarity appears to be independent of its role in regulating myosin II, as Baz localization is not affected in myosin mutants and activated myosin does not reproduce the effects of Rho-kinase in culture. Instead, Rho-kinase can directly phosphorylate the Baz C-terminal coiled-coil domain that is required for Baz association with the cortex. Deletions within the Baz C-terminal domain or replacement of the Baz C-terminus with a heterologous phospholipid binding motif abolish Baz planar polarity in vivo. These results are consistent with a model in which Rho-kinase directly inhibits the association of the Baz C-terminal domain with specific regions of the cell cortex (de Matos Simões, 2010).
Rho-kinase has been shown to phosphorylate mammalian Par-3 in cultured cells, disrupting its interaction with the Par complex proteins Par-6 and aPKC (Nakayama, 2008). The Par complex is necessary for some aspects of epithelial organization but dispensable for others. Par-6 and aPKC are not required for Baz planar polarity in Drosophila, suggesting that the role of Rho-kinase in this process is unlikely to occur through a similar mechanism. This study provides evidence for a different mechanism of regulation by Rho-kinase involving the Baz C-terminal domain, which is phosphorylated by Rho-kinase in vitro and is necessary for Baz planar polarity in vivo. The Baz C-terminus has been shown to bind directly to phosphoinositide membrane lipids including PI(3,4,5)P3, PI(3,4)P2 and PIP (Krahn, 2010). This study shows that Rho-kinase inhibits the association of Baz with phosphoinositide membrane lipids in vitro, consistent with a model in which Rho-kinase directly regulates Baz association with the cortex. Alternatively, Rho-kinase could regulate Baz localization indirectly through other proteins that interact with the Baz C-terminal domain. Despite potential differences in the mechanism, these results demonstrate that the regulation of Par-3 localization or activity by Rho-kinase is a conserved feature of cell polarity in Drosophila and mammals (de Matos Simões, 2010).
The results demonstrate that Rho-kinase is an asymmetrically localized protein that initiates a cascade of events required for the planar polarized distribution of contractile and adherens junction proteins in intercalating cells. The upstream signals that generate localized Rho-kinase activity are not known. Differences between cells conferred by striped or graded patterns of gene expression orient cell movement during axis elongation, and AP patterning genes expressed in stripes are necessary for the asymmetric localization of Rho-kinase. These findings raise the possibility that planar cell polarity may be generated by the local activation of a Rho GTPase signaling pathway. The Drosophila genome contains 21 RhoGEFs and 19 RhoGAPs that are candidate upstream regulators in this process. Rho GTPase pathways are activated by a number of upstream signals including G protein-coupled receptors, receptor tyrosine kinases, cytokine receptors, and cell-cell and cell-substrate adhesion. Identification of the signals upstream of Rho-kinase will help to elucidate the spatial cues that initiate planar polarity in the Drosophila embryo (de Matos Simões, 2010).
The role of Rho-kinase in planar cell polarity is reinforced by the effect of Baz on the localization of contractile and adherens junction proteins. The relationship between Baz and myosin II is complex. In the C. elegans zygote, a contractile myosin network carries PAR-3 to the anterior cell cortex, suggesting a positive relationship between these proteins. In other cell types myosin appears to be dispensable for Baz localization. PAR-3 is required to sustain myosin contractility in C. elegans and Drosophila, and Baz promotes myosin apical localization during C. elegans gastrulation and in the Drosophila follicular epithelium. The ectopic association of myosin with DV cell boundaries in baz mutants, and the complementary distributions of Baz and myosin in several contexts, raise the possibility of inhibitory effects of Baz on myosin. This regulation could also occur indirectly through effects of Baz on apical-basal polarity (de Matos Simões, 2010).
Differential adhesion is sufficient to drive cell sorting in culture and has been proposed to influence tissue morphogenesis in vivo. This study shows that Rho-kinase and Baz regulate the planar polarized localization of the adherens junction protein β-catenin. Rho-kinase has been shown to downregulate adhesion in culture, an activity that is thought to occur through myosin II, which can play positive and negative roles in junctional stabilization. The ability of Rho-kinase to exclude the Baz/Par-3 junctional regulator from the cortex suggests an alternative mechanism for the regulation of adherens junctions by Rho GTPases. These results suggest that Rho-kinase can both promote contractility and inhibit adhesion, providing a single molecular mechanism linking cortical contraction with adherens junction disassembly during tissue morphogenesis (de Matos Simões, 2010).
Force generation by Myosin-II motors on actin filaments drives cell and tissue morphogenesis. In epithelia, contractile forces are resisted at apical junctions by adhesive forces dependent on E-cadherin, which also transmits tension. During Drosophila embryonic germband extension, tissue elongation is driven by cell intercalation, which requires an irreversible and planar polarized remodelling of epithelial cell junctions. This study investigate how cell deformations emerge from the interplay between force generation and cortical force transmission during this remodelling in Drosophila melanogaster. The shrinkage of dorsal-ventral-oriented ('vertical') junctions during this process is known to require planar polarized junctional contractility by Myosin II. This study shows that this shrinkage is not produced by junctional Myosin II itself, but by the polarized flow of medial actomyosin pulses towards 'vertical' junctions. This anisotropic flow is oriented by the planar polarized distribution of E-cadherin complexes, in that medial Myosin II flows towards 'vertical' junctions, which have relatively less E-cadherin than transverse junctions. The evidence suggests that the medial flow pattern reflects equilibrium properties of force transmission and coupling to E-cadherin by alpha-Catenin. Thus, epithelial morphogenesis is not properly reflected by Myosin II steady state distribution but by polarized contractile actomyosin flows that emerge from interactions between E-cadherin and actomyosin networks (Rauzi, 2010).
The data suggest that the anisotropic actomyosin flow may largely depend on the distribution of junctional anchoring points. This requires E-cadherin/β-Catenin complexes at AJs and depends on α-Catenin. E-cadherin/β-Catenin/α-Catenin complexes are planar polarized, such that medial pulses flow towards regions with lower amounts of E-cadherin complexes. The level of E-cadherin along 'vertical' relative to adjacent junctions (E-cadherin anisotropy) is also fluctuating. Moreover, the onset of medial pulses coincided with the time when E-cadherin anisotropy reached a local maximum raising the possibility that E-cadherin anisotropy may orient the actomyosin flow. Reduction of E-cadherin by RNAi causes the disappearance of medial Myo-II. The junctional Myo-II level is consequently strongly reduced and no longer planar polarized. It was reasoned that reducing the levels of α-Catenin by RNAi should attenuate coupling more subtly. α-Catenin RNAi reduces the number of E-cadherin clusters at AJs and disrupts interactions with junctional F-actin. Moreover, the distribution of E-cadherin is no longer planar polarized in α-CateninRNAi embryos. This is associated with a loss of medial and junctional Myo-II planar polarity. Thus, the planar polarized distribution of E-cadherin/β-Catenin/α-Catenin complexes biases the flow of medial Myo-II and junctional polarization (Rauzi, 2010).
In addition to Myo-II contractility, flow requires (1) crosslinkers between filaments to transmit tension within the medial meshwork, and (2) coupling at the cortex to E-cadherin/β-Catenin/α-Catenin complexes. Increased levels of E-cadherin in 'transverse' junctions may change properties of the actin network (for example, crosslinking/viscosity) and inhibit internal transmission of contractile forces and hence prevent D-V oriented flow. To test this, the force balance within the medial actomyosin network was disrupted by focal ablation, and the redistribution of medial clusters was imaged. If increased E-cadherin levels at transverse junctions inhibit tension transmission along the D-V axis, then medial pulses should not flow in this direction following ablation. However, it was observed that Myo-II medial clusters flowed radially and away from the point of ablation towards the junctions in 100% of cases, even towards transverse junctions. Focal ablation of the actin meshwork produces a local hole, which expands radially. This argues that transverse junctions do not inhibit flow per se and that flow directionality emerges from the properties of the actomyosin meshwork integrated over the entire apical surface (Rauzi, 2010).
The mechanical properties of the medial actomyosin network are locally defined by Myo-II contractility (concentration, affinity, duty cycle), tension transmission within the network (crosslinking), and viscous resistance to deformations (interactions between filaments). Moreover, these properties fluctuate owing to protein turnover and interactions. E-cadherin is known to anchor and modify actin dynamics. The results suggest that the polarized distribution of E-cadherin may control the actomyosin flow pattern by spatially modulating mechanical properties of the actin network (Rauzi, 2010).
Current models of epithelial morphogenesis centre on Myo-II steady state distribution and associated contractile forces. The current data show however that cell deformations cannot be simply derived from the Myo-II distribution itself, but from two central features of actomyosin dynamics, namely concentration (pulses) and movement (flow). Pulsed dynamics defines the rhythm and possibly the speed of deformation. Flow pattern, which in the case of intercalation is anisotropic, dictates the orientation of cell deformation. Flows of Myo-II foci have been reported in the one-cell stage C. elegans embryo, pointing to a more general property of actomyosin networks. An important future avenue of research will be to investigate what properties of actin networks control Myo-II flow dynamics in different systems (Rauzi, 2010).
Cytokinesis entails cell invagination by a contractile actomyosin ring. In epithelia, E-cadherin-mediated adhesion connects the cortices of contacting cells; thus, it is unclear how invagination occurs, how the new junction forms, and how tissue integrity is preserved. Investigations in Drosophila embryos first show that apicobasal cleavage is polarized: invagination is faster from the basal than from the apical side. Ring contraction but not its polarized constriction is controlled by septin filaments and Anillin. Polarized cleavage is due instead to mechanical anchorage of the ring to E-cadherin complexes. Formation of the new junction requires local adhesion disengagement in the cleavage furrow, followed by new E-cadherin complex formation at the new interface. E-cadherin disengagement depends on the tension exerted by the cytokinetic ring and by neighboring cells. This study uncovers intrinsic and extrinsic forces necessary for cytokinesis and presents a framework for understanding how tissue cohesion is preserved during epithelial division (Guillot, 2013).
Epithelial cells divide in the plane of the tissue, allowing the equal partitioning of polarity proteins. This study delineated two major events during epithelial cytokinesis that shed light on how this is controlled. Cleavage progresses along the apicobasal axis and is polarized, as it is faster from basal to apical. This is not due to polarized contraction of the ring but to apical anchoring of the ring to E-cad complexes. Second, cleavage occurs in the plane of junction and involves local adhesion disengagement. In contrast to standard cytokinesis, this study delineated intrinsic and extrinsic mechanical processes operating during epithelial cytokinesis. Contractility of the ring itself is dependent on septins and Anillin. Ring contraction is resisted by intercellular adhesion mediated by E-cadherin complexes and by tension from neighboring cells transmitted by adhesion. Thus, E-cad-based adhesion plays a pivotal role in epithelial cytokinesis by anchoring the contractile ring, while its disengagement uncouples intrinsic and extrinsic tensile activity (Guillot, 2013).
In Drosophila embryos, epithelial cells exhibit polarized cleavage furrow ingression. This is likely to be general in epithelial cells, albeit at different magnitudes. MDCK cells too divide from the basal side toward the apex, and neuroepithelial cells in vertebrates partition the basal body first before the more apical part of the cell. Polarized cleavage is not a property unique to epithelial cells, however. Embryonic cleavage in several species exhibit a range of patterns, from completely unilateral cleavage, as reported in jellyfish (Clytia and Beroe) and Ctenophores (Pleurobrachia), to partly asymmetric cleavage in the one-cell-stage C. elegans embryos). In the latter case, polarized ingression of the cleavage furrow is stochastic and correlates with heterogeneities in the recruitment of the actin crosslinker Anillin and of septins. In anillin and septin knockdowns, cleavage becomes symmetric. This contrasts with activators of MyoII, such as Rho kinase, which affects the speed of contraction but not its polarity. Thus, in nonepithelial cells, polarized cleavage is a purely autonomous process governed by heterogeneities in regulators of contractility. This study found, however, that in Drosophila embryos, polarized cleavage is not determined by polarized distribution of Anillin and septins or by differential biomechanical properties of the ring. Septins display a marginal yet significant enrichment basally, and Anillin is slightly enriched apically. However, invagination was still normally polarized along the apicobasal axis in both peanut mutants and anillin RNAi embryos, despite strong reduction in constriction rat. Moreover, no significant difference between apical and basal relaxation kinetics was detected following ablation in wild-types. The ablation kinetics reflects the relative effect of stiffness in the ring and friction internal to the ring and with the cytoplasm. With the caveat that the latter cannot be directly measured and and is assumed to be uniform, these ablation experiments indicate the relative stiffness in the ring. The fact that relaxation is faster (<5 s) than turnover of the internal components of the ring, such as MyoII, substantiates the idea that mostly the elastic relaxation of the ring was measured and not a quasi-static relaxation associated with turnover/movements of ring components (Guillot, 2013).
The rate of constriction was monotonic such that big rings and small rings contracted at a constant rate in wild-types but also in anillin or septin mutants, although it was strongly reduced in the latter cases. This contrasts with reports in C. elegans, where constriction was scaling with ring size, suggesting a mechanism based on disassembly of contractile units whose number scales with ring size. This difference may stem from the fact that cytokinesis is especially rapid in Drosophila embryos (about 150 s). Alternatively, it could reflect the epithelial nature of the divisions reported in this study (Guillot, 2013).
The evidence argues instead that polarized ingression depends largely on apical anchoring of the ring to E-cad complexes. First, E-cad complexes colocalize with the contractile ring for the most part of invagination. Second, ingression is symmetric in either e-cad or α-cat RNAi embryos. Although E-cad complexes, in particular α-cat, can recruit regulators of MyoII, this cannot explain polarized invagination of the ring, since apical and basal relaxations are not significantly different in wild-types and in α-cat RNAi embryos. E-cad complexes transmit actomyosin tension in epithelia. Two sets of observation support the idea that junctions exert pulling forces on the ring due to anchoring. The ring is stretched laterally as it constricts, and this requires apical junctions via e-cad and α-cat. The relative deformation of the ring following ablation is larger apically than basally, and this also requires cell junctions. It is striking that extrinsic and intrinsic regulators of the ring contraction have very different effects on ring dynamics. In the absence of Pnut or Anillin, the ring constriction is reduced but it is still polarized. However, following e-cad or α-cat depletion, ring constriction is normal but symmetric. It is concluded that the mechanical connection of E-cad complexes to the contractile ring causes polarized invagination. It is possible that, in other systems, both intrinsic and extrinsic regulation will operate in parallel to increase the cleavage asymmetry. This may be important in highly columnar epithelial cells or when adhesion is lower and unable to resist the ring tension (Guillot, 2013).
Polarized cleavage effectively separates apical and basal cleavage, adhesion complexes being a barrier separating the apical and lateral domains. The central problem becomes: How does cleavage occur at adherens junctions? This study delineated two critical phases in junctional cleavage. First, the adherens junctions invaginate with the actomyosin ring, consistent with the fact that the ring is anchored to the junctions. During this phase, E-cad intercellular adhesion is stable in the face of the tension exerted by the ring, and E-cad colocalizes with the ring at the point of coupling. Invagination of junctions then stops as E-cad levels decrease in this area. However, ring constriction continues and appears to detach from junctions. This is interpreted as a point of adhesion disengagement. Adhesion disengagement marks the formation of the new vertices and of the new junction between daughter cells. Electron microscopy images show this membrane disengagement. Consistent with this, the membrane still invaginates with the actomyosin ring), although E-cad is still not detected. Closer examination shows that E-cad monomers are present at this late stage of cytokinesis but that adhesion complexes form gradually from this stage onward. It is striking that adhesion is very locally (<1 μm out of ∼40 μm of junction perimeter) and transiently (∼200 s) perturbed during division. In the first 150 s, E-cad clusters immediately adjacent to the cleavage furrow remain in position as the junction invaginates. This suggests that the cortex can be extensively remodeled locally. It likely reflects the fact that tension induces membrane flows with respect to the actin-rich cortex and argues that E-cad-mediated adhesion does not prevent membrane flow during disengagement. Interestingly, local disengagement allows local cell deformation without affecting the overall shape of cell contacts. Consistent with the idea that adhesion is locally disengaged, the amount of E-cad has a strong impact on the timing and depth of junctional cleavage. Increasing E-cad delays disengagement (i.e., the formation of the new junction, inducing strong cell deformations. More generally, this implies that increasing adhesion may provide an efficient mechanism to prevent local cell-cell disengagement when internal tension is used to remodel junctions during morphogenesis. In apical constriction in the Drosophila mesoderm, actomyosin cables pull on the junctional cortex and reduce junction lengths. If adhesion was not strong enough, local disengagement would occur and junctions could not remodel. The fact that adhesion disengagement is local and transient during cytokinesis is also probably key to the overall maintenance of cell polarity and adhesion during epithelial division (Guillot, 2013).
It is proposed that adhesion disengagement is mechanically induced by tension in the cytokinetic ring and by tension from neighboring cells. When the cumulated tension is higher that the adhesive force, disengagement occurs. Consistent with this, disengagement and formation of the new junction is strongly delayed in mutants that reduce the constriction of the cytokinetic ring, namely, in septin mutants and in Anillin knockdown embryos. Likewise, ablation of neighboring cells delays disengagement. It is, however, possible that adhesion is also locally disrupted by either E-cad endocytosis or phosphorylation of β-cat/Arm (Guillot, 2013).
Adhesion complexes transmit cell tension exerted by neighboring cells. Surrounding junctions and, more specifically, MyoII cables oriented toward or near the cleavage furrow strongly affect furrow invagination when E-cad is present at high levels. The invagination in this case is very shallow, suggesting a tug of war between intrinsic (ring contraction) and extrinsic tension (MyoII cables in neighbors). This results in asymmetric furrows in the plane of junctions due to the asymmetric distribution of MyoII cables around the cell. When E-cad is expressed at lower levels, even if surrounding junctions are oriented toward the cleavage furrow, invagination is unaffected and symmetric. It is proposed that E-cad complexes sensitize cells to their mechanical environment. This may provide a mechanism for cells to integrate stress coming from the environment. It will be important to explore how E-cad levels may affect cells responsiveness to extrinsic stress during division by affecting the timing of the formation of the new junction by local disengagement and the resulting cell shape and topology (Guillot, 2013).
The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. Its role in the developing Drosophila embryonic epidermis during the process of dorsal closure has been investigated. By expressing the dominant negative DRhoAN19 construct in stripes of epidermal cells, it has been confirmed that Rho function is required for dorsal closure and it is necessary to maintain the integrity of the ventral epidermis. Defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoAN19 expression. Furthermore, these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoAN19-expressing cells (Bloor, 2002).
Tension generated in the amnioserosa and the leading edge of the lateral epidermis independently contributes to the forces that drive dorsal closure. It has been proposed that nonmuscle myosin II activation generates tension in the leading edge and that this causes a leading edge intracellular actomyosin purse-string to shorten. Signaling downstream of RhoGTPase activates nonmuscle myosin II by modulating the level of myosin regulatory light chain phosphorylation. As such, expression of RhoAN19 in epidermal stripes might disrupt contraction of the leading edge purse-string. Defects in actin and nonmuscle myosin II organization caused by RhoAN19 expression are first observed at germband extension, up to 2 hours before purse-string formation. Thus, while actin and nonmuscle myosin II are localized at the leading edge in wild-type tissue, a purse-string structure is never formed in leading edge cells that express RhoAN19. RhoAN19 expression therefore effectively cuts the leading edge purse-string at multiple sites. This does not necessarily prevent progression of dorsal closure, confirming previous experiments which demonstrate that the integrity of the leading edge is not required for dorsal closure to continue to completion. It is concluded that small independent regions of leading edge in wild-type epidermal stripes can, in conjunction with contraction of the amnioserosa, migrate dorsally with relative normalcy (Bloor, 2002).
The question that arises is how do epidermal cells expressing RhoAN19 move dorsally in the absence of a leading edge purse-string? These cells could hitchhike, i.e. they are pulled dorsally by the amnioserosa or dragged along with neighboring wild-type cells. Although spread and disorganized, dorsal RhoAN19-expressing cells do maintain adhesion with wild-type neighbors and this might then allow passive RhoAN19-expressing cells to move dorsally with wild-type tissue. This is consistent both with the inverse correlation between integrity of the ventral epidermis and the extent to which dorsal closure proceeds, as well as with observations on the distribution of tension at the embryo surface during dorsal closure. Thus, during the time that the epidermis lateral to the leading edge opposes dorsal closure, ventral failure of epidermal integrity (and hole formation) would release the tensional restraints on the remaining lateral epidermis, allowing it to move dorsally with more success. Similarly, in the absence of this release (i.e. the ventral epidermis retains its integrity and opposes dorsal movement of the epidermis), the leading edge is presumably no longer capable of generating sufficient force to drive dorsal closure to completion (Bloor, 2002).
Throughout development, a series of epithelial movements and fusions occur that collectively shape the embryo. They are dependent on coordinated reorganizations and contractions of the actin cytoskeleton within defined populations of epithelial cells. One paradigm morphogenetic movement, dorsal closure in the Drosophila embryo, involves closure of a dorsal epithelial hole by sweeping of epithelium from the two sides of the embryo over the exposed extraembryonic amnioserosa to form a seam where the two epithelial edges fuse together. The front row cells exhibit a thick actin cable at their leading edge. The function of this cable has been tested by live analysis of GFP-actin-expressing embryos in which the cable is disrupted by modulating Rho1 signaling or by loss of non-muscle myosin (Zipper) function. The cable serves a dual role during dorsal closure. It is contractile and thus can operate as a 'purse string,' but it also restricts forward movement of the leading edge and excess activity of filopodia/lamellipodia. Stripes of epithelium in which cable assembly is disrupted gain a migrational advantage over their wild-type neighbors, suggesting that the cable acts to restrain front row cells, thus maintaining a taut, free edge for efficient zippering together of the epithelial sheets (Jacinto, 2002).
To investigate the function of the actin cable during dorsal closure, advantage was taken of rho1 and also zip alleles that produce phenotypes in which the actomyosin cable disassembles part way through the dorsal closure process but in which the overall tissue movement typically does not fail. These mutants offered the opportunity to analyze the effects of cable loss at the cellular level without the complete disruption of tissue architecture. Also, they allowed the use of live GFP-actin embryos to analyze the dynamic cell behavioral response and determine how these behavioral changes influence the capacity of epithelial cells to participate in a coherent forward-sweeping movement (Jacinto, 2002).
Scanning electron micrographs of rho1 and zip embryos that complete dorsal closure reveal significant similarities. In both cases, combinations of amorphic or strong mutations, zip1/zipIIX62 or zip1/zip1 in the case of zip, and rho172O/rho172R in the case of rho1, were used. Both zip and rho1 mutant embryos exhibit the same dramatic defects in head involution, a tissue movement distinct from dorsal closure, but a clear, posterior dorsal hole is observed in none of the rho1 mutants, and in only 59% of the zip/zipIIX62 or 8% of the zip1/zip1 embryos. The remaining zip mutants appear to complete dorsal closure successfully; although, frequently, like their rho1 counterparts, these embryos show puckering or segment misalignments along the closed midline seam. Interestingly, puckering has previously been observed in mutants in which epithelial movement is not properly downregulated at the midline (e.g., puckered), suggesting that the actin cable may be regulating cell spreading during some periods of dorsal closure. It is presumed that the phenotypic variation in zip embryos is due to differences in the maternal contribution to the myosin II RNA and protein pool, which must run out around the stage of dorsal closure (Jacinto, 2002).
Phenotypic similarities between zip embryos that complete closure of the dorsal hole and rho1 mutants are not surprising since rho1 and zip have previously been shown to interact genetically in Drosophila. Moreover, a signaling link has also been revealed in mammalian cell culture studies, with Rho-associated kinase (ROCK), a Rho effector, shown to regulate Myosin function, and thus the assembly and maintenance of cable-like stress fibers, by repression of Myosin Light Chain phosphatase and direct activation of the Myosin Light Chain. It is also likely that Rho1 regulates the cytoskeleton via alternative effectors. The kinase Protein kinase related to protein kinase N (see Rho1 Protein Interactions) has been shown to function downstream of Rho1 during Drosophila dorsal closure, and mDia, the murine homolog of Drosophila Diaphanous, has been shown to bind active Rho1 and contribute to the formation of stress fibers in mammalian cells by promoting actin polymerization. However, whether or not these signaling pathways regulate actomyosin contractility during dorsal closure has yet to be demonstrated. Interestingly, Rho1 also interacts with p120ctn and regulates cadherin-based adherens junctions in the Drosophila embryo, suggesting that the leading edge disorganization seen in rho1 mutants may be not only a consequence of actin cable misregulation but also a result of defective adherens junctions (Jacinto, 2002).
Further similarities between the rho1 and zip embryos were revealed when higher-resolution SEM was used to look at the leading edge cells during the final stages of dorsal closure. In both mutants, the normally straight, and apparently taut, epithelial leading edge now appears to be relatively disorganized and to have lost tension, with front row cells extending increased levels of filopodial and lamellipodial protrusions compared to their wild-type counterparts (Jacinto, 2002).
To observe the dynamic behavior of these cells, time-lapse confocal microscopy was used to analyze the final stages of dorsal closure in living, wild-type embryos versus rho1 and zip embryos, expressing GFP-actin fusion proteins using the GAL4-UAS system. The fusion protein was driven in the epidermis by the epithelial driver e22cGAL4, and only embryos that subsequently closed the dorsal hole are described. As expected from SEM observations, the cytoskeletal architecture of the leading edge that normally characterizes wild-type dorsal closure is lost in both rho1 and zip mutants. The actin cable, which is clearly apparent in the wild-type leading edge from stage 13 onward, fails to assemble or disassembles during the dorsal closure process in rho1 and zip mutants. The disassembly of the actin cable is temporally coincident with a transition from an organized to disorganized leading edge. In both mutants, loss of cable is also coincident with more exuberant filopodial extensions than in wild-type leading edge cells, and these filopodia frequently coalesce to form lamellipodia. In wild-type embryos, lamellipodia are generally more a feature of the final stages of dorsal closure, as opposing epithelial fronts make contact with one another (Jacinto, 2002).
The coalescing of filopodia to form lamellipodia in mutant embryos leads to a broader extent of protrusion per unit length of leading edge cells and an increase of up to 300% in the total protrusion area extending from these cells. It is suggested that the increased level of actin-based protrusions seen in both rho1 and zip embryos at a time when the leading edge cable is disassembling may reflect that the cable serves some function in repressing excessive protrusive activity in these front row cells. The link could simply be due to a greater availability of free actin monomers, but it might also be a consequence of changes in membrane and cortical actin stiffness at the free edge in these cells. In this regard, myosin II has been shown to play a role in maintaining the integrity and stiffness of the cortical cytoskeleton during Dictyostelium morphogenesis. It is not clear from this data whether downregulation of Rho in any way feeds back on Cdc42 activity, but no increase in thickness or apparent contractility of the actin cable is observed in cells expressing dominant-negative versions of Cdc42 (Jacinto, 2002).
These observations indicate that maintenance of a fully formed and functioning actin cable is not an absolute requisite for closure of the dorsal hole. In order to test further how a failure to activate Rho1 and assemble an actin cable might influence a cell's capacity to participate in dorsal closure, the enGAL4 driver was used to express both GFP-actin and either dominant-negative Rho1 (RhoN19) or constitutively active Rho1 (RhoV14) in 4- to 5-cell-wide epithelial stripes. These embryos were then imaged live or, alternatively, fixed and costained with Alexa594-phalloidin to reveal the actin machinery of all the cells, including the intervening wild-type stripes that do not express GAL4. Initial live analysis of these embryos, from the earliest stages of dorsal closure, revealed differences between leading edge cells that are blocked in Rho1 signaling and their wild-type neighbors. Rho1N19-expressing cells fail to assemble an actin cable but do express broad filopodia and lamellipodia. Without a cable, they do not constrict at their leading edge in the way that their wild-type neighbors clearly do. Subsequently, these mutant Rho1N19 stripes of cells sweep forward, apparently released from their usual constraints, overspilling and displacing their wild-type neighbors. Frequently, the dorsal edges of intervening wild-type stripes are enveloped by adjacent Rho1N19-expressing stripes, and this wild-type tissue is consequently trapped back from the leading edge. By the time that the dorsal hole is closed, most of the midline seam epithelium is taken up by Rho1N19-expressing cells that clearly have a migration advantage over their wild-type, cable-assembling neighbors. In the converse experiment in which GFP-actin was expressed and a constitutively active Rho1 construct (Rho1V14) was expressed using the enGAL4 driver, precisely the opposite effect is seen. Now, Rho1V14-expressing cells are more constricted than their wild-type neighbors at early stages, and they are subsequently outcompeted during dorsal closure, such that wild-type stripes tend to dominate the leading edge during dorsal closure. Thus, when dorsal closure is complete, the midline seam epithelium is largely wild-type (Jacinto, 2002).
These results suggest that the actomyosin cable has a dual role to play during dorsal closure. It is a driver of leading edge cell contractility from the earliest stages of dorsal closure, but it is also required for restraining the leading edge epithelial cells and maintaining a coherent and taut epithelial margin during the later phases of dorsal closure, particularly as the epithelial faces are being zippered together. It is proposed that, in addition to the previously described 'purse string' function, actomyosin cables may have a more subtle, but equally important, support role during morphogenetic episodes such as dorsal closure. These cytoskeletal structures, regulated by Rho activity, function to maintain epithelial coherence during coordinated epithelial movements, particularly at free epithelial edges where a taut front enables efficient zippering together and alignment of cells at a zipper seam, in a process that is perhaps analogous to zippering closed a very full luggage bag (Jacinto, 2002).
In a sense, the restraining function of the cable during dorsal closure is analogous to that which operates for the band of actin at the periphery of a tissue culture clone of epithelial cells. In this case, the growth factor, scatter-factor, can trigger disruption of the restraining actin band as well as dissolution of cell:cell junctions, and cells at the periphery are consequently now able to break free of their neighbors (Jacinto, 2002).
The dual role for the actomyosin cable during dorsal closure, operating both as a 'purse string' and as a cell restrainer, is likely to be a finely balanced operation. Modulation of normal Rho1 activity, as in the experiments reported above, results in either overcontractility of cells or their release from leading edge constraints. These data support previous evidence suggesting that multiple cytoskeletal events drive dorsal closure but demonstrate that these events must be finely balanced to ultimately produce the precisely organized zippering required for perfect midline fusion of the two epithelial faces (Jacinto, 2002).
Dorsal closure (DC) is a tissue-modeling process in the developing Drosophila embryo during which an epidermal opening is closed. It begins with the appearance of a supracellular actin cable (AC) that surrounds the opening and provides a contractile force. Amnioserosa cells that fill the opening produce an additional critical force pulling on the surrounding epidermal tissue. This force is not gradual but pulsed and occurs long before dorsal closure starts. Quantitative analysis, combined with laser cutting experiments and simulations, reveals that tension-based dynamics and cell coupling control the force pulses. These constitutively pull the surrounding epidermal tissue dorsally, but the displacement is initially transient. It is translated into dorsal-ward movement only with the help of the actin cable, which acts like a ratchet, counteracting ventral-ward epidermis relaxation after force pulses. This work uncovers a sophisticated mechanism of cooperative force generation between two major forces driving morphogenesis (Solon, 2009).
This work has addressed the cooperative nature of two main forces driving dorsal closure. The results suggest a model that is in contrast to two popular views of DC mechanics. First, they show that force production by AS cells is constitutive and independent of the signal that triggers DC. The previous view that AS cells start to produce their pulling force at the onset of DC was based on the observation that only then do they begin to reduce their apical surfaces, a reduction that was consequently interpreted as being the force-producing process. The data of this study suggest that this reduction in surface area is passive and a consequence of closure, which globally reduces AS tissue surface and therefore prevents AS cells from expanding back to their original size after a contraction (Solon, 2009).
The second modification to the current picture is that the AC acts like a ratchet supporting the AS-mediated force pulses rather than contributing to dorsal-ward LE displacement as an autonomous purse string. AC tension builds up slowly, becoming prominent only much later in the DC process. Efficient DC, however, starts with the first signs of an AC, when the latter is unlikely to produce sufficient force for purse-string activity. This view is also supported by the overall geometry of the AC; its contractile forces act in the anterior-posterior direction, parallel to the LE, and will produce a dorsal-ward force component only because of AC curvature. There is evidence that this force component is not sufficient to independently pull on the epidermis because releasing tension by cutting the AS tissue leads to an initial ventral-ward relaxation of the AC. In this model, the AC does not have to exert much force, because it merely has to shift the preexisting equilibrium of forces between AS and epidermal tissue to counteract the ventral-ward relaxation of the LE after a force pulse. Simulations demonstrate that this scenario is indeed possible (Solon, 2009).
To understand how the AC exerts the proposed ratchet-like function at the molecular level, it will be necessary to understand the behavior of the major AC components that build up tension, actin and the motor protein myosin. The details of subcellular actin/myosin organization are not known, but it is possible to speculate how they may work to create a ratchet-like function. It is important to keep in mind that the AC is a supracellular structure made of many smaller ACs, which form inside the cells of the LE. These produce individual forces that are transmitted via intercellular bonds. Every cell can therefore act as an individual unit. If an AS cell next to the LE contracts, it will not only pull the LE dorsally but will also compress regions of the LE that are in contact with it. This compressive force will act in the anterior/posterior direction and will therefore slightly shorten the LE and as a consequence locally reduce AC tension. The involved LE cells can then compensate for this tension loss by condensing their actin/myosin network along the compression. This can be achieved by myosin-mediated sliding of actin filaments. When the AS cell relaxes, AC tension will increase again because the epidermal tissue will now try to pull the LE back to its original position, which requires it to stretch. This cannot happen if the condensed actin/myosin network does not relax. In this system, every force pulse would locally increase AC tension, which fits well with the observed gradual increase of AC tension over time (Solon, 2009).
At the mechanistic level, the major difference between this model and the previous purse-string model is that the AC manages to shorten only during dorsal-ward LE displacement, when it is supported by compressive forces, whereas in between force pulses it merely maintains its state. It is possible that later in the process, the AC is sufficiently enforced to produce an independent purse-string activity. Unfortunately, during these stages the zippering process creates additional pulling forces, which hamper the respective analysis (Solon, 2009).
Another important question arising from these observations is how AS cell pulsing is produced and controlled at the molecular level. It is conceivable that this may also involve actin and myosin function. It has been shown that Myosin II is critical for AS cell activity during DC, which is consistent with this view. What regulates the pulsed activity of these molecules remains to be shown. In the simplest case one can imagine a cell-autonomous mechanism that leads to regular pulses. Simulations show that such a mechanism works in principle but it cannot reproduce the full behavior of the in vivo system. In contrast, in vivo behavior can be convincingly reconstituted by introducing a tension-based mechanism in the simulations, where contraction initiation simply depends on cells being critically stretched by contracting neighbors. In particular, this model reproduces pulsing arrest in cells surrounding a laser-induced cut. It also reproduces the coupling behavior observed between AS cells in vivo. Such coupling could be achieved by a mechano-sensitive signaling system. At this stage, the action of a complex reaction-diffusion signaling network controlling cell pulsing cannot be fully excluded. However, no evidence was found that would indicate the existence of such a network, such as wave-like propagation of pulsing or any other regularity (Solon, 2009).
The simulations also offer an explanation for the sequential pulsation arrest observed in vivo. DC progression reduces the overall surface of the AS tissue. As a consequence, individual cells can no longer expand their surfaces to the initial size after a contraction, which is reflected by the observed decrease in pulsing amplitude. The simulations show how this quickly leads to a critical loss of surface tension and the arrest of pulsations in the entire tissue. In vivo, AS cells sequentially arrest in a contracted state, which increases the available space for the remaining AS cells, thus compensating for the surface loss and maintaining tissue tension. The simulations show that in a tension-based system, this mechanism will extend AS cell force production. A recent paper added further support to this model. It showed that apoptosis of about 10% of the AS cells contributes to closing efficiency by up to 50% (Toyama, 2008). It is clear that the elimination of AS cells also creates additional space and preserves tissue tension. Apoptosis could thus represent a second mechanism acquired to prolong pulsed force generation by AS cells. Accordingly, this study found that apoptosis starts late only in DC, at a stage when the AS tissue has significantly decreased in size. How apoptosis and the sequential arrest of pulsing are controlled remains to be determined. The latter could involve the diffusible signaling molecule Dpp, which is secreted by the cells of the LE. A Dpp gradient originating from the LE could in principle generate the observed, sequential pulsation arrest, which may explain the behavioral defects of AS cells in Dpp mutants (Fernandez, 2007; Wada, 2007; Solon, 2009).
This work describes a mechanism for displacing and shaping tissues that combines a field of pulsing cells with a ratchet-like function. This may represent a more general principle by which other tissues can also be remodeled; the pulsing cells generate a constitutive force and the location of the ratchet mechanism determines which tissue is affected in what way by the force. A recent paper describing Drosophila gastrulation suggested a similar principle mediating the first step of epidermal tissue invagination. There, a stepwise contraction of the apical cell surfaces occurs, which is maintained by a ratchet mechanism that is intrinsic to the cell and prevents cell relaxation after each contraction step (Martin, 2009). It will be interesting to investigate the dynamics of further tissue movements with the appropriate time resolution to see whether this does indeed represent a general mechanism for the directed movement of cell sheets, one of the hallmarks of morphogenesis (Solon, 2009).
Fluctuations in the shape of amnioserosa (AS) cells during Drosophila dorsal closure (DC) provide an ideal system with which to understand contractile epithelia, both in terms of the cellular mechanisms and how tissue behaviour emerges from the activity of individual cells. Using quantitative image analysis this study shows that apical shape fluctuations are driven by the medial cytoskeleton, with periodic foci of contractile myosin and actin travelling across cell apices. Shape changes were mostly anisotropic and neighbouring cells were often, but transiently, organised into strings with parallel deformations. During the early stages of DC, shape fluctuations with long cycle lengths produced no net tissue contraction. Cycle lengths shortened with the onset of net tissue contraction, followed by a damping of fluctuation amplitude. Eventually, fluctuations became undetectable as AS cells contracted rapidly. These transitions are accompanied by an increase in apical myosin, both at cell-cell junctions and medially, the latter ultimately forming a coherent, but still dynamic, sheet across cells. Mutants with increased myosin activity or actin polymerisation exhibited precocious cell contraction through changes in the subcellular localisation of myosin. thick veins mutant embryos, which exhibited defects in the actin cable at the leading edge, showed similar timings of fluctuation damping to the wild type, suggesting that damping is an autonomous property of the AS. These results suggest that cell shape fluctuations are a property of cells with low and increasing levels of apical myosin, and that medial and junctional myosin populations combine to contract AS cell apices and drive DC (Blanchard, 2010).
The process of DC relies on the coordination of the elongation of epidermal cells and contraction of the AS. Throughout the early stages of this process the AS cells exhibit fluctuations of their apical area. This fluctuating behaviour is driven by transient actin-myosin accumulations at the apical cortex of cells, involving the assembly, contraction and disassembly of large-scale actin and myosin structures. The formation and disassembly of foci could result from the self-organising properties of myosin and actin and the presence of specific regulators, which are known to spontaneously form active networks, asters and rings in vitro. Tension generated by the contraction of neighbouring cells, the sudden loss of tension due to detachment of actin from the cell membrane, spontaneous catastrophic collapse, and the stretching of the apical membrane resulting in the influx of ions such as calcium, could all contribute to the timing and location of fluctuations (Blanchard, 2010).
Differences in the absolute and relative strengths of actin-myosin structures in distinct subcellular populations provide an explanation for the patterns that were observed in wild-type and mutant embryos. The observations show that throughout DC the amounts of myosin increase in two subcellular populations: in a cortical ring and in a medial apical network. The amount of myosin at cell-cell junctions determines the shape of cell membranes: low levels lead to wiggly membranes and increasing levels of junctional myosin lead to the straightening of the membranes. The amount of actin-myosin in the medial population determines the fluctuating behaviour of cells: low levels lead to low-frequency fluctuations and no tissue contraction, as seen in the early phase of DC; intermediate levels lead to high-frequency fluctuations and slow tissue contraction, as seen in the slow phase of DC; and high levels lead to a coherent, but dynamic, sheet of actin-myosin across cells that was strongly contractile, as seen in the wild-type fast phase (Blanchard, 2010).
Increasing the amounts of both junctional and medial myosin in ASGal4/UAS-ctMLCK embryos led to premature apical contraction, precocious straight cell membranes and isotropic cell shapes, which are a signature of high cortical tension. By contrast, in ASGal4/UAS-DiaCA embryos, expressing an activated form of Diaphanous, low junctional myosin levels throughout DC gave rise to wiggly apical membranes. However, these cells showed precocious apical contraction and no discernible apical shape fluctuations. An increase in myosin levels was observed at a sub-apical position, which suggests that in these cells contraction is not only apical but spans the sub-apical and lateral axis. This sub-apical and lateral contraction of AS cells could be an impediment to apical shape fluctuations, as apical contraction must be accommodated by basal or lateral expansion (Blanchard, 2010).
Overactivating myosin led to apical blebbing in AS cells. Blebbing results from an increase in the ratio of cortical tension to cortex-membrane adhesion and is associated with transient actin-myosin accumulations similar to those observed in this study. No evidence was found for blebs in AS cells in wild-type embryos. It is suggested that dynamic actin-myosin structures are the more general property of cells, which in combination with high cortical tension or weak cortex-membrane adhesion can lead to blebs (Blanchard, 2010).
Previous results have shown that neighbours oscillate mainly in anti-phase (Solon, 2009). The current results reveal a more subtle picture, with anti-phase correlation predominantly in one orientation and in-phase correlation perpendicular to this. This combination results in interesting patterns, with rows or patches of cells that become synchronised for short periods of time and represent an emergent property of the system. It is expected that some sort of multicellular pattern is inevitable because of the requirement to maintain a coherent epithelium while cells fluctuate. However, the patterns also suggest that cell fluctuations can become entrained locally and for short periods. Analysis of the dynamics of cell shape suggests that cell contraction is the active process, but that fluctuating behaviour of a cell can be influenced and the timing of active contraction altered by the forces generated by immediate neighbours and more distant cells. Thus, the organisation of apical contractions and expansions at the multicellular scale arises from the feedback in both directions between intrinsic cell behaviour and mechanical context (Blanchard, 2010).
This study was undertaken to understand how changes in actin-myosin behaviour at a subcellular scale resulted in the patterned contraction of the AS. There is a gradual increase in the rate of contraction of individual cells that strongly accelerates with the onset of zippering behaviour. These changes were correlated primarily with a shortening of fluctuation cycle length. The current results suggest that these changes result from an increase in both apical medial and junctional myosin levels. The overall increase in myosin levels and the formation of a continuous actin-myosin network could provide the molecular basis for the transition of the AS to a more solid tissue (Blanchard, 2010).
What causes the increase in apical myosin levels? One possibility is that it is induced by a chemical or mechanical signal from the epidermis. A radial gradient of fluctuations (Solon, 2009) and of the rate of contraction of AS cells (Gorfinkiel, 2009) suggests that the epidermis is providing some information for the patterned contraction of the AS. However, the analysis of tkv mutants shows that even when the mechanical properties of dorsal-most epidermal cells have been altered and Dpp signalling is compromised, AS cells change their fluctuation behaviour in a similar pattern to the wild type and finally contract. This reveals that several processes that are individually redundant ensure DC. DC could result from an AS-autonomous programme of increasing medial and junctional myosin (and/or actin), through changes in the dynamics of actin and myosin activity, of intracellular trafficking or of cell adhesion. Alternatively, apical myosin might increase as a result of the fluctuating behaviour of AS cells and the build-up of tension due to neighbour contractions. Whatever the mechanism, it is likely that an increase in myosin activity involves activation of the Rho GTPase, which has a central role both in integrating mechanical and structural cues and in regulating myosin-based tension (Blanchard, 2010).
AS cells fluctuate at low frequencies for a long period during early DC without any tissue contraction. High-frequency fluctuations drive moderate cell and tissue contraction during the slow phase, before they disappear in the transition to rapid tissue contraction. This raises the question of whether fluctuations have a function, as it is at least theoretically possible that contraction could be achieved in a smooth manner. One can speculate that cell fluctuations would be a way to maintain a basic level of cell activity that could be turned easily into morphogenetically relevant behaviours. Cell fluctuations could, more simply, be an epiphenomenon of the self-organised dynamics of actin and myosin. Alternatively, pulsatile contraction might ensure that differences in apical tension are equilibrated between neighbouring cells, ensuring coordination in the contraction of cells across the tissue. This analysis of neighbour relations suggests that fluctuations allow for a certain degree of coordination between cells. A combination of empirical investigation and modelling will be crucial to understand the importance of fluctuations per se during morphogenesis (Blanchard, 2010).
Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Nonmuscle myosin II regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to push rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter (Barros, 2003).
Class II myosins are barbed end-directed motors that form bipolar filaments. The filaments bind actin and initiate contraction when the two ends of the bipolar filament pull in opposite directions. Myosin's mode of action makes it unlikely that myosin II could transport cargo from one side of the cell to the other, except perhaps by progressive contraction along the cortex. The lack of colocalization of myosin II and Miranda in neuroblasts further implies that myosin II does not transport Miranda directly. The data suggest, first, that myosin II is required to maintain an intact cortical actin cytoskeleton and, second, that active myosin modifies the actin cytoskeleton at the apical cortex to exclude Miranda binding. The C. elegans myosin II may act in a similar fashion, as it appears to limit PAR-3 to the anterior of the zygote (Barros, 2003).
Several alternative approaches were taken to inactivate myosin II in neuroblasts. First, germline clones of Sqh were analyzed. In severe sqh1GLC embryos, levels of the regulatory light chain are greatly reduced from early development, and the heavy chain is found only in inactive aggregates. The actin cytoskeleton is disrupted, and neither Lgl nor Miranda localize to the cortex. Miranda concentrates instead at the spindle microtubules. Therefore, active myosin is necessary from early development to organize the actin cytoskeleton, which is in turn required for Lgl and Miranda to localize to the cortex (Barros, 2003).
Myosin II is activated by phosphorylation of its regulatory light chain by Rho kinase. Myosin was inactivated at the time of neuroblast cell division by inhibition of Rho kinase. Myosin II no longer localizes at the apical neuroblast cortex but instead spreads into the cytoplasm, and basal protein localization is disrupted. Although F-actin and Lgl remain uniformly at the cortex, cell fate determinants are now found around the entire cell cortex, demonstrating that apical cortical myosin is required to confine determinants to the basal half of the cell. Inhibition of Rho kinase also blocks cytokinesis, although the defect in basal protein localization is unlikely to be the consequence of mitotic arrest or a block in cytokinesis. First, basal protein localization is not disrupted in neuroblasts arrested in mitosis by colcemid treatment. Second, mitosis occurs without cytokinesis in pebble mutants, but the resultant polyploid neuroblasts still localize Numb and Prospero asymmetrically. Finally, the loss of asymmetry resulting from Rho kinase inhibition can be rescued by expression of a constitutively active form of the myosin II regulatory light chain (SqhE20E21). It is concluded that myosin II is required to restrict cell fate determinants to the basal cortex (Barros, 2003).
Myosin II localizes to the apical cortex of metaphase neuroblasts. Why is myosin localization/activity asymmetric? Lgl binds myosin II heavy chain directly and inhibits myosin filament formation. This binding is regulated by phosphorylation of Lgl; this phosphorylation inhibits its interaction with myosin II in vitro. If Lgl negatively regulates myosin activity and localization, then myosin should be uniformly distributed in an lgl mutant. Indeed, in lgl1GLC mutants myosin II no longer concentrates apically but is found uniformly around the cortex. Most Miranda protein is released from the cortex and binds microtubules, again suggesting that myosin excludes Miranda from the cortex (Barros, 2003).
Myosin II localizes to the entire cortex in lgl mutants and thereby prevents Miranda binding basally. In Drosophila neuroblasts in which Lgl levels are reduced (zygotic lgl1 mutants), Miranda is released from the cortex. Miranda localization can be rescued by simultaneously reducing the level of myosin II (zip1 zygotic mutants). Reducing the level of active myosin may restore the balance between the levels of Lgl and myosin, enabling the remaining myosin to concentrate apically (Barros, 2003).
How does myosin II restrict neuroblast proteins to the basal side of the cell cortex? Myosin II and Miranda occupy primarily opposite sides at the neuroblast cortex: myosin II is concentrated at the apical cortex while Miranda localizes as a basal crescent. As myosin II shifts to the cleavage furrow, Miranda is segregated into the forming GMC. The apical F-actin compartment may be modified by myosin II to exclude binding of basal proteins like Miranda. Active myosin II requires Rho kinase activity and depends on inactivation of Lgl at the apical cortex by aPKC (Betschinger, 2003). Ectopic expression of a nonphosphorylatable form of Lgl, in which the conserved aPKC-dependent phosphorylation sites are mutated from Serines to Alanines (Lgl-3A), results in mislocalization of Miranda around the neuroblast cortex (Betschinger, 2003). The data support a spatially regulated interaction between myosin II and Lgl. Myosin is apically localized in wild-type neuroblasts, corresponding to the domain in which Lgl is inactivated by aPKC. In lgl mutants, myosin is no longer restricted apically but localizes around the entire cell cortex. Conversely, when nonphosphorylatable Lgl is expressed in neuroblasts, myosin is inhibited throughout the cell and drops off the cortex. It is proposed that myosin II is activated and can form filaments at the apical cortex, where phosphorylated Lgl is inactive and unable to bind myosin II. Myosin may then modify the actin cytoskeleton to prevent the binding of Miranda. At the basal cortex, in the absence of aPKC, Lgl is active and can bind and inhibit myosin. Myosin cannot form filaments, which are required for it to bind to the actin cortex. As a result, Miranda can bind to the basal cortex (Barros, 2003).
At anaphase, myosin II moves to the equator and appears to 'push' cell fate determinants into the daughter cell. This movement is regulated in an Lgl-independent fashion and occurs whether myosin is restricted to the apical cortex or is uniformly cortical (as in lgl mutants). Cortical myosin is essential, however, to efficiently segregate determinants into the GMC at telophase (telophase rescue). In neuroblasts expressing Lgl-3A, myosin II is cytoplasmic, and determinants are not partitioned to the daughter cell. Nonetheless, at telophase, myosin seems to be recruited from the cytoplasm, since it still accumulates to the cleavage furrow. Thus three separate steps of myosin regulation in neuroblasts can be defined. First, myosin forms an apical crescent. This is positively regulated by Rho kinase and negatively regulated by Lgl. Second, cortical myosin moves to the equator. This movement occurs independently of Lgl. Third, cortical and cytoplasmic myosin accumulates at the cleavage furrow, a step that is also Lgl independent. Rho Kinase activation seems to be important for all three steps of myosin II regulation. When Rho kinase is inhibited, myosin falls into the cytoplasm, and there is no cleavage furrow formation (Barros, 2003).
In conclusion, these results demonstrate that myosin II acts downstream of Lgl and the apical protein complex to regulate the segregation of cell fate determinants. Myosin II does not negatively regulate basal protein targeting, as has previously been suggested nor does it transport determinants directly. Instead, it is proposed that myosin II acts in a novel fashion, excluding determinants from the apical cortex and 'pushing' them into the GMC at anaphase and telophase. Myosin II might modify the actin cytoskeleton to prevent determinants binding, although the actual structure formed and the physical change in the actin cytoskeleton remains to be determined (Barros, 2003).
Cell sorting involves the segregation of two cell populations into 'immiscible' adjacent tissues with smooth borders. Echinoid (Ed), a nectin ortholog, is an adherens junction protein in Drosophila, and cells mutant for ed sort out from the surrounding wild-type cells. However, it remains unknown which factors trigger cell sorting. This study dissected the sequence of this process and found that cell sorting occurs when differential expression of Ed triggers the assembly of actomyosin cable. Conversely, Ed-mediated cell sorting can be rescued by recruitment of Ed, via homophilic or heterophilic interactions, to the wild-type cell side of the clonal interface, even when differential Ed expression persists. It was found, unexpectedly, that when actomyosin cable was largely absent, differential adhesion was sufficient to cause limited cell segregation but with a jagged tissue border (imperfect sorting). It is proposed that Ed-mediated cell sorting is driven both by differential Ed adhesion that induces cell segregation with a jagged border and by actomyosin cable assembly at the interface that smoothens this border (Chang, 2011).
This study has dissected the sequence of events in Ed-mediated cell sorting and concludes that both differential adhesion and the induction of actomyosin cable formation are required and act cooperatively to mediate cell sorting. It was also demonstrated that the relocalization of Ed by Ed, Fred and Edδintra, but not Nrg-Ed, to the clonal interface of the wild-type cells is sufficient to dprevent actomyosin cable formation in the wild-type cells. How differential expression of Ed induces actomyosin cable formation only at the Ed+ interface cells (but not the Ed- cells) to generate a polarized response remains unknown. It has been suggested that interfacial tension is the result of cortical tension decreased by adhesion energy at this interface. Moreover, cortical myosin II recruitment is regulated by tension in a positive-feedback loop that could promote actomyosin cable formation (Fernandez-Gonzalez, 2009). Therefore, it is postulated that the reduction of adhesion energy caused by the loss of Ed would increase the interfacial tension so as to induce actomyosin cable formation at that interface. However, although interfacial tension also increases in ed mutant cells no prominent actomyosin cable formation was detected in these cells. Thus, interfacial tension alone is insufficient to explain this polarized effect (Chang, 2011).
Laplante (2011) proposed that, during dorsal closure, asymmetric distribution of Ed is required in the dorsal-most epidermal (DME) cells for the polarized accumulation of actin regulators (such as Enabled, Diaphanous and RhoGEF2) in the actin-nucleating centers (ANCs) of DME cells, and that this in turn promotes actomyosin cable assembly at the leading edge. Ed-mediated cell sorting resembles embryonic dorsal closure, where the DME cell is equivalent to the Ed+ interface cell, the leading edge is equivalent to the interface of ed mutant clones, and the ANC is equivalent to the interfacial tricellular junction of Ed+ interface cells. A similar polarized accumulation of actin regulators, such as Enabled, was also found at the tricellular junction of Ed+ interface cells of ed-RNAi clones. As cells within ed mutant clones cannot have a polarized distribution of Ed and actin regulators to form actomyosin cable, this provides an alternative mechanism for generating a polarized effect. Moreover, as both Nilson's group and the current demonstrated that the intracellular domain of Ed is required for actomyosin cable formation, it is possible that the asymmetric distribution of Ed might, via its intracellular domain, regulate the polarized accumulation of actin regulators at the interfacial tricellular junctions of Ed+ cells that in turn promotes actomyosin cable assembly (Chang, 2011).
The induction of actomyosin cable formation only at the Ed+ interface cells was observed not only when a large number of wild-type cells surrounded a few ed-depleted cells (in a small ed-RNAi clone) but also when a large number of ed-RNAi cells surrounded a few wild-type cells (in a very large ed-RNAi clone). The actomyosin cable at the interface supplies the tension needed to form a smooth border, tension that can be supplied either by the Ed+ interface cells surrounding a small ed-RNAi clone or by the Ed+ interface cells within a large ed-RNAi clone. However, apical constriction was present in ed-depleted cells surrounded by a large number of wild-type cells (in small ed-RNAi clones). Similarly, apical constriction was also detected when a few wild-type cells were surrounded by a large number of ed-RNAi cells (in large ed-RNAi clones). Since significant p-MLC accumulation was not detected in the apically constricted ed-RNAi nor wild-type cells, it is suggested that myosin-mediated contraction is not important in the generation of apical constriction (Chang, 2011).
Ed-mediated cell sorting is similar to the process of dorsal closure. However, during dorsal closure, amnioserosa cells actively undergo pulsed contraction that leads to a reduction in their apical surfaces. This, together with the actomyosin cable acting as a ratchet, pulls the surrounding epidermal cells towards the midline. By contrast, the apical surface of Ed-deficient cells gradually increases when the ed-RNAi clones expand. Moreover, the actomyosin cable of the interface cells acts not as a ratchet but instead as a mechanical fence to smoothly separate wild-type and Ed-deficient cells. Finally, Ed-mediated cell sorting involves the polarized assembly of actomyosin cable only in the wild-type interface cells. This is in contrast to the formation of the anteroposterior boundary in the embryo, where the formation of actomyosin cable by cells on both sides of the boundary is postulated to be the primary mechanism of cell sorting. This study suggests that differential adhesion of Ed alone is sufficient to trigger the segregation of cells into separate populations with jagged borders, but it remains unknown whether differential adhesion mediated by differential expression of as yet unidentified compartment-specific CAMs plays a role in establishing the initial anteroposterior boundary, where actomyosin cable ensures that this boundary remains straight (Chang, 2011).
This work combined genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure, during the morphogenesis of the amnioserosa, the main force-generating tissue during the process of Dorsal Closure in Drosophila. This work reveals that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. In contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, in amnioserosa cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through the control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis (Duque, 2016).
The correct localization of myosin II to the equatorial cortex is crucial for
proper cell division. A collection of genes was examined that causes defects
in cytokinesis and revealed (with live cell imaging) two distinct phases of myosin
II localization. Three genes in the rho1 signaling pathway, pebble (a Rho
guanidine nucleotide exchange factor), rho1, and rho kinase, are
required for the initial recruitment of myosin II to the equatorial cortex. This
initial localization mechanism does not require F-actin or the two components of
the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and
racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin
(diaphanous), and profilin (chickadee) are required to stably
maintain myosin II at the furrow. In the absence of these latter genes, myosin
II delocalizes from the equatorial cortex and undergoes highly dynamic
appearances and disappearances around the entire cell cortex, sometimes
associated with abnormal contractions or blebbing. These findings support a model
in which a rho kinase-dependent event, possibly myosin II regulatory light chain
phosphorylation, is required for the initial recruitment to the furrow, whereas
the assembly of parallel, unbranched actin filaments, generated by
formin-mediated actin nucleation, is required for maintaining myosin II
exclusively at the equatorial cortex (Dean, 2005).
This study has discovered three steps in the myosin II
localization/activation process that involve distinct groups of genes:
(1) an initial recruitment of myosin II to the equatorial cortex that is
independent of F-actin and centralspindlin but requires rho1 signaling;
(2) a secondary stabilization of myosin II at the midzone that requires
F-actin and a second set of genes that are likely involved in building a
specific type of actin network, and (3) the activation of furrowing
once myosin II is localized that depends on centralspindlin (Dean, 2005).
Rho1, its
activating guanidine nucleotide exchange factor pebble, and rho kinase are each
required for the initial recruitment of myosin II to the equatorial cortex. Rho1
has been implicated in two pathways that are important for cytokinesis.
In the first pathway, rho1 signals to F-actin
through the formin diaphanous. However, proteins on this F-actin
pathway, including F-actin itself, are not essential for the initial myosin II
recruitment to the equatorial cortex. However, rho kinase, another
downstream target of rho1, is essential. Because rho kinase phosphorylates the
myosin II RLC, it is possible that
phosphorylation of the RLC is essential for myosin II recruitment to the furrow.
This hypothesis could not be directly tested, because the myosin II heavy
chain forms large aggregates when the RLC is depleted by RNAi (Dean, 2005).
Phosphorylation of the RLC both activates the motor domain and, in some myosins,
increases bipolar thick filament formation. Because
F-actin is not required for myosin II recruitment, activation of the motor is
unlikely to be the mechanism by which phosphorylation of the RLC would cause
recruitment of myosin II to the equatorial cortex. It is quite possible,
however, that the rho kinase-mediated myosin II phosphorylation leads to thick
filament assembly and that this assembly is important for localization of myosin
to the equatorial cortex. Indeed, in Dictyostelium, it is clear that
bipolar thick filament formation is sufficient for myosin II localization to the
midzone of a mitotic cell. The nonactin-based mechanism of recruitment of myosin II filaments remains
unknown (Dean, 2005).
In contrast to the lack
of F-actin involvement in the early recruitment of myosin II to the equatorial
cortex at anaphase, F-actin disruption by Latrunculin A results in a failure to maintain
myosin II in the equatorial region. Interestingly, the downstream rho1 effectors
diaphanous/formin and chickadee/profilin are also necessary for myosin II
maintenance at the equatorial midzone. Although the loss of these genes could
deplete F-actin, phalloidin staining has shown that F-actin is still present in all
of the RNAi-treated cells. In addition, these RNAi-treated
cells still contract, unlike when F-actin is completely disrupted with LatA.
Thus, myosin II appears to be interacting with F-actin in the cortex as it
disperses in dynamic patches throughout the cortex of these diaphanous- or
chickadee-depleted cells (Dean, 2005).
It is suggested that the role of diaphanous/formin and chickadee/profilin in
maintaining the myosin II contractile ring is through the creation of specific
F-actin structures. In particular, formin- and profilin-mediated nucleation
results in unbranched actin filaments because profilin
promotes the barbed-end growth of formin-capped actin filaments. Indeed, electron microscopy has
shown that F-actin in the cleavage furrow mainly consists of unbranched, bundled
filaments. These parallel
filaments contrast with Arp2/3-mediated nucleation, which creates a highly
branched actin filament network. Indeed, Arp2/3, although essential for
lamellipodia formation, is not required for cytokinesis in Drosophila
cells. The hypothesis here
is that once myosin II is recruited to the equatorial cortex of
the cell by a rho kinase-dependent mechanism, possibly localized activation of
RLC phosphorylation, it is retained there because of its higher affinity for
parallel, unbranched actin filaments than to branched actin networks. Consistent
with this hypothesis, myosin II is depleted from the lamellipodia in migrating
cells where Arp2/3 is localized and branched F-actin networks are formed but is enriched in the lamella where F-actin filaments are
more likely to be aligned in parallel bundles. Thus,
it is proposed that high rho1 signaling to Diaphanous at the cleavage furrow
maintains a higher concentration of parallel actin filaments in this region
compared with the rest of the cortex, and these parallel filaments serve to
selectively retain myosin II at the equator to form a stable contractile ring.
In the absence of these parallel actin filaments, myosin II can bind branched
F-actin throughout the cortex, perhaps occasionally organizing them into
parallel bundles that cause increased myosin recruitment corresponding to the
flashes of cortical myosin accumulation, but these interactions are unstable (Dean, 2005).
Live-cell imaging shows that when pavarotti or racGAP50c
are depleted, the cells do not display significant contractions despite
recruiting myosin II to the equatorial cortex. Although there is some modest
membrane contractile activity in these cells, it is clear that significant
contraction or furrowing requires both components of the centralspindlin
complex. It is surprising that only these proteins were found to be necessary
for cortical contraction at sites of myosin II localization. Data from fixed
cells, as well as earlier studies, indicated that Drosophila cells do not
undergo equatorial contractions during mitosis when Diaphanous or Chickadee is
depleted. However, live-cell imaging shows that
when either of these two genes is depleted in S2 cells, not only is myosin II
transiently localized to the equatorial cortex before dispersing, but cells do
indeed display transient equatorial contraction. It is difficult to recognize
these events in fixed cells because of their transient nature and the somewhat
irregular shapes of cells depleted of these proteins. This work highlights the
importance of live-cell imaging in the study of dynamic processes such as
cytokinesis (Dean, 2005).
In addition to the suppression of furrowing, depletion of centralspindlin also
leads to an inability to retain F-actin exclusively at the equatorial cortex
during cytokinesis. This similar phenotype of the centralspindlin complex and
the F-actin affecting proteins suggests that centralspindlin may be an upstream
regulator of F-actin filament formation. Indeed kinase-dead mutants of Pavarotti
have been shown to accumulate at the spindle poles and are associated with an
abnormal accumulation of F-actin near the centrosomes.
Centralspindlin may be acting indirectly by helping to localize an important
actin-affecting protein at the central spindle, or it may act more directly on
the cortex. Because RacGAP50c has been shown to bind Pebble in vitro, it has been
hypothesized that centralspindlin affects the F-actin cortex through rho1
signaling by the localization and/or activation of Pebble. However, RacGAP50c depletion does
not lead to a lack of myosin II recruitment as does Pebble or Rho1 depletion,
and, thus, centralspindlin must act in a rho1-independent manner. For instance, the racGAP activity of
centralspindlin may itself be important for signaling to the F-actin cortex.
Finally, centralspindlin cannot be the major actomyosin ring positioning signal
because myosin II is properly recruited in its absence (Dean, 2005).
A single Rho GTPase family member is capable of initiating several different processes, including cell cycle regulation, cytokinesis, cell migration, and transcriptional regulation. It is not clear, however, how the Rho protein selects which of these processes to initiate. Guanine nucleotide exchange factors (GEFs), proteins that activate Rho GTPases, could be important in making this selection. In vivo, DRhoGEF2, a GEF that is ubiquitously expressed and specific for Rho1, is reiteratively required for epithelial folding and invagination, but not for other processes regulated by Rho. The limitation of DRhoGEF2 function supports the hypothesis that the GEF selects the outcome of Rho activation. DRhoGEF2 exerts its effects in gastrulation through the regulation of Myosin II to orchestrate coordinated apical cell constriction. Apical myosin localization is also regulated by Concertina (Cta), a Galpha12/13 family member that is thought to activate DRhoGEF2 and is itself activated by a putative ligand, Folded gastrulation (Fog). Fog and Cta also play a role in the morphogenetic events requiring DRhoGEF2, suggesting the existence of a conserved signaling pathway in which Fog, Cta, and DRhoGEF2 locally activate Myosin for epithelial invagination and folding (Nikolaidou, 2004).
If the guanine nucleotide exchange factor (GEF) is important in selecting the outcome of activating Rho, then its function should be limited to a subset of those associated with the GTPase. To address this possibility, the in vivo function of DRhoGEF2 was investigated. Two hypomorphic alleles, DRhoGEF2PX6 and DRhoGEF2PX10, in combination with null alleles of DRhoGEF2, give adults that have crumpled and/or blistered wings. Earlier in development, the DRhoGEF24.1/DRhoGEF2PX6 wing discs appear buckled rather than conforming to the stereotypical folding pattern observed in the wild-type. This malformation is not a result of either improper patterning or loss of apico-basal polarity. It must therefore be caused by disruption of another mechanism -- for example, the propagation of a localized signal that brings about folding in specific places. To test this hypothesis, clones of DRhoGEF21.1 cells spanning a fold were generated (Nikolaidou, 2004).
In large mutant clones that are less influenced by physical constraints, the folds fail to follow the line of the fold in wild-type tissue. Bifurcation of folds does not occur in wild-type discs, supporting the idea that the mutant tissue is unable to respond to a localized signal to fold. Although the clonal and DRhoGEF24.1/DRhoGEF2PX6 mutant tissues do appear folded, the irregularity of the folds indicates that this is probably a consequence of passive folding, as is seen in the gastrulation mutants and murine neurulation mutants that fail to invaginate tissue appropriately (Nikolaidou, 2004).
The possibility was investigated that other events involving epithelial invagination or folding might also require DRhoGEF2 activity. One such event is the invagination of a placode to form a salivary gland tube on both sides of the embryo. Combinations of dominant-negative alleles with a putative null allele of DRhoGEF2 showed that in 93% of embryos some or all of the salivary-gland cells fail to invaginate and instead remain on the outside. Because maternally provided DRhoGEF2 is vital for epithelial invagination in gastrulation, this and the above two phenotypes represent three examples of the requirement for DRhoGEF2 in epithelial-layer morphogenesis (Nikolaidou, 2004).
If DRhoGEF2 is participating in the selection of the cell's response to activated Rho, then its function should be limited. Rho is known to play a role in cytokinesis, cell cycle regulation and planar polarity. The large size of clones of DRhoGEF2, equivalent numbers of cells in twin wild-type and mutant clones, and normal polarity of mutant tissue indicate that unlike Rho, DRhoGEF2 is not required for any of these processes, nor is it required for apico-basal polarity. No significant defects were seen in the gross morphology of the nonepithelial tissues of muscles and neurons in late-stage DRhoGEF24.1/DRhoGEF26.5 and DRhoGEF24.1/DRhoGEF25.1 embryos. In addition, the normal cell cycle control shows that the convolution of DRhoGEF2 mutant wing discs is not a result of excessive proliferation (Nikolaidou, 2004).
Although the possibility exists that DRhoGEF2 has a function not addressed, it seems likely that its role is confined to the control of epithelial morphogenesis. This limit of DRhoGEF2 function suggests that it is important in selecting a role for Rho only in epithelial morphogenesis, whereas other GEFs would activate Rho in other processes; for example Pebble activates Rho primarily in cytokinesis, and Trio acts on Rac in neuronal outgrowth (Nikolaidou, 2004).
To study in more detail the mechanism by which DRhoGEF2 affects epithelial morphogenesis, the possible targets of DRhoGEF2 activation have been considered. One of these is myosin II. During gastrulation, Zipper (Zip), the heavy chain of myosin II, appears to accumulate on the apical side of the mesodermal precursors in the ventral furrow (VF). To address the possibility that apical myosin localization is required for other invagination events, salivary-gland formation was analyzed in embryos expressing the myosin light chain, Spaghetti squash (Sqh), as a fusion with green fluorescent protein (Sqh-GFP). Although Sqh-GFP is present at the cortex of all the cells, it is concentrated at the apical surface of salivary-gland precursors that are about to invaginate or are in the process of invaginating. Sqh-GFP does not accumulate apically until invagination, as demonstrated by the lack of apical localization in cells that are
present more anteriorly in the placode but that will invaginate later (Nikolaidou, 2004).
It is not clear if this apical myosin accumulation is present in time to contribute to apical constriction. To resolve this question, the localization of Sqh-GFP was observed in the invaginating VF during gastrulation. In wild-type cells, Sqh-GFP is maintained at the tip of the growing membrane that forms between the nuclei during cellularization, the process immediately prior to gastrulation. At the end of cellularization, Sqh-GFP begins to decrease on the basal side and accumulate on the apical side of the ventral cells, i.e., only those that will constrict apically. This redistribution of myosin precedes apical cellular constriction, suggesting that it contributes to the process. Basally located Sqh-GFP is subsequently lost, and the apical levels increase (Nikolaidou, 2004).
In DRhoGEF2 germline clone-derived (GLC) embryos (i.e., those lacking maternal DRhoGEF2), Zip, the myosin heavy chain, is lost from the basal side of cells in the developing VF, but it accumulates at much lower levels on the apical side than it does in the wild-type. These results imply that a signal through DRhoGEF2 is needed in order for the ventral cells to induce apical Zip localization. In contrast, relocalization of β-heavy spectrin occurred normally in DRhoGEF2 GLC embryos, indicating that cell polarity is maintained in these cells and that at least some forms of protein relocalization, especially that of a protein that is found in close proximity to Zip (Nikolaidou, 2004).
The possibility was considered that myosin localization is also regulated by other components of the DRhoGEF2 signaling pathway. By analogy to the mammalian and C. elegans orthologs and as a result of genetic studies , DRhoGEF2 is thought to participate in a signal transduction pathway, which is called here the DRhoGEF2 signaling pathway, initiated by Folded gastrulation (Fog) and propagated by Concertina (Cta). Mutations in both these genes result in gastrulation defects. In embryos derived from cta mutant mothers, a low level of Zip accumulates on the apical side only of apically constricting cells in the invaginating VF. This is also true in DRhoGEF2 GLC embryos. In contrast, there is no apical myosin apparent in the cells that do not constrict their apical surface. These data clearly link the presence of apical myosin with apical constriction and indicate that in gastrulation this is controlled by the DRhoGEF2 signaling pathway. The link between DRhoGEF2 and Myosin is also supported by the documented genetic interactions between DRhoGEF2 and zip in leg and wing development (Nikolaidou, 2004).
It is not clear how DRhoGEF2 influences the apical accumulation of myosin. It could act via the Rho effector Rho kinase. When activated by Rho in mammalian cells, Rho kinase is responsible for revealing the actin binding site on the regulatory light chain of myosin II. Thus, in DRhoGEF2 mutants, a possible failure in the activation of Rho1 and Rho kinase would result in the inability of myosin to bind actin (Nikolaidou, 2004).
If DRhoGEF2 is required reiteratively for epithelial morphogenesis, it is hypothesized that Fog and Cta might also be used reiteratively. mRNA for fog is expressed in invaginating tissue during gastrulation and salivary-gland formation, suggesting that Fog also participates in invagination of the salivary gland. At present there are conflicting reports regarding the role of fog in salivary gland formation. This study finds that some or all cells fail to invaginate in 90% of the embryos. Because invagination in gastrulation is cell autonomous, it is considered more likely that this phenotype results from a lack of fog in these cells rather than because of earlier developmental defects (Nikolaidou, 2004).
The possibility was addressed that the pathway is also important in wing development. Initial descriptions indicate that Fog and Cta play no role in this process. However, demonstrating a previously undisclosed role for Fog and Cta, combinations of mutations in fog or cta and DRhoGEF2 result in synergistic effects on wing development. Together, these results point to the reiterative use of the DRhoGEF2 signaling pathway in development to bring about epithelial folding or invagination (Nikolaidou, 2004).
Preliminary data indicate that the folds in the wing disc are brought about by apical cell constriction. It is therefore proposed, because both gastrulation and salivary gland invagination also involve apical cell constriction, that this is a major aspect of DRhoGEF2 function. The location of the folds in wing discs is highly stereotypical, which would suggest that specific signals are activated in these locations to initiate folding. One candidate for this signal is Fog, which is perhaps acting in conjunction with a second signal to bring about epithelial folding in the wings. In gastrulation, fog and cta are essential, but their phenotypes are not as strong as that observed after the removal of maternal DRhoGEF2, again indicating the requirement for additional signals that activate DRhoGEF2. The nature of this additional signal, or signals, remains elusive (Nikolaidou, 2004).
Epithelial planar cell polarity (PCP) allows epithelial cells to coordinate their development to that of the tissue in which they reside. The mechanisms that impart PCP as well as effectors that execute the polarizing instructions are being sought in many tissues. The epidermal epithelium of Drosophila embryos exhibits PCP. Cells of the prospective denticle field, but not the adjacent smooth field, align precisely. This requires Myosin II (zipper) function, and it was found that Myosin II is enriched in a bipolar manner, across the parasegment, on both smooth and denticle field cells during denticle field alignment. This implies that actomyosin contractility, in combination with denticle-field-specific effectors, helps execute the cell rearrangements involved. In addition to this parasegment-wide polarity, prospective denticle field cells express an asymmetry, uniquely recognizing one cell edge over others as these cells uniquely position their actin-based protrusions (ABPs; which comprise each denticle) at their posterior edge. Cells of the prospective smooth field appear to be lacking proper effectors to elicit this unipolar response. Lastly, fringe function was identified as a necessary effector for high fidelity placement of ABPs and it was shown that Myosin II (zipper) activity is necessary for ABP placement and shaping as well (Walters, 2006).
Since the prospective denticle field is clearly polarized, it was wondered if smooth field cells were similarly polarized but simply did not express a marker, such as the ABPs, that revealed that polarity. To test this, the formation of ABPs among prospective smooth cells was induced by ectopically expressing the transcription factor svb/ovo in small groups of cells in the ventral epidermis and these cells were marked by co-expression of GFP. Expression of svb/ovo is necessary and sufficient to induce the formation of ABPs, so by expressing svb/ovo in the smooth field, the localization of these protrusions can be visualized within the cell. When svb/ovo is induced in the smooth field, the ectopic ABPs did not preferentially localize to the posterior edge of cells. Instead, they showed a stochastic dispersal around the apical surface of the cell Both anti-phosphotyrosine and phalloidin stains label these misplaced ABPs, indicating that phospho-epitopes as well as actin are present. Out of the thirty-eight ectopic denticles scored, 63% were mis-positioned (nine on an anterior edge, fifteen placed centrally) and only 37% were localized to the posterior edge of the cell (Walters, 2006).
It is also possible that the stochastic ABP localization observed in the svb/ovo-positive cells was not due to the lack of polarity effectors in the smooth field, but instead a simple issue of developmental timing. Since a heat-shock-driven recombinase was used to induce svb/ovo expression, precise control over the timing of the recombination event or svb/ovo induction was not occuring. Attempts were made to remedy this concern by ectopically expressing svb/ovo using Ptc-GAL4. Since patched is expressed well before cell fate specification, any ectopic ABPs that are present should have had ample time to localize to the posterior cell edge. However, even when svb/ovo is expressed at this early time point, ABPs in the smooth field showed no preference for the posterior edge of cells and remained stochastically positioned. These data strongly suggest that smooth cells do not possess a latent ability to place ABPs with the unipolar asymmetry seen among prospective denticle field cells. At the minimum, an effector of unipolar asymmetry must be active only among prospective denticle field cells. Alternatively, unipolar asymmetry is established only late and is restricted to the prospective denticle field. To investigate the unipolar asymmetry further, the phenomena of ABP formation and eventual placement at the posterior edge of denticle field cells was studied (Walters, 2006).
Denticle field cells are aligned and exhibit posterior ABPs at late stages (Stage 14). Since the denticle field is specified between late stage 11 and stage 12, cell alignment and ABP formation were monitored from stage 11 onward. At late stage 12, there was little alignment among cells within each parasegment. However, cell contours evolved through stage 14 (Walters, 2006).
Turning to the development of ABPs, at late stage 12, there was little or no enhancement in actin accumulation at the apical surface of the prospective denticle field cells compared to cells within the smooth field. Early in stage 13, a diffuse actin meshwork appeared at the apical surface of prospective denticle cells. By late stage 13, the apically enriched actin had coalesced into several patches that appear to represent nascent protrusions. Surprisingly, these nascent protrusions were often located away from the posterior edge of a cell. Only later, during stage 14, were ABPs more uniformly at or near each cell's posterior edge. Note that more fully pointed and curved ABPs can be observed at stage 15 (Walters, 2006).
Quantification of actin accumulation within slices across the apical face of a cell throughout these developmental stages supported the notion of a progression to the posterior edge. The intensity of Rhodamine–phalloidin-labeled actin was measured by dividing a prospective denticle field cell into six domains (progressing from A to P) and recording the pixel intensities in each of the domains at the apical face of that cell. The process was repeated for 3 cells in 3 different animals at early stage 13, late stage 13 and stage 14. The results showed that, during early and late stage 13, actin was not biased to any cell edge. At stage 14, actin was enriched dramatically at the posterior edge. It is concluded that actin first accumulates stochastically on the apical face of the cell and then is later positioned at the posterior edge. A core PCP component, frizzled (fz), was tested for its potential role in establishing or maintaining unipolar asymmetry among prospective denticle cells (Walters, 2006).
The frizzled gene is important for polarizing the hairs on the wing and in the abdomen and for ommatidial orientation. Denticle cuticle pattern were examined in the progeny of fzH51/fzP21 mothers crossed with fzH51/TM3 Ubx-LacZ fathers. Most larval cuticles appeared normal, though half of these were expected to be null for fz function. Two of thirty cuticles analyzed did exhibit patterning errors, but these were fusions among segments. This might have been caused by a partially penetrant deficit in canonical Wingless signaling since fz is also used in this pathway (where it is redundant with Dfrizzled2). The ABPs were examined directly in fz-deficient embryos; these were unambiguously identified by the lack of Ubx-LacZ expression. All fz-deficient embryos exhibited normal cell alignment of denticle field cells, and most mutants (six of eight) exhibited normal posterior placement of ABPs. Only two mutants exhibited misplaced protrusions, and in these embryos, the defects were restricted to row 1. Fully penetrant, but similarly restricted defects (to rows 1 and 2) have been reported for the core PCP mutants fz, strabismus, flamingo and even more weakly so for the PCP-specific mutant of disheveled (dsh1. Collectively, the defects observed implicate core PCP genes in denticle field polarization, but the restriction of these defects solely to anterior-most rows suggests a more minor role than expected in executing unipolar asymmetry (Walters, 2006).
Denticle field cells elaborate ABPs, and this has allowed it to be established that these cells are polarized in the plane of the epithelium. Forcing smooth field cells to elaborate ABPs failed to reveal any latent unipolarity within this portion of the parasegment. Nevertheless, the evidence supports the proposition that both fields are polarized, albeit in different ways. This study establishes bipolar enrichment of Myosin II on prospective smooth and denticle field cells. This suggests that the whole ventral epithelium exhibits bipolar PCP, and all cells can discriminate their A/P from their D/V edges. Since this bipolar enrichment emerges during stage 12, it is possible that this reflects de novo establishment of polarity within the epithelium. However, at earlier stages, cells of this epithelium exhibit a strikingly similar bipolar distribution of Myosin II then used for convergence extension. Thus, the bipolar redeployment of Myosin II might reflect a memory of that earlier polarization. This possibility is particularly compelling given that Myosin II orchestrates the rearrangements of cell junctions necessary for convergence extension. Perhaps Myosin II is being engaged similarly at the later stages, and the re-emergence of a bipolar preference is a precondition to accomplish the necessary junctional re-organization for cell alignments. What signals direct this re-emergence are not yet known. In addition, since Myosin II is bipolar among both prospective denticle and smooth cells, though the latter do not align, there must either be cues or effectors specific to the denticle field that initiate the cell alignment process (Walters, 2006).
Note also that the bipolar enrichment of Myosin II yields no clues as to how the denticle field cells uniquely identify their 'P' cell edge and faithfully position the ABPs to this edge. One possibility is that only the prospective denticle field cells have proper effectors to transmute the global bipolarity into asymmetric unipolarity. Since it is difficult to imagine how this might occur, a second possibility is that unipolarity is imparted locally, only across the prospective denticle field. The failure to observe proper ABP placement after ectopic expression of svb/ovo in the prospective smooth field supports this possibility. If unipolarity is imparted locally, then this also places constraints on the timing of the signals for unipolarity. The epithelium is sorted into smooth and denticle fields only late in development as a consequence of the antagonism between Wingless signaling and EGF receptor activation. This results in the establishment of the domain of expression for Svb. Thus, the denticle field is only established after this time, and the idea is favored that unipolarity is assigned after this stage. It is believed that analysis of Fringe supports this contention: fringe comes to be expressed within denticle field cells only after this stage and mutation of fringe interferes with unipolarity across the whole denticle field. This identifies fringe as, at the minimum, an effector of denticle field unipolarity (Walters, 2006).
Among a set of core genes involved in the establishment and maintenance of polarity in other tissues is frizzled, and surpriskingly it did not play a major role in denticle field polarization. Only two of eight embryos exhibited any defects, and in these, only row 1 cells appeared affected. Yet, a role for frizzled signaling in denticle field unipolarity cannot be ruled out due to possible redundancy with DFrizzled 2. In fact, recent work has implicated several core PCP genes in denticle field unipolarity. Mutation of frizzled, dsh, flamingo or strabismus lead to mild defects restricted to rows 1 and 2 reminiscent of what are report here for the minority of fz embryos. In addition, enrichment for Flamingo, Fz and Dsh occurs on the edges of prospective denticle field cells. In aggregate, these data are very suggestive for a role of core PCP genes in ABP polarity. However, the restriction of the phenotype to the very anterior rows of the denticle field leaves open the possibility that these genes do not play as major of a role as they do in, for example, the wing. In addition, while it has been shown that Fz and Dsh are enriched on certain cell edges, it will be of interest to know whether the enrichment is uniquely to one edge of cells, as it is in wing cells. For example, the data show that Zipper and Squash-GFP are enriched to cell edges but that these are likely both A and P edges of each cell. In denticle field cells, if Fz and Dsh exhibit such bipolar enrichment, rather than the unipolar asymmetry observed in wing cells, then their role in the denticle field would be quite distinct from that currently proposed for wing cells (Walters, 2006).
It will be difficult to establish definitively whether Fz or Dsh play more extensive roles in denticle field polarity, especially since this data strongly suggest that effectors for this polarity are likely established late, after the epithelium is sorted into smooth versus denticle field cells. It would not be possible to easily interpret the removal of all function for dsh and frizzled (which likely would entail the removal of both frizzled and Dfrizzled2) because both proteins play earlier and essential roles in Wingless signal transduction. Removing both maternal and zygotic dsh (or fz Dfz2) function might well lead to polarity phenotypes, but these may be secondary to earlier deficits in Wg signaling. This is especially the case as Wg (and Hedgehog as well) plays a major role in establishing the denticle versus smooth field and subdividing parasegments into smaller signaling territories as well as establishing the responses of the cells to those signals (Alexandre, 1999, Gritzan, 1999, Hatini and DiNardo, 2001b and Wiellette and McGinnis, 1999). These considerations also raise a caution in drawing the conclusion from Wg or Hh null mutants that these pathways play any direct role in polarization (Walters, 2006).
Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper testing of this hypothesis. Loss- and gain-of-function approaches were used to examine the role of Diaphanous in Drosophila morphogenesis. Diaphanous has a dynamic expression pattern consistent with a role in regulating cell shape change. Constitutively active Diaphanous was used to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. This hypothesis was tested by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated effects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis (Homem, 2008).
Previous analysis of Diaphanous-related formins (DRF) function in morphogenesis was hampered by mammalian DRF redundancy and the key role of DRFs in cytokinesis. The
genetic tools available in Drosophila were used to partially circumvent these
difficulties. Dia is known to regulate cellularization. The
analysis revealed new roles during morphogenesis. In embryos with severely
reduced Dia function, gastrulation is disrupted. Apical constriction of
central furrow cells is delayed or blocked in a subset of cells, suggesting a
possible role for Dia in regulated apical constriction. Previous work
suggested that an unknown Rho effector regulates actin during this process;
perhaps this is Dia. Adjacent cells, which are stretched during invagination,
become massively multinucleate in dia5M mutants. This may
reflect the fact that the cells of the blastoderm undergo an incomplete form
of cytokinesis, remaining connected to the underlying yolk by actin-lined yolk
canals. These canals normally close at the onset of gastrulation in ventral
furrow cells; this may prevent cell membrane rupture initiated at the yolk
canal under stress. Dia may regulate yolk canal closure; it localizes there at the end of cellularization and similar defects are seen in mutants lacking the septin Peanut, a yolk canal component. Alternately, Dia may stabilize cortical actomyosin or its connections to AJs, with its absence weakening cortical integrity under stress (Homem, 2008).
Dia also plays a key role during germband retraction. The most striking
effect of reduced Dia/Rho1 function is altered amnioserosal cell
protrusiveness. They normally make stable AJs and form a tight tissue boundary
with the epidermis; this is destabilized in dia/Rho1 and
dia5M/Z mutants. Altered protrusive
activity was seen in other contexts: dia5M/Z ectoderm exhibited
cortical blebbing, and dia/Rho1 and dia5M/Z
amnioserosal cells had altered protrusiveness during dorsal closure. Reduced
Dia function destabilized AJs, and the striking increase in cell
protrusiveness in dia/Rho1 mutants was substantially rescued by
increasing Myosin phosphorylation. These data support the hypothesis that Dia
normally helps restrict actomyosin contractility to AJs, and in its absence,
AJs are destabilized and myosin activity outside AJs stimulates protrusiveness. Interestingly, in cultured cells, fully assembled AJs inhibit Rac1 and lamellipodial activity while increasing Rho and contractility, and myosin is required for mouse Dia1-induced AJ strengthening (Homem, 2008).
Given these morphogenetic roles, the mechanisms of action of
Dia was explored, initially hypothesizing that it would primarily affect actin.
DiaCA expression did elevate F-actin levels (reducing Dia did not
dramatically decrease F-actin, but residual Dia may suffice). However,
surprisingly, Dia activation also increased Myosin accumulation and cell
contractility, and some effects of reduced Dia function were rescued by
increasing Myosin activation. This suggests a surprising role for Dia in
regulating Myosin levels/activity. The data also suggest that Dia stabilizes
AJs, supporting earlier work in cultured cells. There
are intimate connections between AJs and cortical actomyosin: AJs help
organize the apicolateral actin ring while cortical actin stabilizes AJs.
Interestingly, in cultured cells Myosin-mediated contraction of actin
filaments is essential for cell-cell contact expansion, and
mouse Dia1 AJ stabilization requires myosin activity, providing further evidence that contractility, AJ stability and Dia activity are mechanistically linked. The actomyosin network may also stabilize AJs by preventing endocytosis. Perhaps Dia, like formin 1, directly binds AJs. Linear actin filaments promoted by Dia at AJs might recruit α-catenin, which then might inhibit Arp2/3-induced actin branching, facilitating formation of a belt of unbranched actin. Thus, Dia is poised to act at the interface between AJs, cortical actin and contractility (Homem, 2008).
The Yin-Yang relationship between adhesion and protrusiveness is a very
interesting one, underlying epithelial-mesenchymal transitions. Stable AJs
probably actively inhibit protrusiveness, and Dia may assist this, acting in a
reinforcing loop that concentrates actomyosin activity at AJs. There are
interesting parallels with the role of Dia in cytokinesis. Dia stabilizes
Myosin at the midzone. In its absence Myosin is delocalized around the cortex,
leading to abnormal protrusiveness. Future work will reveal how Dia fits into the
regulatory network coordinating adhesion and cytoskeletal regulation (Homem, 2008).
From these data, a mechanistic model was developed for the regulation of actin, Myosin and AJs by Dia during cell shape change, in which Dia activation stabilizes actin and active Myosin at AJs. In radially symmetric amnioserosal cells, Dia promotes organization/activation of an apical actomyosin network linked to AJs, inducing precocious apical cell constriction. Activation of endogenous Dia may regulate normal amnioserosal constriction: this now needs to be tested. Dia activation in cells where AJs are planar polarized, such as epidermal cells, promotes actomyosin organization preferentially at cell borders where AJs are enriched. This leads to cell widening or helps cells resist elongation, generating groove-cell-like morphology. Once again, Dia may be normally activated specifically in groove cells to modulate their shape (Homem, 2008).
How does Dia activation activate Myosin? It does not occur primarily
through SRF, which triggers Myosin accumulation but not cell shape changes.
Myosin activation via MLCK partially mimicked DiaCA, suggesting
that Myosin activation is an important part of the process. However, MLCK did
not precisely mimic DiaCA, suggesting that Dia acts preferentially
at specific sites such as AJs rather than globally activating Myosin. The dual
effects on actin and Myosin suggests the speculative possibility that feedback mechanisms exist to coordinate the Rho-regulated actin and Myosin pathways. Recent work revealed that active Dia1 can activate RhoA by binding the Rho-GEF LARG, and strong genetic interactions were seen between dia and the LARG relative RhoGEF2, making this idea more plausible. Future work is needed to test this hypothesis (Homem, 2008).
Cells heal disruptions in their plasma membrane using a sophisticated, efficient, and conserved response involving the formation of a membrane plug and assembly of an actomyosin ring. This paper describes how Rho family GTPases modulate the cytoskeleton machinery during single cell wound repair in the genetically amenable Drosophila embryo model. Rho, Rac, and Cdc42 were found to rapidly accumulate around the wound and segregate into dynamic, partially overlapping zones. Genetic and pharmacological assays show that each GTPase makes specific contributions to the repair process. Rho1 is necessary for myosin II activation, leading to its association with actin. Rho1, along with Cdc42, is necessary for actin filament formation and subsequent actomyosin ring stabilization. Rac is necessary for actin mobilization toward the wound. These GTPase contributions are subject to crosstalk among the GTPases themselves and with the cytoskeleton. Rho1 GTPase was found to use several downstream effectors, including Diaphanous, Rok, and Pkn, simultaneously to mediate its functions. These results reveal that the three Rho GTPases are necessary to control and coordinate actin and myosin dynamics during single-cell wound repair in the Drosophila embryo. Wounding triggers the formation of arrays of Rho GTPases that act as signaling centers that modulate the cytoskeleton. In turn, coordinated crosstalk among the Rho GTPases themselves, as well as with the cytoskeleton, is required for assembly/disassembly and translocation of the actomyosin ring. The cell wound repair response is an example of how specific pathways can be activated locally in response to the cell's needs (Abreu-Blanco, 2013).
Actin and myosin II recruitment and organization at the wound
edge are key and conserved elements of the single cell wound
repair process in different organisms. In epithelial cells and
Xenopus oocytes, wounding induces a strong cytoskeletal
response dependent on calcium and Rho family GTPase
signaling. In particular, the contractile actomyosin
ring formed in Xenopus oocyte wounds is accompanied by
the flow of cortical F-actin filaments toward the wound from
neighboring regions. Myosin II also accumulates as foci
at the wound edge, and its recruitment is independent of
F-actin and cortical flow, although its subsequent assembly
into a continuous ring does require an intact F-actin network. Interestingly, this study found that actin and myosin II are both
actively mobilized toward the wound in the Drosophila
syncytial embryo, in a process dependent on cortical flow (Abreu-Blanco, 2013).
This mobilization of myosin II is necessary for proper actomyosin
ring assembly and function. In the context of
Drosophila cell wound repair, Rac coordinates the actin and
microtubule network that influences actin and myosin recruitment,
consistent with its known functions in regulating the
polymerization of both actin and microtubules. The sharp
actomyosin ring formed at the leading edge of the wound, as
well as the accompanying halo of dynamic actin, provides
the contractile force driving rapid wound closure. This study found
that Rho1 is necessary for myosin II activation, leading to its
association with actin: actin fails to accumulate as a ring at
the wound edge when myosin is disrupted. This is consistent
with Rho's known role in increasing the phosphorylation of
the myosin II regulatory light chain via its downstream effector
Rok. Rho1, along with Cdc42, also functions upstream of
the assembly and constriction of the actomyosin ring, where
they likely trigger actin filament formation and subsequent
actomyosin ring stabilization (Abreu-Blanco, 2013).
In addition to Rho family GTPases regulating actomyosin
ring assembly, the actin and myosin II cytoskeleton reciprocally
mediates GTPase function. For example, the assembly/
disassembly of the actin network in Xenopus oocyte wounds
has been shown to disrupt the local activation and inactivation
patterns of Rho and Cdc42, which are required for wound healing
progression. In the Drosophila model, actin is required
for the translocation, refinement, and maintenance of the Rho
family GTPase arrays, while myosin II is required for their
recruitment and organization. The actomyosin ring acts as
scaffold for signaling molecules that, in turn, are responsible
for the polymerization of actin and activation of myosin II.
Stabilization of the actin network by Jasplakinolide treatment
provides strong evidence that the actomyosin array and the
underlying signals translocate together. A striking observation
from this model is that Rho1, the first Rho family GTPase to be
recruited, accumulates at the wound site even when the actin
and myosin II cytoskeleton is severely disrupted, indicating
that its recruitment is independent of cytoskeleton integrity.
Cdc42 and Rac1, which accumulate after Rho1, are sensitive
to disruption of actin and myosin II. Thus, Rho
family GTPases are required in Drosophila syncytial embryos
for proper wound healing through reciprocal regulation with
the dynamic and integrated actin and myosin II networks (Abreu-Blanco, 2013).
Rho family GTPases localize at the wound edge with a precise
and characteristic organization pattern. This study shows the recruitment of Rac GTPases to the wound: Rac1 and Rac2 accumulate as graded ring-like arrays at the wound
edge. By analyzing the distribution of the endogenous Rho
GTPases, it was found that Rho, Cdc42, and Rac (Rac1/2) form
partially overlapping concentric arrays that correlate with their
specific functions during wound repair. Another interesting
observation is that Rho is recruited to the wound before the
onset of actin and myosin II recruitment and followed by
Cdc42 and Rac (Rac1/2) with a 90 s delay. The localization of active Rho GTPases was analyzed using a combination
of activity biosensors and downstream effectors. In the
Drosophila model, active Rho1 localizes as a discrete array
internal to the actomyosin ring. This is in contrast to that
observed in Xenopus oocyte wounds, where active Rho and
Cdc42 arrays are formed as discrete concentric rings overlapping
with myosin II and actin, respectively. Although the
timing and configuration of the GTPase arrays is different in
the two cell wound models, they both achieve the same end
result: organization of a highly contractile actomyosin ring
and a dynamic surrounding zone of actin and myosin assembly/
disassembly, thereby ensuring that a region of highly
contractility enriched in myosin II is followed by one of low
contractility where actin can assemble. Indeed, the organization
of Rho GTPases as local arrays is not only restricted to
contractile ring structures such as those observed in wound
repair and cytokinesis, but is a common strategy in different
biological processes. In the context of cell migration, coordinated
zones of Rho family GTPases at the cell's leading
edge regulate the waves of cellular protrusion and retraction
necessary for migration. In this scenario, Rho activation
occurs first, followed by Cdc42 and Rac1 with a 40 s delay.
Rho accumulates at the front of the cell concomitant with protrusions
and its levels are reduced during retraction, while
Rac1 accumulates behind the Rho array with its levels remaining
high during the retraction phase. These patterns of activation
and organization correlate with their proposed activities:
Rho modulates contraction and polymerization, whereas
Cdc42 and Rac1 regulate adhesion dynamics (Abreu-Blanco, 2013).
Negative feedback among Rho GTPases is a conserved
theme in multiple cellular and developmental processes.
In the Xenopus wound model, Rho has been shown to negatively
modulate the integrity of the Cdc42 array, while inhibition
of Cdc42 activity strongly suppresses local Rho activation.
Moreover, Abr, a protein with Rho/Cdc42 GEF and Cdc42 GAP
activity has been shown to accumulate at the Rho zone where
its GAP activity is required to locally suppress Cdc42 activity,
allowing Rho and Cdc42 to segregate into their respective
zones. It was not possible to specifically deplete Cdc42 in
the Drosophila syncytial embryo wounding assays, so its effects on Rho1 and Rac were not specifically addressed in this
model. Nonetheless, the results support the idea of GTPase
crosstalk playing a role in the control of GTPase array organization,
segregation, and levels. A notable example of crosstalk
in this context comes from the expansion of the Rac1 wound-induced
array in embryos where Rho1 levels were depleted,
suggesting that high levels of Rho1 at the wound edge inhibit
Rac1 accumulation at the interior of the actomyosin ring,
thereby allowing Rac1 to control the dynamic actin halo
surrounding the wound. These intricate and highly regulated
interactions among Rho family GTPases allow them to efficiently
execute their multiple functions in the cell (Abreu-Blanco, 2013).
A current challenge in the field is to determine how Rho
GTPases are maintained locally at high levels that dynamically
adjust during wound closure. Recent studies in the Xenopus
model propose a mechanism of GTPase treadmilling wherein
Rho and Cdc42 are subject to rapid local activation and
inactivation, which is different for each GTPase: the Rho
activity zone is shaped by trailing edge inactivation, whereas
Cdc42 undergoes variable inactivation across the array.
The development of biologically active photoactivatable Rho
family GTPase reporters will be necessary to determine the
relevance of this option in the Drosophila cell wound model.
An alternate possibility is that Rho family GTPases and their
regulators are anchored at the plasma membrane in an actin-dependent
manner. Putative candidates for anchoring proteins
are the ERM family proteins, which are known to interact
and regulate Rho GTPases in different cellular process (Abreu-Blanco, 2013).
It was surprising to find that Rho1 utilizes multiple downstream
effectors simultaneously during wound repair, begging
the question of how this specificity is achieved, maintained,
and tweaked dynamically. Considering the complexity of
wound repair, cytoskeletal responses are likely regulated via
several signaling pathways that converge on the contractile
ring. These pathways require precise coordination to provide
and integrate the different components required for the
dynamic assembly and disassembly of contractile ring
machinery as the wound is drawn closed. Rho family GTPases,
rather than switching the behavior of the entire cell, would
need to be capable of locally modulating the dynamics of the
cytoskeleton from one part of the cell to another. This specificity
associated with simultaneous recruitment and function
of downstream effectors is likely to be a key regulatory feature
for dynamic orchestration of cell wound repair. Future challenges
include defining the molecular composition of these
signaling modules and delineating the combinations and specific
subcellular localizations of Rho GTPases, GEFs, GAPs,
upstream regulators, and downstream effectors that are
needed for the proper functioning of these pathways (Abreu-Blanco, 2013).
Elkhatib, N., Neu, M. B., Zensen, C., Schmoller, K. M., Louvard, D., Bausch, A. R., Betz, T. and Vignjevic, D. M. (2014). Fascin plays a role in stress fiber organization and focal adhesion disassembly. Curr Biol 24: 1492-1499. PubMed ID: 24930964L
Migrating cells nucleate focal adhesions (FAs) at the cell front and disassemble them at the rear to allow cell translocation. FAs are made of a multiprotein complex, the adhesome, which connects integrins to stress fibers made of mixed-polarity actin filaments. Myosin II-driven contraction of stress fibers generates tensile forces that promote adhesion growth. However, tension must be tightly controlled, because if released, FAs disassemble. Conversely, excess tension can cause abrupt cell detachment resulting in the loss of a major part of the adhesion. Thus, both adhesion growth and disassembly depend on tensile forces generated by stress fiber contraction, but how this contractility is regulated remains unclear. This study shows that the actin-bundling protein Fascin crosslinks the actin filaments into parallel bundles at the stress fibers' termini. Fascin prevents myosin II entry at this region and inhibits its activity in vitro. In fascin-depleted cells, polymerization of actin filaments at the stress fiber termini is slower, the actin cytoskeleton is reorganized into thicker stress fibers with a higher number of myosin II molecules, FAs are larger and less dynamic, and consequently, traction forces that cells exert on their substrate are larger. It was also shown that fascin dissociation from stress fibers is required to allow their severing by cofilin, leading to efficient disassembly of FAs (Elkhatib, 2014).
Epithelial tissues can elongate in two dimensions by polarized cell intercalation, oriented cell division, or cell shape change, owing to local or global actomyosin contractile forces acting in the plane of the tissue. In addition, epithelia can undergo morphogenetic change in three dimensions. This study shows that elongation of the wings and legs of Drosophila involves a columnar-to-cuboidal cell shape change that reduces cell height and expands cell width. Remodeling of the apical extracellular matrix by the Stubble protease and basal matrix by MMP1/2 proteases induces wing and leg elongation. Matrix remodeling does not occur in the haltere, a limb that fails to elongate. Limb elongation is made anisotropic by planar polarized Myosin-II, which drives convergent extension along the proximal-distal axis. Subsequently, Myosin-II relocalizes to lateral membranes to accelerate columnar-to-cuboidal transition and isotropic tissue expansion. Thus, matrix remodeling induces dynamic changes in actomyosin contractility to drive epithelial morphogenesis in three dimensions (Diaz-de-la-Loza, 2018).
Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. This study analyzed the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. Early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which this study shows to regulate actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis (Krueger, 2019).
Cell shape morphogenesis from spherical to polygonal occurs in epithelial cell formation in metazoan embryogenesis. In syncytial Drosophila embryos, the plasma membrane incompletely surrounds each nucleus and is organized as a polygonal epithelial-like array. Each cortical syncytial division cycle shows circular to polygonal plasma membrane transition along with furrow extension between adjacent nuclei from interphase to metaphase. This study assessed the relative contribution of DE-cadherin and Myosin II at the furrow for polygonal shape transition. Polygonality initiates during each cortical syncytial division cycle when the furrow extends from 4.75 to 5.75 microm. Polygon plasma membrane organization correlates with increased junctional tension, increased DE-cadherin and decreased Myosin II mobility. DE-cadherin regulates furrow length and polygonality. Decreased Myosin II activity allows for polygonality to occur at a lower length than controls. Increased Myosin II activity leads to loss of lateral furrow formation and complete disruption of polygonal shape transition. These studies show that DE-cadherin-Myosin II balance regulates an optimal lateral membrane length during each syncytial cycle for polygonal shape transition (Dey, 2020).
Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. This study reports the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using a system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain were prepared and crystallized. The x-ray crystal structure was solved by molecular replacement, and the resulting model was refined against diffraction data to 2.2 A resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins (Caldwell, 2019).
Zygotically expressed myosin II is required for leg disc eversion. Depletion of Spaghetti squash, the myosin regulatory light chain, results in curved, thickened upper legs and defective wings. Depletion of SQH often results in a complete disorder in the usually perfect hexagonal packing of the ommatidia of the eye, as well as the development of an anterior notch on the eye. The time at which SQH is withheld is critical in full penetrance of the eye phenotype (Edwards, 1996).
During organogenesis, different cell types need to work together to induce functional multicellular structures. To study this process, use was made of the genetically tractable fly retina, with a focus on the mechanisms that coordinate morphogenesis between the different epithelial cell types that make up the optical lens. This work shows that these epithelial cells present contractile apical-medial MyosinII meshworks, which control the apical area and junctional geometry of these cells during lens development. This study also suggests that MyosinII meshworks drive cell shape changes in response to external forces, and thus they mediate part of the biomechanical coupling that takes place between these cells. Importantly, this work, including mathematical modelling of forces and material stiffness during lens development, raises the possibility that increased cell stiffness acts as a mechanism for limiting this mechanical coupling. It is proposed that this might be required in complex tissues, where different cell types undergo concurrent morphogenesis and where averaging out of forces across cells could compromise individual cell apical geometry and thereby organ function (Blackie, 2020).
Morphogenesis is largely driven by changes in the shape of individual cells. However, how cell shape is regulated in developing animals is not well understood. This study shows that the onset of TGFbeta/Dpp signaling activity correlates with the transition from cuboidal to columnar cell shape in developing Drosophila melanogaster wing disc epithelia. Dpp signaling is necessary for maintaining this elongated columnar cell shape and overactivation of the Dpp signaling pathway results in precocious cell elongation. Moreover, evidence is provided that Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, it was demonstrated that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. These results identify a cell-autonomous role for Dpp signaling in promoting and maintaining the elongated columnar shape of wing disc cells and suggest that Dpp signaling acts by regulating Rho1 and MRLC (Widmann, 2009).
Cell extrusion was observed when Dpp signaling was locally reduced in tkva12 bsk- clones, but not when it was reduced throughout the dorsal compartment by expression of Dad. This indicates that cell extrusion is a consequence of the sharp boundary of Dpp signaling at the clone border. One of the first morphological consequences of the loss of Dpp signaling in tkva12 bsk- clones was the apical constriction of mutant cells and surrounding control cells. Apical constriction correlated with increased staining intensities of F-actin and P-MRLC, a marker for active non-muscle myosin II, at the apicolateral side of tkva12 bsk- and neighboring wild-type cells. The formation of a similar actin-myosin ring has been previously demonstrated during the extrusion of apoptotic cells, and it has been proposed that contraction of this ring squeezes cells out of the epithelium. It is currently unclear whether these increased staining intensities reflect an increase in the total amount of F-actin and P-MRLC in tkva12 bsk- mutant clones, or whether they are instead merely a consequence of the apical constriction of cells. Nevertheless, these findings are consistent with the view that contraction of an actin-myosin ring might contribute to the extrusion of tkva12 bsk- cells. Apical cell constriction was paralleled with cell shortening along the apical-basal axis. Based on the observation that reduction in Dpp signaling throughout the wing disc pouch resulted in apical-basal cell shortening, but not in apical cell constriction, it is speculated that cell shortening, and thus the formation of an inappropriate cell shape, might be an initial event leading to the extrusion of tkva12 bsk- cells. If so, cell extrusion might not represent a specific response to eliminate slow-growing or apoptotic cells, but rather represents a general response to inappropriate cell function or morphology. In the wild type, cell extrusion might be instrumental in maximizing tissue fitness by removing cells with inappropriate function or morphology (Widmann, 2009).
The basal membrane of tkva12 bsk- cells and neighboring control cells, identified by PSβ-integrin labeling, became apposed. Since this led to a reduction in the lateral contact between mutant and neighboring control cells, this apposition might help to dislodge tkva12 bsk- cells from the remaining epithelium, and thereby, might aid the extrusion process. It is also noted that extruded tkva12 bsk- cells displayed features reminiscent of epithelial-to-mesenchymal transition (EMT). In particular, a strong decrease in E-cadherin, a hallmark of EMT and actin-rich processes were observed in extruded tkva12 bsk- cells. Interestingly, a role for Dpp/BMPs in preventing EMT has also been identified in vertebrates. Mouse BMP7, which is related to Dpp, for example, is required for counteracting EMT associated with renal fibrosis. Decreased E-cadherin levels have also recently been reported following the extrusion of cells deficient for C-terminal Src kinase from Drosophila epithelia, indicating that this might be a more common consequence of cell extrusion (Widmann, 2009).
Reduced apical-basal cell length was observed when Dpp signaling was severely reduced, either in clones or throughout the wing disc pouch; however, apical cell constriction, fold formation and cell extrusion were only detected by clonal reduction of Dpp signaling. Instead, cells were apically widened and did not extrude when Dpp signaling was reduced throughout the dorsal compartment. These experiments therefore allowed the effects of sharp boundaries of Dpp signaling at clone borders to be separated from cell-autonomous functions of Dpp signaling. They demonstrate that the cell-autonomous function of Dpp signaling is not to prevent apical cell constriction, folding and cell extrusion, but rather to maintain proper columnar cell shape. Moreover, three further observations suggest that Dpp signaling has an instructive role that drives cell elongation. (1) In the wild type, an increase in Dpp signal transduction activity correlated with apical-basal cell elongation in second instar larval discs. (2) In wing discs of late third instar larvae, Dpp signal transduction activity correlated with apical-basal cell length along the anteroposterior axis. (3) Activation of Dpp signaling, by expressing the constitutively active Dpp receptor TkvQ-D, resulted in precocious cell elongation and apical cell narrowing during early larval development. These findings indicate that Dpp signaling is an important trigger for the cuboidal-to-columnar transition in cell shape that occurs during mid-larval development (Widmann, 2009).
How does Dpp signaling promote the apical-basal elongation of wing disc cells? Compartmentalization of Rho1 activity has been recognized as being important for shaping cells and tissues. In the wild-type wing disc, Rho1 protein is enriched and the activity of the Rho1 sensor is increased at the apicolateral side, and more moderately at the basal side, of elongated cells. By contrast, Rho1 activity is more uniform in cuboidal cells, and overexpression of RhoGEF2, which leads to uniform distribution of this protein and presumably also uniform Rho1 activity, resulted in a cuboidal cell shape. Rho1, when present at the apicolateral side of cells, might therefore have a function in stabilizing or promoting cell elongation. Since the apicolateral increase in Rho1 sensor activity correlated with an increase of P-MRLC at a similar location, this function of Rho1 might be mediated by myosin II. The observation that a decrease in the bulk of Rho1 activity, either through expression of Rho1N19 or rho1dsRNA, resulted in cell elongation rather than in cell shortening, further suggests that the compartmentalization of Rho1 activity is important for shaping wing disc cells. Future studies will need to examine the morphogenetic consequences of locally modulating the activity of Rho1 (Widmann, 2009).
The results provide strong evidence for a functional link between Dpp signaling and Rho1-myosin II. Shortening of cells with compromised Dpp signaling could be rescued by a decrease in Rho1 or MRLC activity. In particular, the expression of MbsN300, an activated form of a subunit of myosin light chain phosphatase, which in wild-type wing discs did not significantly alter cell length, did rescue the shortening of Dpp-compromised cells. This indicates a specific interaction between Dpp signaling and Mbs-myosin II. The data further suggest that Dpp signaling controls apical-basal cell length by compartmentalizing Rho1 protein abundance and/or activity. (1) In late third instar wing discs, apicolateral enrichment of Rho1 protein and Rho1 sensor activity directly correlated with the local level of Dpp signal transduction activity. (2) Rho1 protein abundance and Rho1 sensor activity were decreased at the apicolateral side of cells when Dpp signal transduction was compromised by expression of Dad. (3) Rho1 protein and Rho1 sensor activity were increased at the apicolateral side and also at the basal side of cells when Dpp signal transduction was activated during early development by expression of TkvQ-D (Widmann, 2009).
Local activation of Rho1 and myosin II can lead to contraction of actin-myosin filaments, which can increase the cortical tension that is important for the shaping of cells during various developmental processes. By compartmentalizing Rho1 activity, Dpp signaling might promote both apical-basal cell elongation and apical cell narrowing. An increase in tension at the apicolateral cell cortex might promote apical cell narrowing. At the same time, a relative decrease in cortical tension laterally, compared with that on the apicolateral side, might allow cells to elongate through intrinsic cytoskeletal forces and/or extrinsic forces imposed by the growth of the epithelium. In this model, Dpp signaling directs the cuboidal-to-columnar shape transition of wing disc cells by increasing the Rho1 and myosin II activities at the apicolateral side of cells. The local increase of Rho1 and myosin II activities might then shift the balance of tension between the apicolateral cell cortex and the lateral cell cortex towards an increased tension at the apicolateral cell cortex (Widmann, 2009).
The results identify a Dpp-Brk-Rho1-myosin II pathway controlling cell shape in the wing disc epithelium. The elimination of Brk function in mad- mutant cells allowed these cells to maintain a normal columnar cell shape, indicating that Dpp controls epithelial morphogenesis through repression of Brk. Since Brk acts as a transcriptional repressor, the link between Brk and Rho1 is most probably established through an unknown Brk-repressible gene. The identification of genes transcriptionally repressed by Brk will thus be important for determination of how Dpp signaling controls Rho1 and thereby, epithelial cell shape. The finding that Dpp signaling has a cell-autonomous morphogenetic function indicates that Dpp signaling provides a connection between cell-fate specification, cell growth and the control of morphogenesis. It, thereby, might help to facilitate the coordination of these processes during wing disc development (Widmann, 2009).
Given the evolutionary conserved functions of Rho and myosin II, it is anticipated that the mechanisms regulating columnar cell shape, which are describe in this study for the wing disc, will also operate in a wide range of other epithelia. Moreover, the role of TGFβ/Dpp signaling in patterned morphogenesis appears to be conserved in vertebrates, raising the possibility that Rho and myosin II are common mediators of TGFβ/Dpp signaling (Widmann, 2009).
Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained as AJ are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through DE-cadherin endocytosis by limiting the Cdc42/Par6/aPKC complex activity. This study demonstrates that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin containing Rab11-positive recycling endosomes in Drosophila post-mitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 may regulate the formation of recycling endosomes thorough localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin containing vesicular trafficking during AJ remodeling in live epithelia (Yashiro, 2014).
Classically, it has been assumed that adhesive differences are a primary means of sorting cells to their respective territories. Yet it is becoming clear that no single, simple mechanism is at play. In the few instances studied, an emergent theme along developmental boundaries is the generation of asymmetry in cell mechanical properties. The repertoire of ways in which cells might establish and then put mechanical asymmetry to work is not fully appreciated since only a few boundaries have been molecularly studied. This study characterize one such boundary in the develop leg epithelium of Drosophila. The region of the pretarsus / tarsus is a known gene expression boundary that also exhibits a lineage restriction. This study show that the interface comprising this boundary is strikingly aligned compared to other cell interfaces across the disk. The boundary also exhibits an asymmetry for both Myosin II accumulation as well as one of its activators, Rho Kinase. Furthermore, the enrichment correlates with increased mechanical tension across that interface, and that tension is Rho Kinase-dependent. Lastly, interfering with actomyosin contractility, either by depletion of myosin heavy chain or expression of a phosphomimetic variant of regulatory light chain causes defects in alignment of the interfaces. These data suggest strongly that mechanical asymmetries are key in establishing and maintaining this developmental boundary (Ly, 2017).
A key component comprising a developmental boundary is the special
mechanical properties imposed to its interfaces. Insights into these
properties have been gained from the few tissues that have been studied,
such as rhombomere boundaries in the vertebrate, but especially the study of
several boundaries in Drosophila. The latter studies in Drosophila
have afforded much higher resolution so far than study of rhombomeres.
Still, relatively few boundaries overall have been studied, and that
makes it difficult to draw any generalizations for how the underlying
mechanics makes the boundary. This paper reports initial studies on the
late-arising developmental boundary necessary for leg segmentation. The
pretarsal / tarsal boundary was more aligned than the canonical AP
compartment boundary. The rail exhibits an asymmetry in actomyosin
accumulation as well as one of its activators, Rho Kinase. This is shown to result in increased tension along the boundary, which is important in aligning its interfaces (Ly, 2017).
Polarized actomyosin enrichment leads to increased
cell bond tension along the pretarsal / tarsal interfaces. The fold
increase of tension compared with the orthogonal rung interfaces is in
line with differences observed in several other tissues, such as the
Antero-posterior and dorso-ventral compartment boundaries. Here, along the pretarsal /
tarsal interface, actomyosin contractility generates a very smooth,
arcing boundary. The alignment is significant, as it is even more
aligned than the well-studied AP compartment boundary. In itself, this
fact strongly suggests that study of the pretarsal / tarsal boundary
will complement the information obtained though study of other
developmental boundaries (Ly, 2017).
The data revealing enrichment of the Myosin II regulatory light chain as
well as Rho Kinase along rail interfaces strongly implicates
contractility in alignment, and the degree of mis-alignment observed in
zip mutants supports this contention. Furthermore,
treatment with a Rho Kinase inhibitor reduced actomyosin enrichment and
released tension along the rail, rapidly generating a less aligned state. In addition, since removal of the Rho Kinase inhibitor led to
the rapid re-establishment of alignment, the data collectively
argue that asymmetric contractility can drive this alignment event.
Still, Rho Kinase inhibitors can affect other protein kinases, such as Atypical Protein Kinase (aPKC). Thus, even
though a quite selective Rho Kinase inhibitor was used, it is still
possible that another kinase also contributes to alignment, perhaps
targeting a factor in addition to the myosin regulatory light chain (Ly, 2017).
The expression of a phosphomimetic form of the Myosin II regulatory
light chain generated defects along the rail. The precise mechanism
involved awaits live-imaging the formation of the aligned interface.
Without that capability in this epithelium, it cannot be determined whether
the phosphomimetic form of the Myosin II generated defects due to
decreased cycling between on and off states along interfaces normally
enriched for myosin, or to increased activity along the normally
depleted (rung) interfaces. Nevertheless, regulated
contractility is certainly important to alignment (Ly, 2017).
Actomyosin enrichment and the resultant increased tension is a theme
observed repeatedly along cell interfaces. Interestingly, the outcome of
that increase in tension can be quite different in different
circumstances. In some cases, tension stabilizes cell interfaces, as has
been observed along the parasegment boundary of the embryonic
epithelium, as well as the AP and DV compartment boundaries in
developing imaginal disk epithelia. While actomyosin enrichment leads to
stabilization in those cases, in other instances, enrichment and the
associated increased tension drives interface shrinkage. Those shrinkage
outcomes are crucial to the directed junctional remodeling events
necessary for convergence extension. Similar shrinkage events are also observed in tissues at
steady-state. For example, across the epithelial field in the developing
wing, junctional shrinkage events maintain the proper geometry of cell
packing. Just how actomoysin enrichment and
junctional tension can be directed toward two quite diametrically
opposed outcomes, shrinkage or stabilization, is unclear at present.
This issue will only be resolved by examining more boundaries of each
class, and by identifying more components that act along those
interfaces (Ly, 2017).
In fact the pretarsal / tarsal boundary described in this study has several
features in common with another interface described previously. In the late embryonic epidermis, well-after convergence and extension, a select set of cells within each parasegment organizes into
aligned columns. Those aligning cell columns exhibit
enrichments similar to those described here along the smooth, arcing
pretarsal / tarsal rail. In addition, in both cases the cells that
constitute the boundary assume elongate, rectilinear shapes. A comparison
of the mechanics underlying these two alignment events could potentially
reveal how actomoysin enrichment and junctional tension can be directed
toward stabilization (Ly, 2017).
Besides exhibiting alignment, some boundary interfaces, such as the AP
and DV compartment boundaries, are resilient to challenges from
neighboring cells, whether from cell division or intercalation. The
mechanical basis for this is becoming more clear. The pretarsal / tarsal
boundary develops a late-acting lineage-restriction, so it is interesting to consider the degree to which increased tension contributes to the restriction. Interestingly, in depleting or
manipulating Myosin II activity the pretarsal/tarsal boundary became
very irregular. Yet, no evidence was found for 'invasions'
from one territory to the other, at least not in these fixed
preparations. This suggests that tension is not sufficient for this
restriction in the leg. Perhaps like the DV compartment in the wing a
combination of mechanical tension, as seen here, plus oriented divisions
and cell elongation contribute to boundary integrity. Alternatively, the affinity properties of the pretarsal versus
tarsal cells may well contribute to the lineage restriction (Ly, 2017).
Finally, it is noted that the interfaces flanking the rail are also aligned
to a significant degree. This differs from the situation observed along
the AP compartment boundary where the adjacent interfaces were used as
examples of relatively unaligned interfaces. That
raises the interesting question of whether the interfaces flanking the
rail are actively aligned. For instance, machinery similar to that
deployed along the rail might align the -1 and + interfaces.
Alternatively, the flanking interfaces might be aligned only passively,
as a consequence of the geometry enforced by the rail interface on the
other cell interfaces. If there is an active process aligning the
flanking interfaces, MyoII would appear to be minimally involved. No significant enrichment of MyoII was observed along the -1 interface
compared to the adjacent rung, and although these interfaces retained some tension, the level
was much reduced along the -1 and + interfaces compared to the rails (Ly, 2017).
It is not yet known how the polarized enrichments are first established
along the pretarsal / tarsal boundary. There is a fairly well-understood
gene regulatory hierarchy that establishes the pretarsal and tarsal
territories during the mid third instar period of development. The initially rough borders
between the two territories are subsequently refined by further
cross-regulatory interactions. Thus, it is no surprise that interfering
with the transcriptional regulator, C15, can cause defects along the
boundary. In addition, among the factors that are genetically regulated by
this transcriptional circuitry are Fasciclin II and the leucine rich
proteins, Capricious and Tartan (Caps; Trn).
However, it is not known how direct that regulation might be. Moreover,
neither removing Fasciclin II, nor both Caps and Trn, generated
phenotypes that seemed clarifying. This suggests
that key factors remain to be defined. A similar limitation extends to
the parasegmental, AP and DV boundaries. While the Wingless, Hedgehog
and Notch pathways, respectively, have been implicated at those
boundaries, the analyses still leave open the possibility that control by
each of those pathways is indirect. Unraveling the direct links from
cell signaling to the mechanics of tissue boundaries remains an
important goal in studying morphogenesis (Ly, 2017).
Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin-mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. This study used optogenetics to quantitatively control myosin-II levels at the basal surface of invaginating cells during Drosophila gastrulation. While basal myosin-II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre-invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube-like structure. Collectively, these data demonstrate the requirement of myosin-II polarization and basal relaxation throughout the entire invagination process (Krueger, 2018).
Stable intercellular bridges called ring canals form following incomplete cytokinesis, and interconnect mitotically or meiotically related germ cells during
spermatogenesis. Ring canals in Drosophila melanogaster males are surprisingly different from those previously described in
females. Mature ring canal walls in males lack actin and appear to derive directly from structural proteins associated with the
contractile ring. Ring canal assembly in males, as in females, initiates during cytokinesis with the appearance of a ring of
phosphotyrosine epitopes at the site of the contractile ring. Following constriction, actin and myosin II disappear. However, at least
four proteins present at the contractile ring remain: the three septins (Pnut, Sep1 and Sep2) and anillin. In sharp contrast, in ovarian
ring canals, septins have not been detected, anillin is lost from mature ring canals and filamentous actin is a major component. In both
males and females, a highly branched vesicular structure, termed the fusome, interconnects developing germ cells via the ring canals
and is thought to coordinate mitotic germ cell divisions. In males, unlike females, the fusome persists and enlarges
following cessation of the mitotic divisions, developing additional branches during meiosis. During differentiation, the fusome and its
associated ring canals localize to the distal tip of the elongating spermatids (Hime, 1996).
Drosophila Rho-associated kinase (Rok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Rok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain (Winter, 2001), and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton (Winter, 2001).
Rok signaling regulates the phosphorylation of nonmuscle myosin regulatory light chain (MRLC), and hence the activity of myosin II. Does the phosphorylation state of MRLC modify the multiple hair phenotype of dishevelled mutants? Use was made of a series of mutant spaghetti squash (sqh) transgenes (sqh codes for the Drosophila MRLC) with point mutations in the primary (Ser-21) and secondary (Thr-20) phosphorylation sites, changing them either to glutamic acid (phosphomimetic), or to nonphosphorylatable alanine.
Can the phosphorylation state of MRLC also modulate Fz/Dsh signaling? An examination was made to determine whether the phosphomimetic and nonphosphorylatable forms of MRLC could directly modify the dsh1 multiple hair phenotype. Introducing one copy of sqhE20E21 reduces the number of multiple hair cells in dsh1 mutants by 5-fold. sqhE21, or sqhA20E21, also suppresses the dsh1 phenotype by more than 2-fold. In contrast, introduction of sqhA21 into the dsh1 background enhances the multiple hair phenotype. The involvement of MRLC in the Fz/Dsh pathway was also examined using the Fz-overexpression assay. Reducing the wild-type sqh gene dosage from two to one, by introducing a single copy of the sqhAX3 null allele, results in a 2-fold suppression of the multiple hair phenotype caused by Fz overexpression. These results support the notion that MRLC functions in the PCP pathway to restrict F-actin bundle assembly to a single site (Winter, 2001).
MRLC phosphorylation in response to Rok activation would be predicted to modify the conformation and elevate the catalytic activity of its associated heavy chain, Zipper (Zip). Does Zip also participate in regulating actin distribution/wing hair number in response to Fz/Dsh? Loss of one copy of the zip gene enhances the dsh1 phenotype by 4.5-fold, consistent with the genetic interaction data between fz/dsh and sqh. These results suggest that myosin II functions positively downstream of Fz/Dsh in regulating actin prehair development (Winter, 2001).
The localization of Zip protein in wing cells further supports its role downstream of Fz/Dsh. At the apical surface of the pupal wing cell, Zip is asymmetrically localized to the distal portion of the cell, where prehair growth initiates. This distal localization appears to coincide, temporally, with prehair initiation. To test whether Zip localization could be modified by Fz/Dsh signaling, Zip distribution at the apical surface was examined in dsh1 mutants. Instead of being concentrated in the distal region of the cell, Zip is concentrated in the center of the cell, where prehairs form in dsh1 mutants (Winter, 2001).
Does reduction in myosin II/Zip activity also result in the multihair phenotype? Use was made of the hypomorphic zip02957, since zip and sqh null mutations appear to be cell lethal in the wing. As is the case with rok, some homozygous zip02957 wing cells possess multiple F-actin prehairs (Winter, 2001).
Tests were performed to see if the gene crinkled (ck) is involved in the Fz/Dsh signaling pathway regulating wing hair number because (1) ck mutant cells in the wing lead to multiple hair and split hair phenotypes, and (2) ck encodes the Drosophila myosin VIIA protein. Mutations in mouse myosinVIIA lead to stereocilia disorganization and the formation of multiple bundles of stereocilia (Winter, 2001 and references therein).
Reduction of ck activity potently suppresses the dsh1 multiple hair phenotype. This result contrasts with the result that zip1 enhances the dsh1 multiple hair phenotype, and suggests that the two myosin heavy chains have opposing effects in regulating prehair assembly (Winter, 2001).
Both myosin heavy chain genes were tested for their ability to interact with the hs-fz induced multiple hair phenotype, and again it was found that they have opposing effects. Surprisingly, loss of one copy of zip slightly but significantly enhances the late hs-fz multiple hair phenotype, while loss of one copy of ck markedly suppresses this phenotype. These results are the reverse of what one would expect based on their interactions with dsh1, and suggests the possibility that there is a signal from Fz to Ck that is independent of Dsh, or that the multiple hair phenotypes resulting from hypo- or hyper-activity of the Fz/Dsh pathway arise via distinct biochemical mechanisms (Winter, 2001).
To further assess the nature of the relationship between the two myosins, the effect of raising or lowering the activity of MRLC on the ck phenotype was tested. The multiple hair phenotype in animals homozygous for a weak ck mutation is enhanced by one copy of the sqhE20E21 transgene (and hence, a probable increase in myosin II activity), but not by a sqhA20A21 transgene. Taken together, these experiments suggest that a balance between the activities of myosin II and myosin VIIA is important in regulating wing hair number (Winter, 2001).
Unlike other characterized PCP mutants that affect both orientation and number of wing hairs, the primary defect in Drok2 clones appears to be the presence of multiple hairs per cell, with little or no wing hair orientation defect. This suggested that Rok and what lies downstream are involved in transmitting a subset of the Fz/Dsh signal. Supporting this idea, it was found that tubP-Drok and sqhE20E21 suppress the multiple hair phenotype of dsh1, but not the hair misorientation phenotype. Additional data supporting this conclusion comes from observing the site of prehair initiation. Prehairs emerge aberrantly from the center of dsh1 mutant cells, rather than from the distal vertex as seen in wild type cells. Such mispositioning of prehair initiation correlates with the failure to acquire the proper distal orientation. While tubP-Drok expression suppresses multiple prehair formation, it does not affect the site of F-actin initiation in dsh1. Finally, the hair orientation defect resulting from Fz overexpression (via hs-fz) at 24 hours is suppressed by reducing dsh gene dosage but not that of RhoA, rok, sqh or ck. Taken together, these observations suggest that separate mechanisms allow Fz/Dsh to independently regulate the number and the orientation of prehairs, and that only the former involves Rok signaling (Winter, 2001).
The data presented in this study suggest that the Rok/myosin II pathway is involved in regulating the number -- but not orientation -- of the wing hair. What then are the components that regulate wing hair orientation? One possibility is that a bifurcation of the pathway occurs at the level of RhoA, with a separate effector pathway regulating wing hair orientation. In the eye, the JNK pathway has been implicated in functioning downstream of RhoA in regulating ommatidial polarity. However, the function of the JNK pathway in the wing has not been described, and a signaling pathway that regulates transcription is unlikely to encode the requisite spatial information necessary for selection of the site of prehair initiation. Therefore, it is likely that a separate signal from or upstream of RhoA may control the selection of the F-actin assembly site, and therefore the orientation of the wing hair (Winter, 2001 and references therein).
By what mechanism do myosins restrict F-actin bundle formation? In light of the finding that myosin II is concentrated at the site of prehair formation, it seems plausible that myosin II is actively involved in either the recruitment of F-actin to the prehair site, or that it directly participates in the assembly of actin bundles, or both. Studies of mammalian myosin II provide a precedent for a role in the formation of F-actin bundles. Phosphorylation of MRLC promotes a conformational change in myosin II from a folded to an extended state that readily forms multivalent bipolar filaments capable of binding multiple actin filaments. This is thought to result in F-actin bundling and stress fiber formation (Winter, 2001 and references therein).
It appears that in the developing wing, the level of MRLC phosphorylation/myosin II activity must be within an optimal range to establish the formation of a single hair. It is possible that the efficiency of F-actin bundle formation is regulated by MRLC phosphorylation in a manner similar to the control of stress fiber formation. If one further assumes that there are certain bundling substrates present only at limiting concentrations (e.g., F-actin itself), then one would predict that the assembly of one F-actin bundle would reduce the probability of forming a second bundle. When MRLC phosphorylation falls below some threshold level (e.g., in rok mutant cells), the efficiency of primary bundle formation is reduced, and thus the concentration of the limiting substrate remains at sufficient levels to support the assembly of secondary bundles/prehairs. Conversely, if MRLC is hyperphosphorylated (e.g., in Fz-overexpressing cells), the bundling efficiency may increase such that the threshold concentration for bundle formation would be reduced, thereby increasing the probability of assembling multiple bundles/prehairs. Future studies will be required to determine the detailed mechanisms involved (Winter, 2001).
In addition to nonmuscle myosin II, which resembles the myosin II from skeletal muscle, there exists a large class of unconventional myosins that have different properties and potential functions in nonmuscle cells. For instance, several different classes of unconventional myosins are expressed in inner ear epithelium with different subcellular localization. Mutations in three of the unconventional myosins, myosin VI, VIIA, and XV, cause hearing/balancing defects in mice, two of which when mutated in humans result in deafness. Of particular interest in the context of this study is myosin VIIA, mutations of which are responsible for mouse shaker-1 and human Usher's syndrome 1B. Loss-of-function ck (Drosophila Myosin VIIA) mutants exhibit a multiple hair and split wing hair phenotype. ck exhibits strong genetic interactions with components of the signal transduction pathway defined in this study, and has the opposite effects as that of myosin II. The seemingly antagonistic relationship between myosin II and myosinVIIA may suggest a mechanism in which the balance of the activities or stoichiometry of these two myosins is critical for the common process they regulate. For example, myosin II and myosin VIIA may share some common, limiting component(s) required for their activity. Thus, by reducing the myosin VIIA dose, myosin II has a larger share of the common component(s) and thus its activity is upregulated (Winter, 2001 and references therein).
Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2013).
In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2013).
This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2013).
Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2013).
This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2013).
Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6-10 cells, traversing a network of large germline-derived nurse cells within the ovary. This study shows that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Non-muscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. It is proposed that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues (Aranjuez, 2016).
This study reports the role of Spectrins during epithelia morphogenesis using the Drosophila follicular epithelium (FE). α-Spectrin and β-Spectrin are shown to be are essential to maintain a mono-layered FE, but, contrary to previous work, Spectrins are not required to control proliferation. Furthermore, spectrin mutant cells show differentiation and polarity defects only in the ectopic layers of stratified epithelia, similar to integrin mutants. These results identify α-Spectrin and integrins as novel regulators of apical constriction-independent cell elongation, as α-spectrin and integrin cells fail to columnarize. Finally, increasing and reducing the activity of the Rho1-myosin-II pathway enhances and decreases multi-layering of α-spectrin cells, respectively. Similarly, higher myosin-II activity enhances the integrin multi-layering phenotype. This work identifies a primary role for α-Spectrin in controlling cell shape, perhaps by modulating actomyosin. All together, it is suggested that a functional Spectrin-Integrin complex is essential to balance adequate forces, in order to maintain a mono-layered epithelium (Ng, 2016).
This study found that in the germline α-Spec is not a major regulator of the Hippo pathway. Mutations in hippo, β-Spec or α-Spec result in a stratified FE, but contrary to previous interpretations, and unlike Hippo, spectrins are not required for the FCs to exit mitosis. The suggestion that Spec mutant FCs over-proliferate is thought to be an over-interpretation from the multilayering phenotype, as α-Spec cells were not checked for mitotic markers in a previous report. Again unlike hippo, α-Spec mutant PFCs only show defects in differentiation when they are located in the ectopic layers of the stratified FE, and oocyte polarity is largely unaffected in mutant egg chambers. It was recently shown that a β-Spec allele with a premature stop codon at amino-acid 1046 partially phenocopies hippo, with strong defects in FE integrity, actin organization and oocyte polarity. The null β-SpecG113 mutant allele behaves similarly to α-Spec mutants, showing Hnt defects mainly in ectopic layers, but Fas3 mislocalization in monolayers. More importantly, β-SpecG113 FCCs exit mitosis properly. The differences observed between the two β-Spec alleles are likely to be due to the fact that β-SpecG113 is a null allele (Ng, 2016).
In conclusion, α-Spec and β-Spec FCCs do not phenocopy hippo mutants when the cells are part of a monolayer, and they seem to adopt a partial hippo-like differentiation phenotype only when positioned at ectopic layers, even though α-Spec and β-Spec cells never divide after S6. Thus, the main function of the spectrin cytoskeleton in FCs is not proliferation control or regulation of the Hippo pathway, although an interaction between spectrins and Hippo might occur once the FCs are within an aberrantly organized FE. The function of spectrins in FCs is in contrast with other tissues, where α- and β-Spec appear to regulate growth through Hippo (Ng, 2016).
Similar to Hippo, α-Spec and β-Spec are required for the FE to maintain a monolayer. There is an increase in the multilayering phenotype in egg chambers with large clones from S3/6 to S7/8 and 100%, respectively. Also, the presence of control cells in α-Spec mosaic epithelia aids the mutant cells to maintain a monolayer from S6, as there is a higher percentage of S7-9 egg chambers with multilayers when the FE contains large α-Spec clones than when the mutant clone is only at the posterior end. The control of FE architecture appears to be mediated by the lateral spectrin network. Loss of α-Spec seems to disrupt both lateral (α/β) and apical (α/βH) spectrin-based membrane skeleton (SBMS) in the FE, as β and βH subunits are no longer localized laterally and apically in α-Spec cells, but no multilayering was reported for βH-Spec egg chambers, in which a loss of apical α-Spec was observed, suggesting that the loss of the lateral α/β is responsible for the FE stratification. Also, βH-Spec is mislocalized in sosie mutants, but the FE architecture is maintained (Ng, 2016).
Incipient SJs are first detected between the FCs with the completion of proliferation at S6. This study shows that the localization of several SJ components is affected in α-Spec FCCs, suggesting that spectrins are required for proper SJ formation. This is further supported by other observations. (1) Fas3 localization is affected in β-Spec FCCs. (2) Neuroglian (an SJ component) is required for maintaining the stability of the FE. (3) The reduction of both α- and β-Spec leads to mislocalization of Dlg, Neuroglian and Fas2 in neuromuscular junctions. (4) It has been suggested that the SBMS and ankyrin associate with SJ components (Ng, 2016).
As the mislocalization of SJ components in Spec mutant FCCs is observed in monolayers, and thus prior to the onset of stratification, it is speculated that Spec-dependent distribution of SJ components might contribute to the Spec function in the epithelium. This idea is supported by Crumbs overexpression, which leads to defects in SJs and ZA, and multilayering of the ectoderm cells, and by dpak (Pak - FlyBase) FCs, which mislocalize Fas3 and show multilayering and columnarization defects. Furthermore, the aberrant accumulation of Fas2 at the lateral membrane of Tao FCs prevented membrane shrinking in the cuboidal-to-squamous transition. However, fas3, fas2 and cora mutant cells do not show shape defects or multilayering. Thus, if SJ components contribute to the α-Spec phenotype at all, it might be not because they are absent in α-Spec mutant cells, but because they are not properly distributed (Ng, 2016).
Transitions between squamous, cuboidal and columnar epithelial cell shapes are common during development, and contribute to the morphogenesis of tissues. This study demonstrates a cell-autonomous role for α-Spec in promoting the cuboidal-to-columnar shape transition of the FCs. It is important to point out that the FE undergoes lateral elongation without apical constriction, which might allow phenotypes to be interpreted in a simpler manner. This morphogenetic FC behavior is similar to that of vertebrate neuroepithelia, where cell elongation precedes apical constriction, and it would be interesting to study the function of Spec in the columnarization of these cells (Ng, 2016).
Although the molecular mechanism of apical constriction-independent cell elongation is unknown, a primary role for the SBMS is thought to lie in facilitating changes in cell shape, which is further supported by the cell shape defects in α-Spec gut epithelia, perhaps by contributing to the proper distribution of adhesion molecules. This function of the SBMS in membrane biology is conserved in other cells, as spectrins stabilize the plasma membrane during blastoderm cellularization, and control photoreceptor morphogenesis through the modulation of membrane domains. The spectrin cytoskeleton might also impact on FE columnarization by interacting with the actomyosin cytoskeleton. It is known that apical-basal elongation in cytoplasmic actin-binding protein drebrin E (drebrin 1) depleted human Caco2 cells is impaired, as a possible consequence of the lack of interaction between drebrin E with spectrins and actomyosin. Also, the elongation of neuroepithelial cells depends on the assembly of an actomyosin network in the apical junctional complex, regardless of whether cells are constricting or not . In Drosophila wing discs, the Rho1-Myosin II pathway at the apicolateral membrane seem to regulate the cuboidal-to-columnar shape transition, whereas in the germline, Rok and sqh mutant FCs fail to adopt a normal shape. Finally, SBMS seems to modulate cortical actomyosin contractility in the eye, and possibly in the FE. Together, these data suggest that Myosin II activity is aberrant in α-Spec mutant FCs, contributing to defects in columnarization and FE architecture (Ng, 2016).
Increasing Rho1 and Sqh activities enhances the Spec multilayering phenotype, whereas reducing Myosin II activity decreases it. In addition to this functional link between the SBMS and the Rho-Myosin pathway, this study also shows that mys cells fail to columnarize, and that an extra copy of sqh increases the mys multilayering phenotype. It has been shown that integrins regulate the Rho-Myosin pathway to induce actomyosin-generated forces. Thus, as is the case for spectrins, integrins might also control cell shape and epithelia morphogenesis by modulating the actomyosin activity (Ng, 2016).
How the SBMS and integrins might modulate actomyosin is unknown, and one possible mechanism is by regulating Myosin II activity directly. However, an alternative mechanism is proposed. Spectrins can bind F-actin, and integrins and spectrins interact with proteins involved in the association of F-actin with the membrane. Furthermore, α-Spec and integrins regulate the actin cytoskeleton through Rac. Previous studies have shown that both β-Spec and mys mutant FCs display similar defects in the basal level of F-actin, which are recapitulated in α-Spec mutant cells. Thus, any defects in actin organization in mys and Spec mutant FCs could in turn result in defects in the activity of Myosin II (Ng, 2016).
Regardless of whether integrins and spectrins regulate F-actin or myosin, or both, spectrins and integrins might act together. The SH3 domain of α-Spec interacts with Testin ortholog (Tes), a component of integrin-dependent focal adhesions, and mammalian αII-Spec stabilizes β3-integrin anchorage, suggesting α-Spec as a physical link between focal adhesions and F-actin. In the FE, this study observed that α-Spec and αPS1 colocalize in the lateral, and possibly apical, membrane. In addition, it was shown that the localization of α-Spec in mys clones, and the localization of βPS in α-Spec mutant clones, is majorly unaffected. Furthermore, expression of a constitutively active integrin that reduces multilayering of mys FCCs, failed to rescue α-Spec multilayers. Thus, it is proposed that α-Spec and integrins act independently of each other, but as part of the same functional complex regulating the actomyosin cytoskeleton and tissue architecture (Ng, 2016).
An early event following oncogenic mutations in an epithelium is the escape of the daughter cells from the monolayered epithelium, forming disorganized masses. Spindle orientation has been linked to tumor-like growth in various tissues, and this study found that there is a good correlation between spindle misorientation and 'tumor-like masses' at the FE: hippo, mys and α-Spec FCCs show misaligned spindles and severe multilayering, whereas Notch FCCs, which overproliferate, do not show multilayering or spindle orientation defects. However, perpendicular divisions alone are insufficient to promote stratification, and a mechanism, depending on lateral cell-cell adhesions, is in place to avoid multilayering as a sole consequence of spindle misorientation. It is proposed that spindle misorientation contributes to FE disorganization, but that this 'safeguard' mechanism is somehow inactive in hippo, mys and Spec mutant FCCs. What other aspect of the mutant phenotypes might then be linked to multilayering? A clue might come from the Spec mutant and mys FCCs. First, there is an increase in the α-Spec multilayers after S6, when both FCs and egg chambers undergo various morphogenetic changes. Second, the volume of the germline surrounded by large α-Spec FCCs appears smaller. And third, Myosin II activity is increased in α-Spec and mys mutant cells. In this interpretation of the results, a proper distribution of Myosin II activity in a Spec- and integrin-dependent manner allows the right amount of forces to be distributed across the membrane and the epithelium. Thus, it is possible that proper cell-cell interactions, adequate force balance and precise spindle orientation are key to maintaining a monolayered epithelium, especially upon the mechanical stress induced by morphogenesis (Ng, 2016).
Axis elongation in Drosophila occurs through polarized cell rearrangements driven by actomyosin contractility. Myosin II promotes neighbor exchange through the contraction of single cell boundaries, while the contraction of myosin II structures spanning multiple pairs of cells leads to rosette formation. This study shows that multicellular actomyosin cables form at a higher frequency than expected by chance, indicating that cable assembly is an active process. Multicellular cables are sites of increased mechanical tension as measured by laser ablation. Fluorescence recovery after photobleaching experiments show that myosin II is stabilized at the cortex in regions of increased tension. Myosin II is recruited in response to an ectopic force and relieving tension leads to a rapid loss of myosin, indicating that tension is necessary and sufficient for cortical myosin localization. These results demonstrate that myosin II dynamics are regulated by tension in a positive feedback loop that leads to multicellular actomyosin cable formation and efficient tissue elongation (Fernandez-Gonzalez, 2009).
Myosin II directs polarized cell rearrangements in the Drosophila embryo through the contraction of specific boundaries between cells. Time-lapse imaging shows that adjacent myosin-positive edges often contract simultaneously. Contraction of these structures leads to multicellular rosettes that form predominantly during the period of rapid intercalation in stage 8. This may explain why rosettes were overlooked in previous studies at stage 7, when the germband has only reached one-third of its final length (Fernandez-Gonzalez, 2009).
Multicellular contractile structures presumably represent intracellular myosin filaments that are functionally associated across cell boundaries through connections mediated by adherens junctions. These structures are referred to here as actomyosin cables. Actomyosin cables appear to represent sites of increased tension, as cell boundaries in cables were aligned perpendicular to the anterior-posterior (AP) axis. This study quantified the extent of alignment in living embryos using a computer algorithm to identify cell boundaries in confocal images. Alignment was measured as the fraction of AP edges (cell-cell interfaces oriented at 75°-105° relative to the AP axis) that were directly connected to at least one other AP edge. Wild-type embryos displayed a sharp increase in alignment at the onset of intercalation that plateaued at ~40% of all AP edges. Embryos zygotically mutant for eve or maternally mutant for bcd, nos, and torso-like (tsl) are defective for elongation and showed significantly reduced alignment, while alignment occurred normally in twist snail mutants that fail to generate mesoderm but are able to elongate. These results indicate that the timing of alignment correlates with intercalary behavior and does not require external forces from the ventral furrow (Fernandez-Gonzalez, 2009).
The spatial distribution of myosin II was analyzed in time-lapse movies of living embryos that express fluorescently-tagged myosin regulatory light chain from its endogenous promoter. Myo:mCherry localized predominantly to interfaces between anterior and posterior cells. One-third of all AP edges were myosin-positive, and nearly two-thirds of the myosin-positive edges were associated with multicellular cables. Analysis of single-edge behaviors over time revealed that alignment often precedes asymmetric myosin redistribution (Fernandez-Gonzalez, 2009).
This study provides evidence that myosin II is organized into multicellular contractile structures that form nonrandomly in intercalating cells and sustain increased mechanical tension. Mechanical tension is sufficient to promote cortical myosin localization, and conversely, relieving tension leads to a rapid decrease in cortical myosin. These studies demonstrate that myosin II not only generates tension, but myosin II dynamics can also be regulated by tension, generating a positive feedback loop that allows cells to dynamically respond to changes in their mechanical environment (Fernandez-Gonzalez, 2009).
External forces have been shown to recruit myosin to the cortex during cell division and apical constriction. In intercalating cells, myosin is distributed in a planar polarized fashion in response to striped patterns of gene expression that concentrate contractile proteins in specific cortical domains. It is proposed that the recruitment of myosin by tension amplifies these initial subtle asymmetries, reinforcing contractile activity and increasing the number of cells engaged in contractile behavior. This positive feedback loop could explain the formation of multicellular actomyosin cables that promote rosette formation and efficient tissue elongation (Fernandez-Gonzalez, 2009).
How is mechanical tension translated into myosin II stabilization at the cortex? Evidence from the literature suggests three models of mechanotransduction. (1) Forces have been shown to influence gene expression in normal cells as well as during tumorigenesis, suggesting that tension could lead to changes in the expression of myosin II regulatory proteins. Tension has been shown to promote β-catenin-dependent expression of Twist, a transcription factor that regulates apical myosin localization. However, the rapid recruitment of myosin in response to ectopic forces in intercalating cells suggests that the effect of tension is likely to be independent of transcription. (2) A second possibility is that increased tension at the plasma membrane could alter the trafficking of secreted signaling proteins. Such a mechanism has been proposed to occur during Drosophila mesoderm invagination, in which mechanical tension potentiates the activity of the secreted Twist target gene Fog. However, Twist and Fog are not expressed or required in intercalating cells, suggesting that myosin localization during intercalation occurs through a different mechanism. (3) Mechanical tension can alter signaling pathways directly through force-dependent changes in protein interactions. Myosin itself could act as the mechanosensor in this context, as tension favors the ADP-bound form of myosin II in vitro, stabilizing its association with actin. Mechanical tension alters the activity of several myosin and kinesin motors and may represent a general mechanism regulating motor protein function (Fernandez-Gonzalez, 2009).
Multicellular actomyosin cables are characteristic of many developmental processes including epithelial closure, tracheal tube invagination, and neural plate bending and elongation. The role of mechanical tension in regulating myosin dynamics could serve to promote contractile activity and maintain the integrity of contractile cables in the presence of interruptions caused by cell shape changes, cell division, or cell death. In the Drosophila embryo, spatially regulated mechanical forces may also act as a long-range signal to allow cells to maintain planar polarity despite the transient nature of local cell interactions during morphogenesis (Fernandez-Gonzalez, 2009).
Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. This study quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. Mechanical tension was found to be approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632, an inhibitor of Rho-kinase whose main effector is Myosin II. Simulations using a vertex model demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. These results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries (Landsberg, 2009).
A long-standing hypothesis to explain the maintenance of compartment boundaries is based on differential cell adhesion (or cell affinity). Cell adhesion molecules required for the maintenance of compartment boundaries, however, have not been identified. More recently, it has been proposed that actin-myosin-based tension is important for keeping the dorsoventral compartment boundary of the developing Drosophila wing smooth and straight. However, whether a similar mechanism operates at the anteroposterior compartment boundary (A/P boundary) is unclear. Moreover, a physical measurement of differential mechanical tension at compartment boundaries has not been reported. Furthermore, whether and how differential mechanical tension governs cell sorting at compartment boundaries is not well understood (Landsberg, 2009).
To test whether actin-myosin-based tension is increased at the A/P boundary, the levels of Filamentous (F)-actin and nonmuscle Myosin II (Myosin II) were quantified. The A/P boundary in the wing disc epithelium was particularly well defined by the cell bonds located at the level of adherens junctions, indicating that mechanisms maintaining the boundary operate at this cellular level. F-actin and the regulatory light chain of Myosin II (encoded by spaghetti squash, sqh) were increased at these cell bonds along the A/P boundary. Cell bonds displaying elevated levels of Myosin II correlate with decreased levels of Par3 (Bazooka in Drosophila), a protein organizing cortical domains, at the dorsoventral compartment boundary and during germ-band extension in Drosophila embryos. Likewise, Bazooka was decreased at cell bonds along the A/P boundary, indicating a common mechanism of complementary protein distribution of Myosin II and Bazooka. The level of E-cadherin, a component of adherens junctions, was not altered along the A/P boundary (Landsberg, 2009).
To identify signatures of increased tension in the vicinity of the A/P boundary, the morphology of cells were quantitatively analyzed at the level of adherens junctions. Line tension and mechanical properties of cells have been proposed to contribute to cell shape and to influence angles between cell bonds. Line tension associated with adherens junctions, here termed cell bond tension, can be defined as the work, per unit length, performed as a cell bond changes its length. Cell bond tension results from actin-myosin bundles and other structural components at junctional contacts that generate tensile stresses. Wing discs from late-third-instar larvae were stained for E-cadherin and engrailed-lacZ, a marker for the posterior compartment. Cell bonds were identified, and morphological parameters were analyzed. Adjacent anterior and posterior cells (A1 and P1, respectively) displayed a significantly enlarged apical cross-section area compared to cells farther away from the compartment boundary, indicating that apposition of anterior and posterior cells alters specifically the properties of A1 and P1 cells. Angles between adjacent cell bonds along the A/P boundary were larger compared to angles between bonds of the remaining cells and were significantly smaller in mutants for Myosin II heavy chain (encoded by zipper; zip2/zipEbr). Thus, the unique morphology of A1 and P1 cells depends on Myosin II. These data are consistent with an increased Myosin II-based tension of cell bonds located along the A/P boundary (Landsberg, 2009).
Cells on opposite sides of the A/P boundary differ in gene expression. The homeodomain-containing proteins Engrailed and Invected as well as the Hedgehog ligand are only expressed on the posterior side. The Hedgehog signal is transduced exclusively on the anterior side. Hedgehog signal transduction and the presence of Engrailed and Invected are required to maintain this compartment boundary. Whether the altered cell morphology at the A/P boundary could be reproduced by ectopically juxtaposing Hedgehog signaling and non-Hedgehog signaling cells was tested. Clones of cells that expressed Hedgehog from a transgene and that were also mutant for the gene smoothened (encoding an essential transducer of the Hedgehog pathway) were generated. In the P compartment, which is refractory to Hedgehog signal transduction, clones displayed a normal morphology. In the A compartment, a response to Hedgehog that is secreted by the clones is elicited in the surrounding wild-type cells. These clones had a rounder appearance, and at the clone border, but not away from it, apical cross-section area and bond angles were increased. Similarly, juxtaposing cells expressing engrailed and invected with cells that are mutant for these genes resulted in increased apical cross-section area and increased bond angles at the clone border. It is concluded that the morphology that is characteristic of cells at the A/P boundary can be imposed on cells within a compartment by juxtapositioning cells with different activities of Hedgehog signal transduction or Engrailed and Invected (Landsberg, 2009).
Ablating cell bonds generates cell vertex displacements, providing direct evidence for tension on cell bonds. Individual cell bonds were ablated by using a UV laser beam focused in the plane of the adherens junctions. Single-cell bonds were cut, and the displacement of vertices of neighboring cells, visualized by E-cadherin-GFP, was recorded. The P compartment was visualized by expression of GFP-gpi under control of the engrailed gene via the GAL4/UAS system. The increase in distance between the two vertices of the ablated cell bond and the initial velocity of this vertex separation were analyzed. The ratio of initial velocities in response to cell bond ablation is a measure of the tension ratio on these cell bonds. Initial velocity and extent of vertex separation were indistinguishable between anterior (A/A) and posterior (P/P) cell bonds located away from the A/P boundary. This was also the case when specifically cell bonds between the first and second row of anterior cells were ablated. By contrast, ablation of bonds between adjacent anterior and posterior cells (A/P cell bonds) gave rise to a larger vertex separation. This result was not due to the fact that A/P cell bonds have a preferred orientation. Moreover, the initial velocity of ablated A/P bonds was 2.37-fold higher compared to the mean of initial velocities of A/A and P/P bonds. This value provides an estimate of the ratio λ of cell bond tension along the A/P boundary relative to the average tension of cell bonds. In the presence of the Rho-kinase inhibitor Y-27632, the ratio of initial velocity of vertex separation of A/P cell bonds relative to A/A cell bonds was reduced to 1.46. Given that Myosin II is the main effector of Rho-kinase, these results strongly suggest that Myosin II-based tension acting on cell bonds is locally increased along the A/P boundary (Landsberg, 2009).
To quantify λ by an independent method, the displacement field was calculated after laser ablation. Using a vertex model, two populations of adjacent cells were introduced and cell bond ablations were simulated, varying λ between 1 and 4. When λ = 2.5, the vertex displacement, and in particular the anisotropy of displacements, in the simulations closely matched the vertex displacements in the experiment. In the vertex model, λ = 2.5 also resulted in increased bond angles at the interface of the two cell groups, similar to the A/P boundary in the wing disc. Thus, on the basis of two different methods, the data demonstrate that cell bond tension is increased approximately 2.5-fold along the A/P boundary compared to the remaining tissue (Landsberg, 2009).
To test whether a 2.5-fold increase in cell bond tension is sufficient to maintain a compartment boundary, the vertex model was used to simulate the growth of two adjacent cell populations for λ = 1, 2.5, and 4. For λ = 1, the interface between two growing cell populations became increasingly irregular. By contrast, for λ = 2.5 and 4, a well-defined interface was maintained. Moreover, corresponding changes in cell bond tension at borders of simulated clones resulted in the morphology and sorting behavior of cell patches that resembled those of experimental cell clones compromised for Hedgehog signal transduction or Engrailed and Invected activity. The roughness of the interface in the simulations decreased with increasing λ, showing that cell bond tension is sufficient to maintain straight interfaces between growing cell populations. For λ = 2.5, the roughness of the interface was still larger than the roughness of the A/P boundary in wing discs. This suggests that additional mechanisms might contribute to further reduce the roughness of the A/P boundary. Also, because of the uncertainty of the mechanical properties of A1 and P1 cells, which differ from those of the remaining cells, the value of λ, inferred from laser ablation of cell bonds, might be underestimated. Remarkably, the roughness of the A/P boundary could be altered in mutant conditions. In zip2/zipEbr mutant wing discs, the roughness of the compartment boundary was significantly larger than in controls, demonstrating a role for Myosin II in maintaining a sharp and straight A/P boundary (Landsberg, 2009).
In summary, by applying physical approaches and quantitative imaging, this work for the first time demonstrates and quantifies an increase in tension confined to the cell bonds along the A/P boundary. Moreover, simulations show that this increase in tension suffices to maintain a stable interface between two proliferating cell populations. Genetic studies demonstrated that cells of the two compartments differ in their expression profiles and signaling activities. It has therefore been proposed that biophysical properties of cells within the P compartment differ from those within the A compartment, and that such differences could drive cell sorting. When quantifying cell morphology and vertex displacements after laser ablation, no differences were detected in the biophysical properties of cells between the two compartments. However, the two rows of abutting A and P cells show clear differences in biophysical properties from other cells. Most importantly, the cell bond tension along the A/P boundary is increased. Cell divisions in the vicinity of the A/P boundary were randomly oriented in the epithelial plane. Thus, taken together with the simulations, these results suggest a sorting mechanism by which an increased cell bond tension guides the rearrangement of cells after cell division to maintain a straight interface. Increased cell bond tension and the roughness of the A/P boundary depend on Rho kinase activity and Myosin II, indicating a role for actin-myosin-based tension in this process. Because cell bond tension also depends on cell-cell adhesion, differences in the adhesion between A1 and P1 cells as compared to the remaining cells might also contribute to sorting. The heterotypic, but not homotypic, interaction of molecules presented on the surface of A and P cells might trigger the local increase in cell bond tension. Hedgehog signal transduction and the presence of Engrailed and Invected might control the expression of these heterotypically interacting molecules. These data indicate an important role for cell bond tension directing cell sorting during animal development (Landsberg, 2009).
Mitotic cells assume a spherical shape by increasing their surface tension and osmotic pressure by extensively reorganizing their interphase actin cytoskeleton into a cortical meshwork and their microtubules into the mitotic spindle. Mitotic entry is known to interfere with tissue morphogenetic events that require cell-shape changes controlled by the interphase cytoskeleton, such as apical constriction. However, this study shows that mitosis plays an active role in the epithelial invagination of the Drosophila tracheal placode. Invagination begins with a slow phase under the control of epidermal growth factor receptor (EGFR) signalling; in this process, the central apically constricted cells, which are surrounded by intercalating cells, form a shallow pit. This slow phase is followed by a fast phase, in which the pit is rapidly depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. It was found that mitotic cell rounding, but not cell division, of the central cells in the placode is required to accelerate invagination, in conjunction with EGFR-induced myosin II contractility in the surrounding cells. It is proposed that mitotic cell rounding causes the epithelium to buckle under pressure and acts as a switch for morphogenetic transition at the appropriate time (Kondo, 2013).
The invagination of epithelial placodes converts flat sheets into the three-dimensional structures that form complex organs, and it is a key morphogenetic process in animal development. A major mechanism of invagination is apical constriction, which is driven by actomyosin contraction. However, not all constricted cells invaginate, and some cell internalization occurs without apical constriction, suggesting that additional mechanisms of inward cell movement contribute to invagination (Kondo, 2013).
To obtain three-dimensional information about cell behaviour during invagination, live imaging was performed of the Drosophila tracheal placode. Ten pairs of tracheal placodes, each of which is composed of about 40 cells, are formed in the ectoderm at mid-embryogenesis, and each placode initiates invagination simultaneously. Using an adherens junction marker, DE-cadherin-green fluorescent protein (E-cad-GFP), it was found that the adherens junctions of the central placode cells slowly created a depression by apical constriction, which became the tracheal pit. After 30 to 60 min of slow movement (slow phase), the tracheal pit was suddenly enlarged, and the tracheal cells were rapidly internalized (fast phase) and eventually formed L-shaped tube structures (Kondo, 2013).
After the fast transition, all the tracheal cells and surrounding epidermal cells entered mitosis 16, the final round of embryonic mitosis. It was noticed that the fast invagination was always associated with the mitotic entry of central cells that were frequently the first to enter mitosis 16. Intriguingly, mitotic rounding of the central constricted cells occurred simultaneously with the rapid depression of their apices, followed by chromosome condensation 10 min later. In this study, this atypical mitotic rounding associated with apical depression in an internalized cell is called 'rounding', to distinguish it from canonical surface mitosis (surface cell rounding) (Kondo, 2013).
To determine whether cell rounding is required for invagination, zygotic mutants were examined of the cell-cycle gene Cyclin A (CycA), which fail to enter mitosis 16, and double parkeda3 (dupa3), which show a prolonged S phase 16 and delayed entry into mitosis 16. Tracheal invagination was initiated normally in the CycA and dupa3 mutants, but proceeded more slowly than in controls, indicating that entry into mitosis 16 is required for proper timing of the fast phase (Kondo, 2013).
Although delayed, the accelerated invagination in the CycA or dupa3 mutants eventually occurred, allowing the formation of tube structures and suggesting that additional mechanisms are involved. After invagination, fibroblast growth factor (FGF) signalling is activated in the tracheal cells to induce branching morphogenesis through chemotaxis. To examine the contribution of FGF signalling to invagination, mutants of the FGF ligand branchless (bnl) or the FGF receptor breathless (btl) were analyzed. These mutants invaginated normally, indicating that chemoattraction to FGF is dispensable for invagination (Kondo, 2013).
Next, to assess FGF's role in the mitosis-defective condition, double mutants were analyzed for CycA and bnl or CycA and btl, and it was found that they showed slower invagination than CycA single mutants. Furthermore, the invagination in these double mutants was incomplete, in that the cells failed to form L-shaped tubular structures. Therefore, FGF signalling is critical for invagination when mitosis is blocked, serving a back-up role. Tracheal-specific CycA expression rescued the defects in invagination speed and tube structure in the CycA btl mutants. In addition, mitosis of cells outside the pit was occasionally observed that occurred before the mitosis of the central apically constricted cells and was not correlated with the fast invagination phase. Thus, mitosis of the surrounding epidermal cells is dispensable for tracheal invagination. Taken together, it is concluded that mitotic entry of central cells is a major mechanism for accelerating tracheal invagination (Kondo, 2013).
To distinguish the role of cell rounding from that of cell division in the fast phase, the microtubule inhibitor colchicine was used to arrest the cell cycle after cell rounding. Colchicine treatment after mitosis 15 induced M-phase arrest at mitosis 16, but the fast invagination movement accompanied by cell rounding was not affected. This result indicates that cell rounding, but not cell division, is responsible for the acceleration phase of the tracheal invagination (Kondo, 2013).
Mitosis of cells in the columnar epithelium normally occurs at the apical surface after surface rounding. It was next asked how the apical surface of the central cells becomes depressed during internalized cell rounding. One possible model explains internalized cell rounding as cell-autonomously controlled by the association of the cells with the basement membrane or underlying mesodermal cells. However, genetic removal of basement-membrane adhesion by the maternal and zygotic mutation of βPS-integrin (also known as mys) did not compromise the speed of invagination, and snail-twist double-mutant embryos, which lack mesodermal cells, still showed tracheal invagination with internalized cell rounding. These results suggest that anchoring to the basal side is probably not required (Kondo, 2013).
A second model proposes that the apical depression of the rounding cells is driven by local planar interactions among the tracheal cells. Before and during tracheal invagination, myosin II is enriched at the cell boundaries tangential to the centre of the placode and regulates cell intercalation. It was noted that the myosin II level in the central cells was lower than in the surrounding, intercalating cells. Nevertheless, the apices of the central cells were constricted during the slow phase, strongly suggesting that the surrounding cells exerted centripetal pressure on the central cells through myosin II cables. Myosin II cables fail to form in EGFR signalling mutants (such as rho, the rhomboid endopeptidase required for EGF ligand maturation, and Egfr), and apical constriction is impaired in these mutants. The first few cells undergoing mitosis 16 in the tracheal placode of rho or Egfr mutants showed surface cell rounding with expanded apices, indicating that EGFR signalling is required to couple the mitotic cell rounding with fast apical depression. It is speculated that the columnar shape of the central cells resists centripetal movements, resulting in the accumulation of inward pressure during the slow phase. The existence of such resistance was supported by the results of a physical perturbation experiment using a pulsed ultraviolet lase. The cell rounding associated with mitotic entry would release the stored inward pressure by means of cytoskeletal remodelling that causes rapid depression of apical surface together with the active shortening of cell height, leading to rapid buckling of the apical surface and the fast phase of invagination (Kondo, 2013).
Even with the loss of both EGFR and FGF signalling, the tracheal placodes form moderately invaginated structures, compared to the flat tracheal placode observed in the rho-bnl-CycA triple mutant at the same stage, indicating that cells needed to undergo mitosis 16 to induce invagination, independent of EGFR and FGF signalling. In rho bnl double mutants, although the cells undergoing the earliest mitoses showed surface cell rounding, some of the subsequent mitotic events were coupled to apical depression and internalized cell rounding. Unlike the earlier mitotic events on the surface, the internalized rounding cells in the rho bnl embryos showed constricted apices and were surrounded by apically rounded cells before mitosis. Internalized rounding with a constricted apical surface were shared properties of cells in mitoses leading to invagination, in both control and rho bnl embryos. It is suggested that the first few cells undergoing surface cell rounding compress the adjacent interphase cells and restrict their apical area, so that they are forced to move internally after rounding, causing the epithelial layer to buckle and invaginate (Kondo, 2013).
Although invagination was largely blocked in the rho-bnl-CycA triple mutants, any double mutant combination permitted invagination to some degree, indicating that three qualitatively distinct mechanisms, mitotic cell rounding, myosin II contractility (EGFR) and active cell motility (FGFR), can independently trigger invagination. In the normal context of wild-type development the combination of cell rounding and EGFR signalling may optimize the timing and speed of invagination, and then invaginated tracheal sacs subsequently respond to FGF emanating from several target tissues guiding branching morphogenesis (Kondo, 2013).
These observations demonstrates a new role for mitosis in tissue morphogenesis to generate mechanical force through cell rounding, independent of cell division. This is distinct from previously described invagination mechanisms involving cell-autonomous constriction by the apical activation of actomyosin contractility, which is incompatible with mitosis. Mitosis 16 outside the tracheal placode occurs in clusters on the ectoderm surface, but does not lead to invagination, suggesting that the tracheal placode is sensitized to invaginate upon mitosis, independent of EGFR and FGFR signalling. Future research to uncover the properties of the tracheal placode that enables it to respond to clustered mitosis will explain not only this new mode of morphogenesis, but also the homeostasis mechanisms of epithelial architecture (Kondo, 2013).
Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, Actin5C was identifed as a key molecule. The results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII; Zipper) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter (see Model for Drosophila retinal lumen formation). Together, these results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen (Nie, 2014; Pubmed
Several in vitro and limited in vivo experiments have shown that neurons maintain a rest tension along their axons intrinsically. They grow in response to stretch but contract in response to loss of tension. This contraction eventually leads to the restoration of the rest tension in axons. However, the mechanism by which axons maintain tension in vivo remains elusive. The objective of this work is to elucidate the key cytoskeletal components responsible for generating tension in axons. Toward this goal, in vivo experiments were conducted on single axons of embryonic Drosophila motor neurons in the presence of various drugs. Each axon was slackened mechanically by bringing the neuromuscular junction toward the central nervous system multiple times. In the absence of any drug, axons shortened and restored the straight configuration within 2-4 min of slackening. The total shortening was approximately 40% of the original length. The recovery rate in each cycle, but not the recovery magnitude, was dependent on the axon's prior contraction history. For example, the contraction time of a previously slackened axon may be twice its first-time contraction. This recovery was significantly hampered with the depletion of ATP, inhibition of myosin motors, and disruption of actin filaments. The disruption of microtubules did not affect the recovery magnitude, but, on the contrary, led to an enhanced recovery rate compared to control cases. These results suggest that the actomyosin machinery is the major active element in axonal contraction, whereas microtubules contribute as resistive/dissipative elements (Tofangchi, 2016).
Epithelial-mesenchymal transitions play key roles in development and cancer and entail the loss of epithelial polarity and cell adhesion. This study used quantitative live imaging of ingressing neuroblasts (NBs) in Drosophila melanogaster embryos to assess apical domain loss and junctional disassembly. Ingression is independent of the Snail family of transcriptional repressors and down-regulation of Drosophila E-cadherin (DEcad) transcription. Instead, the posttranscriptionally regulated decrease in DEcad coincides with the reduction of cell contact length and depends on tension anisotropy between NBs and their neighbors. A major driver of apical constriction and junctional disassembly are periodic pulses of junctional and medial myosin II that result in progressively stronger cortical contractions during ingression. Effective contractions require the molecular coupling between myosin and junctions and apical relaxation of neighboring cells. Moreover, planar polarization of myosin leads to the loss of anterior-posterior junctions before the loss of dorsal-ventral junctions. It is concluded that planar-polarized dynamic actomyosin networks drive apical constriction and the anisotropic loss of cell contacts during NB ingression (Simoes, 2017).
Most epithelial tubes arise as small buds and elongate by regulated morphogenetic processes including oriented cell division, cell rearrangements, and changes in cell shape. Through live analysis of Drosophila renal tubule morphogenesis this study shows that tissue elongation results from polarised cell intercalations around the tubule circumference, producing convergent-extension tissue movements. Using genetic techniques, it was demonstrated that the vector of cell movement is regulated by localised Epidermal growth factor (EGF) signalling from the distally placed tip cell lineage, which sets up a distal-to-proximal gradient of pathway activation to planar polarise cells, without the involvement for PCP gene activity. Time-lapse imaging at subcellular resolution shows that the acquisition of planar polarity leads to asymmetric pulsatile Myosin II accumulation in the basal, proximal cortex of tubule cells, resulting in repeated, transient shortening of their circumferential length. This repeated bias in the polarity of cell contraction allows cells to move relative to each other, leading to a reduction in cell number around the lumen and an increase in tubule length. Physiological analysis demonstrates that animals whose tubules fail to elongate exhibit abnormal excretory function, defective osmoregulation, and lethality (Saxena, 2014: PubMed).
Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).
To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flowerlose (fwelose) This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fweloseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwelose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).
Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-loser mixing is increased during competition. This could affect the outcome of cell competition by increasing the surface shared between losers and winners. Clone splitting was used as a readout for loser–winner mixing. Two non-exclusive mechanisms can drive clone splitting: cell death followed by junction rearrangement, or junction remodelling and cell–cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwelose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition or during Minute-dependent competition. Altogether, this suggested that winner–loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell–cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser–loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser–loser junctions and in winner–winner junctions than in winner–loser junctions. The preferential stabilization of winner–loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser–winner mixing through cell–cell intercalation (Levayer, 2015).
It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc–tub-dmyc interfaces compared with WT–WT and WT–tub-dmyc interfaces (Fig. 3a, b). Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fweloseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M-/+ cells. Thus differences in PIP3 levels might be responsible for winner–loser mixing. Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum , whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner–loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser–winner mixing and required for loser cell elimination (Levayer, 2015).
It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).
Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).
Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).
The Drosophila tracheal system consists of an interconnected network of monolayered epithelial tubes that ensures oxygen transport in the larval and adult body. During tracheal dorsal branch (DB) development, individual DBs elongate as a cluster of cells, led by tip cells at the front and trailing cells in the rear. Branch elongation is accompanied by extensive cell intercalation and cell lengthening of the trailing stalk cells. Although cell intercalation is governed by Myosin II (MyoII)-dependent forces during tissue elongation in the Drosophila embryo that lead to germ-band extension, it remained unclear whether MyoII plays a similar active role during tracheal branch elongation and intercalation. This study used a nanobody-based approach to selectively knock down MyoII in tracheal cells. The data show that, despite the depletion of MyoII function, tip cell migration and stalk cell intercalation (SCI) proceed at a normal rate. This confirms a model in which DB elongation and SCI in the trachea occur as a consequence of tip cell migration, which produces the necessary forces for the branching process (Ochoa-Espinosa, 2017).
During musculoskeletal system development, mechanical tension is generated between muscles and tendon-cells. This tension is required for muscle differentiation and is counterbalanced by tendon-cells avoiding tissue deformation. Both, Jbug/Filamin, an actin-meshwork organizing protein, and non-muscle Myosin-II (Myo-II) are required to maintain the shape and cell orientation of the Drosophila notum epithelium during flight muscle attachment to tendon cells. This study shows that halving the genetic dose of Rho kinase (Drok), the main activator of Myosin-II, enhances the epithelial deformation and bristle orientation defects associated with jbug/Filamin knockdown. Drok and activated Myo-II localize at the apical cell junctions, tendon processes and are associated to the myotendinous junction. Further, it was found that Jbug/Filamin co-distribute at tendon cells with activated Myo-II. Finally, it was found that Jbug/Filamin and Myo-II are in the same molecular complex and that the actin-binding domain of Jbug/Filamin is necessary for this interaction. These data together suggest that Jbug/Filamin and Myo-II proteins may act together in tendon cells to balance the tension generated during development of muscles-tendon interaction, maintaining the shape and polarity of the Drosophila notum epithelium (Manieu, 2018).
Tissue mechanics play a crucial role in organ development. They rely on the properties of cells and the extracellular matrix (ECM), but the relative physical contribution of cells and ECM to morphogenesis is poorly understood. This study analyzed the behavior of the peripodial epithelium (PE) of the Drosophila leg disc in the light of the dynamics of its cellular and ECM components. The PE undergoes successive changes during leg development, including elongation, opening and removal to free the leg. During elongation, it was found that the ECM and cell layer are progressively uncoupled. Concomitantly, the tension, mainly borne by the ECM at first, builds up in the cell monolayer. Then, each layer of the peripodial epithelium is removed by an independent mechanism: while the ECM layer withdraws following local proteolysis, cellular monolayer withdrawal is independent of ECM degradation and driven by myosin-II-dependent contraction. These results reveal a surprising physical and functional cell-matrix uncoupling in a monolayer epithelium under tension during development (Proag, 2019).
Collective cell movements play a central role in embryonic development, tissue repair, and metastatic disease. Cell movements are often coordinated by supracellular networks formed by the cytoskeletal protein actin and the molecular motor non-muscle myosin II. During wound closure in the embryonic epidermis, the cells around the wound migrate collectively into the damaged region. In Drosophila embryos, mechanical tension stabilizes myosin at the wound edge, facilitating the assembly of a supracellular myosin cable around the wound that coordinates cell migration. This study shows that actin is also stabilized at the wound edge. However, loss of tension or myosin activity does not affect the dynamics of actin at the wound margin. Conversely, pharmacological stabilization of actin does not affect myosin levels or dynamics around the wound. Together, these data suggest that actin and myosin regulation during embryonic wound closure in Drosophila are largely independent, thus conferring robustness to the embryonic response to wounds (Kobb, 2019).
Epithelial sheets undergo highly reproducible remodeling to shape organs. This stereotyped morphogenesis depends on a well-defined sequence of events leading to the regionalized expression of developmental patterning genes that finally triggers downstream mechanical forces to drive tissue remodeling at a pre-defined position. However, how tissue mechanics controls morphogenetic robustness when challenged by intrinsic perturbations in close proximity has never been addressed. Using Drosophila developing leg, this study shows that a bias in force propagation ensures stereotyped morphogenesis despite the presence of mechanical noise in the environment. Knockdown of the Arp2/3 complex member Arpc5 specifically affects fold directionality while altering neither the developmental nor the force generation patterns. By combining in silico modeling, biophysical tools, and ad hoc genetic tools, these data reveal that junctional myosin II planar polarity favors long-range force channeling and ensures folding robustness, avoiding force scattering and thus isolating the fold domain from surrounding mechanical perturbations (Martin, 2021).
Myosin II is required during oogenesis for follicle cell migration and cytoplasmic "dumping" of nurse cell contents (Wheatley, 1995 and Edwards, 1996), it is required during early development for the posterior migration of nuclei (Wheatley, 1995), it is required for dorsal closure late in embryonic development (Young, 1993) and it is required for development of the eye in larvae and for the eversion of the leg disc and proper formation of the wing during pupation (Edwards, 1996).
Embryos that lack
functional myosin display defects in dorsal closure, head involution, and axon patterning. Analysis of
cell morphology and myosin localization during dorsal closure in wild-type and homozygous mutant
embryos demonstrates a key role for myosin in the maintenance of cell shape and suggests a model
for the involvement of myosin in cell sheet movement during development. These experiments, in
conjunction with the observation that cytokinesis also requires myosin, suggest that the processes of
cell shape change in morphogenesis and cell division are intimately and mechanistically related (Young, 1993).
Drosophila is an ideal metazoan model system for analyzing the role of nonmuscle myosin-II during development. In Drosophila, myosin function is required for cytokinesis and morphogenesis driven by cell migration and/or cell shape changes during oogenesis, embryogenesis, larval development and pupal metamorphosis. The mechanisms that regulate myosin function and the supramolecular structures into which myosin incorporates have not been systematically characterized. The genetic screens described here identify genomic regions that uncover loci that facilitate myosin function. The nonmuscle myosin heavy chain is encoded by a single locus, zipper. Contiguous chromosomal deficiencies that represent approximately 70% of the euchromatic genome were screened for genetic interactions with two recessive lethal alleles of zipper in a second-site noncomplementation (SSNC) assay for the malformed phenotype. Malformation in the adult leg reflects aberrations in cell shape changes driven by myosin-based contraction during leg morphogenesis. Of the 158 deficiencies tested, 47 behave as second-site noncomplementors of zipper. Two of the deficiencies are strong interactors, 17 are intermediate and 28 are weak. Finer genetic mapping reveals that mutations in cytoplasmic tropomyosin and viking (collagen IV) behave as second-site noncomplementors of zipper during leg morphogenesis and that zipper function requires a previously uncharacterized locus, E3.10/J3.8, for leg morphogenesis and viability (Halsell, 1998).
Collagen IV is a basement membrane collagen; its function has been demonstrated during morphogenesis in C. elegans and Drosophila. Mutations in the emb-9 locus [Collagen 1(IV)] and the let-2 locus [Collagen 2(IV)] cause defects during the late morphogenetic stage that result in embryonic lethality. In Drosophila, two Collagen IV genes, localize to the SSNC interval uncovered by Df(2L)sc19-5. Mutations in viking act as SSNCs of zip during leg morphogenesis. Levels of Collagen IV are detectable throughout the life cycle of the fly, with high levels detected until the onset of pupation. During leg morphogenesis, the basal lamina detaches from the disc epithelium, and proteolysis of Collagen IV is thought to make the basement membrane more extensible, thus facilitating leg elongation. This site-directed cleavage of Collagen IV occurs in response to ecdysone. Identification of Stubble as a necessary gene product during leg imaginal disc morphogenesis also demonstrates a role for proteolysis during this process. The Stubble locus encodes a type II transmembrane serine protease; Stubble interacts genetically with road1 and zipEbr. However, since the existing viking mutations are P-element insertions, it is more likely that these mutations affect the expression of Collagen IV, rather than its proteolysis, during metamorphosis. Basal cytoplasmic extensions have been observed in the imaginal discs of several insects and are thought to be important for morphogenetic movements. Perhaps mutations in viking interfere with such processes in the Drosophila leg imaginal disc; if so, this effect would occur prior to the observed proteolysis (Halsell, 1998 and references).
rib and raw mutations prevent cells in a number
of tissues from assuming specialized shapes, resulting
in abnormal tubular epithelia and failure of morphogenetic
movements such as dorsal closure. Mutations of
zipper, which encodes the nonmuscle myosin heavy chain,
suppress the phenotypes of rib and raw, suggesting that
rib and raw are not directly required for myosin function.
Abnormal formation of the actin cytoskeletal structures
underlying embryonic cuticular hairs suggests possible
roles for rib and raw in organizing the actin cytoskeleton.
The actin prehair structures are absent in rib
mutants and abnormally shaped in raw mutants, indicating
that the two genes have different functions required
for organizing the actin cytoskeleton (Blake, 1999).
The fact that zip mutations suppress many of the mutant
phenotypes of rib and raw is inconsistent with the
hypothesis that either rib or raw is directly required for
contraction of the actin cytoskeleton by myosin. Nevertheless,
the suppression of the rib and raw phenotypes
by zip mutations might be observed if the rib and raw
products regulate myosin contraction either by repression
or by controlling the direction of contraction. Alternatively,
both the effect of the mutations on cell shape
and the suppression by zip mutations could be observed
if rib and raw contribute to remodeling of the actin cytoskeleton by involvement in the organization of the actin
filaments. The counteraction of rib and raw mutant phenotypes
by zip mutations could then occur if the normal
activities of the rib and raw products on the cytoskeleton
oppose to some extent the activity of myosin (Blake, 1999).
The effect of the rib and raw mutations on hairs and denticles
of the embryonic cuticle offers support for the hypothesis
that the gene products are active in organizing actin.
In late embryogenesis, bundles of filamentous actin
form epidermal extensions around which cuticular structures
are secreted. Some of these cuticular structures are
the external apparatus of sensory organs and others are
nonsensory projections, the dorsal and lateral cuticular
hairs and ventral denticles. The denticles and hairs, both
sensory and nonsensory, have various shapes and orientations
and are organized in stereotypical, segmentally repeated
patterns. The actin cytoskeletal supports of the cell
extensions can be observed in stage 16 and 17 embryos by
staining with rhodamine labeled phalloidin. Both rib and raw mutations
alter the morphology of the F-actin structures, but mutations
of each gene have different effects on the structures (Blake, 1999).
In normal embryos actin bundles form a prehair in the
cells that secrete sensory hairs, but no prehairs
form in rib embryos. rib mutants lack hairs
and denticles almost completely, leaving only a
few isolated cuticular hairs and denticles. The remaining
hairs are either much longer than normal or are abnormally
curved. At junctions of three or more cells, rib embryos
display intense actin spots, some of which could be sockets
of sense organs. In addition, F-actin, which in wild-type
epidermal cells accumulates in the cytoplasm and
subsequently dissipates, remains in the cytoplasm of rib
epidermal cells. The observation of cytoplasmic F-actin
accumulation that disappears prior to formation of the
actin prehair is consistent with the possibility that actin filaments
begin to form in the cytoplasm and are recruited
into the prehair structures. The rib product is apparently
required for the formation of the larger actin structures
from smaller actin filaments that form in the cytoplasm (Blake, 1999).
The cells of raw mutant embryos do form projections,
albeit abnormally shaped ones. In raw mutants, hairs are
generally disorganized in appearance, may be
inappropriately clustered, and are often forked or
branched. These are the same types of abnormalities
described for embryos mutant for forked (f) and singed (sn), two genes that encode
actin bundling proteins. Thus, the raw product could have a role in bundling
or otherwise organizing actin filaments.
The formation of the F-actin prehair structures might
be independent of the activity of myosin. Zygotic expression
of zip is not obviously required for formation of actin
prehairs and predenticles since both form normally in zip
mutants. If myosin does not affect the organization
of actin into prehair structures, zip mutations would
not be expected to alter the phenotype of rib mutants with
respect to the failure of formation of prehairs. However,
zip mutation suppresses the rib phenotype, causing a substantial increase
in the number of denticles and hairs present on the
embryonic cuticle of rib;zip mutants, as compared to rib mutants. Thus, zip counteracts the effect of rib mutations
for each of the rib phenotypes. The suppression of
the cuticular hair phenotype of raw mutations by zip is
less obvious, but the severity of the branching and forking
characteristic of the raw prehair phenotype is reduced in
raw;zip double mutants. The fact that a zip mutation
causes actin prehairs and predenticles to form more
normally in rib and raw mutants indicates that myosin antagonizes
the formation of the actin structures (Blake, 1999).
Although rib and raw have similar effects on the ability
of cells to elongate, the differences in the effects of
mutations of the two genes on the actin structures that underly
cuticular hairs suggest that the two gene products
have different functions with respect to the actin cytoskeleton.
Distinct functions for rib and raw products are
consistent with the observation that raw;rib double mutants
are far more defective than embryos mutant for either
of the genes individually. In
the double mutants many of the affected tissues are greatly
reduced in size and the embryos are generally very delicate. The extreme phenotype of the double mutant could
be the result of separate defects in the actin cytoskeleton.
The evidence presented provides further support
for the hypothesis that rib and raw products have
functions necessary for cytoskeletal activity, either in a
structural or regulatory capacity. The data also indicate
that the gene products are not required for myosin to apply
force to the actin cytoskeleton. Because the products are
essential for formation of the actin models of cuticular
hairs and denticles, they could function directly in organizing
actin filaments. Defects in reorganization of actin
filaments of the cortical cytoskeleton could also explain
the abnormal cell shapes associated with rib and raw mutants.
However, as is the case with other rib and raw phenotypes,
lack of zygotic zip activity suppresses the effect
of mutations of the genes on hair and denticle formation in
the embryonic cuticle. Therefore the rib and raw products
could also act by repressing myosin or controlling its activity
in some other way. Analysis of the rib and raw
products will likely be necessary to resolve the issue (Blake, 1999).
A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. The regulation of myosin activity has been explored by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, a screen was performed for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. A single missense mutation in the zipperEbr allele gives rise to its sensitivity to second-site noncomplementation. The Rho signal transduction pathway has been identified as necessary for proper myosin function. A lethal P-element insertion interacts genetically with zipper. Subsequently this second-site noncomplementing mutation has been shown to disrupt the RhoGEF2 locus. Two EMS-induced mutations, previously shown to interact genetically with zipperEbr, disrupt the RhoA locus. Further, their molecular lesions have been identified and it has been determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, it has been shown that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. These studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan (Halsell, 2000).
To identify loci encoding gene products that collaborate with nonmuscle myosin during morphogenesis, second-site noncomplementation screens were performed for the malformed adult leg phenotype (mlf). Depletion of myosin during leg imaginal disc morphogenesis results in mlf. A collection of 268 single, lethal P-element insertional mutations on the second chromosome were screened for genetic interactions with the zipEbr allele. Fourteen insertions failed to complement zipEbr. The strength of the genetic interaction is arbitrarily defined on the basis of the percentage of flies of the appropriate genotype that exhibit the malformed phenotype: weak interactions show penetrance of 10%-25% while intermediate interactions are 25%-75% penetrant. Eleven of the lethal P-element insertions identified are weak interactors. Three of the insertions are intermediate interactors. Two of these intermediate interactors are not second-site noncomplementing loci but are new zipper alleles, exhibiting intraallelic complementation. The third intermediate interacting mutation, l(2)04291, causes mlf flies in trans to zipEbr with a penetrance of 38% (Halsell, 2000).
The P-element insertion of l(2)04291 disrupts the RhoGEF2 locus. Genomic DNA flanking the P-element insertion was recovered by plasmid rescue, and by sequencing flanking DNA it was discovered that the P element lies within an intron that interrupts the 5' UTR of the RhoGEF2 gene (Barrett, 1997; Hacker, 1998). To further confirm that the genetic interaction observed with zipEbr results from a mutation in RhoGEF2, two EMS-induced mutant RhoGEF2 alleles, 1.1 and 4.1, were tested in the malformed leg assay. Both alleles interact with zipEbr; the penetrance of the malformed phenotype in double heterozygous flies is 33% with the RhoGEF21.1 allele and 27% with the RhoGEF24.1 allele, comparable to that seen with the original P-insertional allele (Halsell, 2000).
In addition to the malformed legs observed in flies double heterozygous for mutant RhoGEF2 and zipEbr, malformed wings were observed at comparable frequencies. Between 80% and 97% of the flies exhibiting a malformed leg phenotype also exhibit malformed wings. In contrast, most other loci that interact with zipper do not exhibit significant wing defects. Malformed wings are rarely observed when the legs are wild type. Taken together, these data indicate a requirement for RhoGEF2 during myosin-driven leg and wing imaginal disc morphogenesis (Halsell, 2000).
RhoAE3.10 genetically behaves as a severe allele, yet molecularly results from a single amino acid change that converts a cysteine at position 189 to a tyrosine residue. This missense mutation causes severe effects because it alters the first residue, cysteine, in the CaaX box. The CaaX box is a common feature of members of the Ras-superfamily of small GTPases. Functionally, the cysteine residue is the site of a post-translational prenylation modification. Subsequent to this modification further lipid modifications may occur, and in most cases, the final three amino acids are removed. These modifications are required for proper association of the small GTPase and the membrane; without this association, the GTPase is nonfunctional. These functional relationships have been demonstrated for numerous Ras superfamily members, including Rho. Site-directed mutagenesis that changes the CaaX box cysteine to serine of the S. cerevisiae RhoA homolog, Rho1, results in the failure of the mutated Rho1 protein to repartition from the cytosolic compartment to the membrane. Further, these Rho1 mutant cells fail to grow. In mammalian tissue culture, CaaX box-mutated RhoB cannot be lipid modified, and these cells lose their ability to become transformed in sensitized backgrounds. Therefore, it is likely that the RhoAE3.10-encoded protein cannot be post-translationally modified, resulting in a complete loss of RhoA function. Similarly, the nonsense mutation at residue 180 in the J3.8 allele would remove the CaaX box and an additional nine amino acids and, therefore, would also behave as a severe RhoA allele (Halsell, 2000).
However, on the basis of the differences observed in their genetic interactions with Df(2R)Jp1 and their levels of reduced viability in trans to zipEbr, RhoAE3.10 appears to be a more severe allele than RhoAJ3.8. It is hypothesized that the protein encoded by RhoAE3.10 may have a partial dominant-negative effect because it does not repartition properly. On the other hand, the premature stop codon in RhoAJ3.8 may give rise to an unstable gene product. Since appropriate antibodies directed against Rho are not yet available, this alternative cannot be adequately evaluated (Halsell, 2000).
Studies reveal that multiple processes require myosin function throughout Drosophila development, including oogenic cell migrations, larval cytokinesis, and imaginal disc morphogenesis. Strong or null alleles of zipper are embryonic lethal, fail during dorsal closure, and give rise to embryos with dorsal cuticular holes. Additionally, myosin immunolocalization studies suggest that myosin is required during stages not yet tested functionally, including embryonic cellularization and gastrulation. RhoGEF2 and RhoA also function at least during a subset of the morphogenetic processes that require myosin (Halsell, 2000 and references therein).
Mutations in the Drosophila RhoGEF2 gene have been identified by three distinct means: phenotypic suppression of ectopically expressed RhoA (Barrett, 1997); genetic screens for maternally encoded molecules required during early Drosophila embryogenesis (Hacker, 1998), and genetic screening for molecules required for myosin function (this study). Maternal depletion of RhoGEF2 results in defects during gastrulation (Barrett, 1997; Hacker, 1998). Specifically, embryos lacking maternal RhoGEF2 fail during apical constriction of ventral furrow cells. Interestingly, myosin localizes to the apical ends of these ventral furrow cells. This observation coupled with the genetic interaction between RhoGEF2 and myosin during leg morphogenesis suggests that RhoGEF2 may exert some of its effect during gastrulation via the activity of myosin in these cells (Halsell, 2000 and references therein).
RhoA mutations are recessive embryonic lethals. Zygotic depletion of RhoA results in an anterior dorsal hole in the cuticle. This defect has been characterized as a dorsal closure phenotype. Dorsal closure is an embryonic morphogenetic event in which the lateral epidermis moves over the dorsal side of the embryo, ultimately fusing along the midline. If dorsal closure fails, then cuticular holes result. Typically, these holes are more posteriorly localized than those observed in RhoA mutants. However, certain zipper alleles give rise to cuticular holes that extend from the posterior one-third of the embryo to the anterior end. These extensive cuticular holes are consistent with the head involution defects observed in zipper mutants and may reflect combined defects in head morphogenesis and dorsal closure. Therefore RhoA loss-of-function mutations may more accurately represent a particular sensitivity in head morphogenesis to perturbation rather than being dorsal closure mutants per se (Halsell, 2000 and references therein).
Nonetheless, RhoA function during dorsal closure has been implicated by analysis of embryos expressing dominant negative RhoA transgenes. In wild-type embryos, the leading-edge cells and the adjacent lateral cells elongate during dorsal closure. When dominant-negative RhoA is driven in the leading edge by utilizing the GAL-4 UAS system, stretching of the leading cells initiates but is ultimately lost, and the lateral cells never elongate. The Jun-kinase signal transduction cascade acts during dorsal closure and induces expression of the TGFß gene, decapentaplegic (dpp), in the leading-edge cells. Leading-edge dpp expression is a prerequisite for elongation of the flanking lateral cells. In the dominant-negative RhoA embryos, dpp expression is wild type, therefore the authors suggest that RhoA acts upstream of a separate transcriptional pathway. Three observations suggest that RhoA may function directly upstream of myosin in the leading edge. (1) It has been shown that RhoA signaling is necessary for myosin-driven cell shape changes during leg imaginal disc morphogenesis. (2) zipper mutants lose myosin in the leading-edge cells, and, subsequently, the leading-edge cells fail to elongate. (3) Myosin is delocalized in leading-edge cells expressing dominant negative RhoA. Taken together, these results suggest that RhoA signaling may have a direct cellular output at the level of myosin activity in the leading-edge cells and may not exert its effect via a transcriptional pathway (Halsell, 2000 and references therein).
Numerous pharmacological, cell culture, and biochemical studies implicate the Rho subfamily of GTPases as signal transducers upstream of actin cytoskeleton rearrangements and myosin regulation. In Drosophila, injection of mutant forms of Rho or Cdc42 proteins induces gross malformations in the actomyosin cytoskeleton, disrupting a specialized embryonic cytokinesis known as cellularization. When dominant-negative Rac1 is expressed at later stages of embryogenesis, the actomyosin cytoskeleton is disrupted in the leading-edge cells during dorsal closure. In Swiss 3T3 cells, the Rho GTPase induces the formation of actin stress fibers. Further, it has been demonstrated that contractility of the actin cytoskeleton, presumably mediated by myosin, is required for stress fiber formation and that this contractility is downstream of Rho signal transduction (Halsell, 2000 and references therein).
In metazoans, nonmuscle myosin and smooth muscle-based contractility depend on the phosphorylation state of the noncovalently bound regulatory light chain. Molecularly, activated Rho may modulate the phosphorylation state of the regulatory light chain. Biochemical analysis reveals that activated Rho binds and activates a variety of effectors, including a group of serine/threonine kinases known as Rho kinase/ROK and p160ROCK/ROKß. In vitro biochemical assays reveal that Rho kinase can phosphorylate the regulatory light chain at its activating sites and induce myosin activity. Further, Rho kinases phosphorylate the myosin binding subunit of myosin phosphatase and thus repress its activity; the net result is a further increase in the phosphorylation state of the regulatory light chain (Halsell, 2000 and references therein).
Genetic screens for morphogenesis defects in C. elegans have also identified mutations in loci encoding Rho signal transduction components. Mutations in the C. elegans Rho kinase locus, let-502, disrupt embryonic elongation, while mutations in the regulatory subunit of the myosin phosphatase gene, mel-11, suppress the let-502 morphogenetic defect (Wissmann, 1997). These results suggest that Rho signal transduction is upstream of myosin-driven morphogenesis in C. elegans. This hypothesis cannot be tested directly because myosin mutations that affect cell sheet morphogenesis have not been identified in C. elegans. Nonmuscle myosin is encoded by more than one locus and functional redundancy of these loci may preclude the isolation of morphogenetic myosin mutations (Halsell, 2000 and references therein).
ribbon is thought to be required for generating specialized cell shapes. For instance, during dorsal closure, leading edge cells of the lateral epidermis fail to elongate in rib mutants. rib mutants also show abnormal dilation of salivary gland lumina in late embryogenesis, suggesting that either rib is also required at late stages to maintain organ shape or loss of early rib function indirectly causes the late lumenal dilation. rib appears to control cell shapes by regulating the cytoskeleton. During dorsal closure, a band of actin and myosin forms at the dorsal margin of leading edge cells. In rib embryos, the actin band is narrower and myosin heavy chain (MHC) is absent from leading edge cells. Thus, rib may be required for the localization or organization of cytoskeletal components. zip encodes a nonmuscle MHC and is required in many of the same tissues as rib; however, strong loss-of-function mutations in zip suppress the distended lumenal phenotype of rib salivary glands, suggesting that rib does not positively regulate myosin activities. Instead, rib may repress myosin contraction or regulate the direction of contraction, perhaps by providing a balancing force to the direction of basal myosin contractions. These studies reveal a role for rib in coordinating directed cell migration, a process that clearly involves actin/myosin dynamics. Thus, rib may modulate actin/myosin behavior for cell movement and cell shape during both tissue formation and tissue homeostasis. If rib is responding to signaling pathways, rib could be a critical factor linking signaling events to changes in the cytoskeleton (Bradley, 2001).
Nonmuscle myosin-II is a key motor protein that drives cell shape change and cell movement. The function of nonmuscle myosin-II has been analyzed during Drosophila embryonic myogenesis. Nonmuscle myosin-II and the adhesion molecule, PS2 integrin (Myospheroid), colocalize at the developing muscle termini. In the paradigm emerging from cultured fibroblasts, nonmuscle actomyosin-II contractility, mediated by the small GTPase Rho, is required to cluster integrins at focal adhesions. In direct opposition to this model, it has been found that neither nonmuscle myosin-II nor RhoA appear to function in PS2 clustering. Instead, PS2 integrin is required for the maintenance of nonmuscle myosin-II localization and the cytoplasmic tail of the ßPS integrin subunit is capable of mediating this PS2 integrin function. Embryos that lack zygotic expression of nonmuscle myosin-II fail to form striated myofibrils. In keeping with this, a PS2 mutant that specifically disrupts myofibril formation is unable to mediate proper localization of nonmuscle myosin-II at the muscle termini. In contrast, embryos that lack RhoA function do generate striated muscles. Finally, nonmuscle myosin-II localizes to the Z-line in mature larval muscle. It is suggested that nonmuscle myosin-II functions at the muscle termini and the Z-line as an actin crosslinker and acts to maintain the structural integrity of the sarcomere (Bloor, 2001).
The myogenic function of nonmuscle myosin-II has been analyzed by using Drosophila genetics to manipulate the levels of nonmuscle myosin-II heavy chain, PS2 integrin, and RhoA GTPase in vivo in the developing larval muscles.
Both nonmuscle myosin-II and PS2 colocalize
at muscle termini. However, in contrast to models based on
cultured fibroblasts, there is no evidence for either
nonmuscle myosin-II or RhoA function in PS2 clustering.
Instead, the maintenance of nonmuscle
myosin-II localization at muscle termini is dependent on
the presence of PS2 integrin and the cytoplasmic tail of
the ßPS integrin subunit is sufficient for this. Further,
nonmuscle myosin-II maintenance at the muscle termini is compromised in ifSEF, a ßPS2 integrin subunit mutant that specifically disrupts myofibril formation. Through the analysis of actin distribution in the musculature of living wild-type and mutant embryos, it has been demonstrated that RhoA-independent nonmuscle myosin-II function is required for the proper sarcomeric organization of the muscle cytoskeleton. Finally, since nonmuscle myosin-II localizes to the Z-line in late larval muscle, it has been suggested that nonmuscle myosin-II functions at both the muscle termini and the Z-line to maintain the structural integrity of the sarcomere (Bloor, 2001).
When fibroblasts in culture attach to ECM substrates
through their cell surface integrin receptors, they dramatically
redistribute these receptors such that they become
clustered at focal adhesions. Integrin ligand binding also
induces the actin cytoskeleton to rearrange into stress
fibers, which terminate at focal adhesions and connect to
the cytoplasmic domains of the clustered integrins. Pharmacological
inhibitors of contractility block this complex
cellular response, providing evidence that nonmuscle myosin-II driven
contractility is essential for integrin clustering. At least a
subset of nonmuscle myosin-II based contractility is dependent
on RhoGTPase-mediated phosphorylation events. This suggests a model for focal
adhesion and stress fiber formation in which, when diffuse
cell surface integrins bind ECM ligand, they associate with
actin filaments and activate Rho. In turn, Rho activates
nonmuscle myosin-II, driving the formation of nonmuscle
myosin-II filaments and increasing contractility. This increases
the tension exerted on the actin cytoskeleton causing
actin filaments to bundle and align into stress fibers (Bloor, 2001).
Bundling drives actin-associated cell surface integrins into
clusters and focal adhesions are formed.
The localization of PS2 integrin at Drosophila muscle
termini is an ideal system in which to test this model in
vivo. Indeed, consistent with the possibility that RhoA-mediated
nonmuscle myosin-II contractility drives PS2 integrin clustering in the larval musculature, genetic evidence implicates RhoA-dependent activation of nonmuscle
myosin-II in multiple morphogenetic pathways during Drosophila
development. Furthermore, previous studies have shown that an
uncharacterized intracellular mechanism is capable of driving
integrin localization to the muscle termini. Despite this, this study shows that
genetic depletion of either nonmuscle myosin-II or RhoA
fails to disrupt PS2 localization (Bloor, 2001).
Maternally contributed RNAs and proteins support the
early stages of Drosophila embryogenesis. Both zip and
RhoA are maternally expressed and, in the absence of
zygotic expression, this maternal contribution is sufficient
for development to proceed normally until stage 14. Subsequent to this, depletion of maternal gene product results in defects in epidermal morphogenesis. Since the localization of PS2 integrin to the muscle termini occurs during stages
15 and 16, it seems likely that maternally contributed gene product is depleted
from zip and RhoA mutant embryos prior to PS2 clustering.
Thus, the presence of PS2 at muscle termini in zip and
RhoA mutants suggests that neither nonmuscle myosin-II
nor RhoA are required for PS2 clustering. However, the
absolute amount of these gene products required to localize
PS2 may be lower than that required for continued epidermal
morphogenesis. Alternatively, these gene products may
perdure longer in the mesoderm than in the developing epidermis (Bloor, 2001).
It is possible to eliminate the maternal contribution of a
gene by generating mutant clones in the female germline.
However, germline clones of zip and RhoA null mutations
fail to make eggs. A novel technique was used to address the role
of maternal nonmuscle myosin-II in PS2 localization. p127-l(2)gl, a nonmuscle myosin-II heavy chain binding protein, was overexpressed in a zip2 mutant background. The
p127-l(2)gl protein binds and sequesters maternal non-muscle
myosin-II heavy chain, effectively titrating the
available levels of nonmuscle myosin-II and antagonizing
its function. In these embryos, the epidermal
morphogenesis defects associated with zip2
mutations are enhanced; however, muscle attachment and PS2 localization
are unaffected. Muscle abnormalities are, however, observed in these and in zip zygotic null embryos: a variable subset of ventral muscles is deleted. Interestingly the affected muscles, VA1, VA2, and VA3, are derived from
two muscle progenitors that arise from the same cluster of
mesodermal cells. One progenitor divides to produce the muscle founder cells for VA1 and VA2. Subsequently, the other progenitor divides to produce
the VA3 muscle founder and an adult muscle founder cell. The lineage and temporal
relationships between these cells are reflected in the frequency
at which these muscles are deleted: VA3 is more
commonly deleted than VA1 and VA2, which are always
either both present or both deleted. Although it is unclear
whether these are the last progenitors to divide, it seems
likely that defects in nonmuscle myosin-II-dependent cytokinesis are the basis of this phenotype. The fact that depletion of nonmuscle myosin-II can affect myogenic events occurring during stages 11 and 12 without affecting the localization of PS2 that occurs during stages 15 and 16 further supports the contention that nonmuscle myosin-II is not required for PS2 localization (Bloor, 2001).
An alternative approach to the analysis of gene function is the ectopic expression of dominant negative constructs. Although the interpretations of such experiments are not always unambiguous, ectopic expression of the dominant negative RhoAN19 construct has implicated RhoA function in biological processes not revealed by maternal and zygotic mutational analyses. For example, driving RhoAN19 expression in the early mesoderm disrupts invagination of this tissue, phenocopying embryos that lack both maternal and zygotic DRhoGEF2 expression. In this study, the 24B GAL4 driver was used to express UAS-RhoAN19 in the mesoderm during later stages of development (Bloor, 2001).
24B-driven expression can be detected by stage 10 and strong expression occurs from stage 13 onward. Thus, this approach will compromise endogenous RhoA function during late stages of mesoderm development. Indeed 24B-driven
UAS-RhoAN19 expression affects the development of the
visceral mesoderm and causes defects in somatic muscle
patterning similar to those seen in zip mutant embryos. However, no defects were detected in somatic muscle attachment or PS2 localization in these embryos.
Thus, while it is not absolutely certain that maternally
supplied nonmuscle myosin-II or RhoA do not contribute to PS2 localization, these experiments provide strong evidence against this possibility (Bloor, 2001).
To exert tension on an underlying substrate, a cell must
form a strong transmembrane connection between the
substrate and its contractile cytoskeleton. At Drosophila
muscle attachments this connection is mediated by localized
PS2 integrin. As such, these structures superficially resemble focal adhesions,
a point further emphasized by the localization of the
focal adhesion protein integrin-linked kinase (ILK) to these
sites. In contrast to the recruitment of intracellular proteins to focal adhesions, it has been shown that the
initial localization of nonmuscle myosin-II to Drosophila
muscle termini is not dependent on integrin clustering.
Similarly, the localization of PAK and ILK are both independent of PS2 integrin.
Furthermore, PS2 is also not required for the formation of
an electron-dense hemiadherens junction at the muscle
termini. However, among the proteins known to localize to the muscle termini, nonmuscle myosin-II is so far unique in that only it is dependent on
PS2 integrin for its continued localization at these sites (Bloor, 2001).
Interestingly, the localization of nonmuscle myosin-II becomes
PS2-dependent at the same developmental stage at
which the PS2-dependence of muscle adhesion becomes
apparent. When integrins bind ECM ligand, they are thought to undergo a
conformational change that displaces the cytoplasmic tail
of the integrin alpha subunit, exposing protein binding sites on
the cytoplasmic tail of the ß subunit, a process known as
outside-in signaling. It is possible that the accessibility of
the ßPS cytoplasmic tail for protein-protein binding regulates
PS2-dependent nonmuscle myosin-II localization.
This is supported by the observation that, in the absence of
endogenous PS2, the ßPS cytoplasmic tail is sufficient to
keep nonmuscle myosin-II at the muscle termini. Excitingly,
biochemical studies show that peptides derived from
the cytoplasmic tail of the vertebrate integrin ß3 subunit are
able to interact with the tail of nonmuscle myosin-II. In addition, this interaction has recently been demonstrated in cultured cells, suggesting that the maintenance of nonmuscle myosin-II localization might occur through a direct binding reaction with the ßPS cytoplasmic tail (Bloor, 2001).
PS2 integrin is required in muscle both for attachment to
the epidermis and for the generation of sarcomeric ultrastructure.
These data suggest that the sarcomeric function of
PS2 is due, at least in part, to its role in maintaining
nonmuscle myosin-II at the muscle termini. A possible
function for nonmuscle myosin-II at the muscle termini is
to physically link PS2 integrin to the muscle cytoskeleton
by directly binding both PS2 and actin. One prediction of
this model is that, in the absence of nonmuscle myosin-II,
the muscle cytoskeleton will detach from the muscle
termini, but that PS2 will continue to mediate adhesion of
the muscle sarcolemma to the tendon matrix of the muscle-attachment
site. Such a phenotype is observed in embryos
mutant for ilk, the gene that encodes ILK. However, this disconnection of the muscle cytoskeleton from the muscle termini is clearly distinct from that
observed in the muscles of zip mutant embryos, where
muscle actin remains connected to the muscle termini, but
fails to organize into sarcomeres. While this does not rule
out a role for nonmuscle myosin-II in connecting PS2 and
actin, it certainly argues against nonmuscle myosin-II being
the primary component of this link (Bloor, 2001).
An alternative function for nonmuscle myosin-II is suggested
by the demonstration that nonmuscle myosin-II
localizes to the Z-line in the somatic muscles of third instar
larvae. Both the Z-line and the muscle termini are ultrastructural
elements that transmit tensile stress during muscle contraction. It is possible that nonmuscle myosin-II might function as an actin-crosslinking protein at these
sites to help maintain their structural integrity. This role is
generally assumed to be a function of alpha-actinin, an actin
crosslinking protein that is the major component of muscle
termini and Z-lines in both vertebrates and invertebrates.
Mutations in the single Drosophila alpha-actinin gene do cause
terminal defects and sarcomeric abnormalities. Surprisingly though, embryos that lack both maternal and zygotic alpha-actinin expression hatch and lethality
does not occur until the end of the first larval instar. Furthermore, the organization of actin into a striated pattern of I-bands is unaffected in alpha-actinin mutants. This implicates other actin-binding proteins in the maintenance of actin organization at the muscle termini and Z-line and the data
suggest that nonmuscle myosin-II maybe one such protein (Bloor, 2001).
There is no clear model of how nonmuscle myosin-II
might fulfill this function. Intriguingly, myosin heads from
a single filament have been shown to be able to bind parallel
actin thin filaments of opposite orientation. One speculation is that nonmuscle myosin-II might be capable of such behavior, but how such cross-links
would occur at cellular levels of ATP is not clear. One
possibility is that nonmuscle myosin-II in the muscle
termini and Z-line is in a 'catch' muscle state in which
tension is maintained without turnover of ATP. By this scenario,
nonmuscle myosin-II would not function in contraction,
but would serve as an effective actin crosslinker. A noncontractile
function for nonmuscle myosin-II in myofibrillogenesis would explain why RhoA appears to have no role in this process. It is interesting to note that, in Dictyostelium, a contraction-independent function of non-muscle myosin-II has been shown to be important for the generation of cortical tension (Bloor, 2001).
Finally, PS2 has been shown to be present at the sarcolemma
above the Z-line in cultured Drosophila myotubes. It is therefore possible that PS2 somehow functions to maintain nonmuscle myosin-II within the
Z-line. Indeed, adhesion between Z-line-associated hemiadherens
junctions and the muscle basement membrane fails in the absence of PS2. This suggests that the integrity of the sarcomeric muscle cytoskeleton requires it to be connected to the ECM at both the muscle termini and the Z-line and that this connection is mediated by nonmuscle myosin-II and PS2 integrin (Bloor, 2001).
Drosophila Myosin binding subunit
(MBS), the homolog of mammalian MBS, was identified to study the roles of myosin phosphatase in morphogenesis. Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC: Spaghetti squash). Myosin phosphatase's regulatory myosin-binding subunit, MBS, is responsible for regulating the myosin phosphatase catalytic subunit in response to upstream signals and for determining myosin phosphatase's substrate specificity (Mizuno, 2002).
Embryos defective for both maternal and zygotic MBS demonstrate a failure in dorsal closure. In the mutant embryos, the defects are mainly confined to the leading edge cells which fail to fully elongate. Ectopic accumulation of phosphorylated MRLC is detected in the lateral region of the leading edge cells, suggesting that the role of MBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-associated kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of MBS with mutations in the components of the Rho signaling cascade also indicates that MBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis (Mizuno, 2002).
To examine whether defects in the dorsal closure in the embryos lacking MBS or overexpressing wild-type Rho kinase are due to an aberrant activation of nonmuscle myosin II, the genetic interactions with zipper (zip), which encodes the heavy chain of nonmuscle myosin II, were analyzed. About 25% of the progeny from crossing the females transheterozygous with DMBSP2 and Df(3L)th117 to the males heterozygous for DMBSE1 are embryonically lethal. It was expected that a reduction in the gene dosage of zip+ would suppress the defects in the MBS mutant or Rho-kinase-expressing embryos. When DMBSP2/Df(3L)th117 females are mated with males heterozygous for both DMBSE1 and zipEbr, half of the embryos defective for both maternal and zygotic MBS should be heterozygous for zipEbr. As expected, the embryonic lethality was reduced to nearly half that of the corresponding cross. Similarly, the heterozygosity for zipEbr considerably suppresses lethality due to ectopic wild-type Rho kinase expression. These results strongly suggest that either loss of MBS+ or overexpression of wild-type Rho-kinase causes hyperactivation of nonmuscle myosin II through increasing the levels of phosphorylation of MRLC (Mizuno, 2002).
zipEbr is a point mutation reported to be highly sensitive to genetic backgrounds. About 70% of the flies transheterozygous between zipEbr and zip02957 have malformed wings with varying degrees of severity. Although zipEbr is recessive, a considerable percentage of the flies heterozygous for both zipEbr and the mutations in the components of the Rho signaling pathway such as DRho1 and DRhoGEF2 produced similar defects. A half reduction of Drok, which encodes Rho-kinase, also dominantly enhances zipEbr. This indicates the involvement of the Rho signaling pathway and its effector, Rho-kinase, in the myosin function of adult wing morphogenesis. When the flies are also heterozygous for DMBSE1, wing malformation is significantly suppressed, suggesting that MBS functions antagonistically to the Rho signaling pathway (Mizuno, 2002).
The global cell movements that shape an embryo are driven by intricate changes to the cytoarchitecture of individual cells. In a developing embryo, these changes are controlled by patterning genes that confer cell identity. However, little is known about how patterning genes influence cytoarchitecture to drive changes in cell shape. This paper analyzes the function of the folded gastrulation gene (fog), a known target of the patterning gene twist. Analysis of fog function therefore illuminates a molecular pathway spanning all the way from patterning gene to physical change in cell shape. Secretion of Fog protein is apically polarized, making this the earliest polarized component of a pathway that ultimately drives myosin to the apical side of the cell. fog is both necessary and sufficient to drive apical myosin localization through a mechanism involving activation of myosin contractility with actin. This contractility driven form of localization involves RhoGEF2 and the downstream effector Rho kinase. This distinguishes apical myosin localization from basal myosin localization; the latter does not require actinomyosin contractility or FOG/RhoGEF2/Rho-kinase signaling. Furthermore, once localized apically, myosin continues to contract. The force generated by continued myosin contraction is translated into a flattening and constriction of the cell surface through a tethering of the actinomyosin cytoskeleton to the apical adherens junctions. Therefore, this analysis of fog function provides a direct link from patterning to cell shape change (Dawes-Hoang, 2005).
A myosin-YFP transgene (mYFP-myosin IIDN) was constructed in
which the YFP moiety has replaced the actin-binding motor head domain of the
myosin heavy chain, zipper. Based on equivalent modifications in Dictyostelium, mYFP-myosin IIDN homodimers should completely lack actin binding and contractility, and the 'single headed' wild-type myosin/mYFP-myosin IIDN heterodimers should have severely decreased actin binding and contractility. Consistent with this, it was found that YFP-containing myosin isolated from mYFP-myosin IIDN expressing Drosophila
embryos shows reduced actin binding when compared with wild-type myosin in a
standard spin down assay. However, no dominant-negative activity of this transgene during embryogenesis was detected, presumably because of the high levels of endogenous myosin (Dawes-Hoang, 2005).
To analyze the localization of this mYFP-myosin IIDN, the Gal4 system was used to express the transgene uniformly in embryos that also carry
wild-type copies of zipper. For comparison the following were examined: (1) a fully functional myosin-GFP fusion, in which GFP is fused to the myosin light chain, sqhGFP, and (2) the endogenous myosin II of wild-type embryos. No differences were found between the localization patterns of sqhGFP and
endogenous myosin, and only the endogenous myosin will be referred to (Dawes-Hoang, 2005).
When cells divide during later stages of development, the non-functional
mYFP-myosin IIDN shows a localization similar to endogenous myosin. Both localize to the contractile ring as it forms, constricts and then disappears following the completion of cell cleavage. Similarly, during cellularization,
mYFP-myosin IIDN localizes to the cellularization front in a manner similar to endogenous myosin. As reported for sqhGFP, the
mYFP-myosin IIDN tends to form aggregates in the interior of the
embryo. In time-lapse movies, the aggregates associate with the cellularization front, which 'clears' them from the outer edges of the embryos as cellularization
proceeds, but they do not fully integrate into the regular hexagonal array of
mYFP-myosin IIDN associated with the advancing furrows (Dawes-Hoang, 2005).
The first differences between functional and non-functional myosin are
observed at the onset of gastrulation. Unlike endogenous myosin, mYFP-myosin
IIDN fails to localize apically at the onset of ventral furrow
formation and throughout later stages of apical constriction and invagination. The ability of these cells to undergo normal ventral furrow formation despite a lack of apically localized mYFP-myosin IIDN presumably reflects the
activity of endogenous zipper. Both endogenous myosin and mYFP-myosin
IIDN are lost from the basal side of the invaginating ventral
furrow cells. This basal loss is slightly delayed and patchy for mYFP-myosin
IIDN, but otherwise proceeds normally (Dawes-Hoang, 2005).
The requirement for actin binding and subsequent actin-dependent
contractile activity therefore appears to distinguish two functionally
different modes of myosin localization: an actin-independent mode of
localization during cellularization and cytokinesis, and a second mode during
gastrulation where localization to the apical side of the cell is dependent
upon actin binding/contractility. It is possible that the mYFP-myosin
IIDN is defective in ways other than its ability to interact with
actin. However, equivalent constructs in Dictyostelium do not effect any other
aspects of myosin function, including RLC phosphorylation or filament assembly.
Therefore, although such secondary effects can not be entirely ruled out,
the defects seen are most likely a result of the inability to interact with
actin and at the very least distinguish two different types of myosin
localization to the apical and basal sides of the cell. They also highlight the
potential importance of actin-myosin interaction and contractility as a target
for fog signaling (Dawes-Hoang, 2005).
Proper control of epithelial morphogenesis is vital to development and is often disrupted in disease. After germ band extension, the cells of the Drosophila ventral embryonic epidermis are packed in a two-dimensional polygonal array. Although epithelial cell rearrangements are being studied productively in several tissues, the ventral epidermis is of particular interest as the final cell arrangement is, uniquely, far from equilibrium. Over the course of several hours, a subset of cells within each parasegment adopts a rectilinear configuration and aligns into parallel columns. Live imaging shows that this is accomplished by the shrinkage of select cell interfaces, as three-cell junctions are converted to four-cell junctions. Additionally, it was shown that non-muscle Myosin II and the polarity proteins Discs large (Dlg) and Bazooka are enriched along cell interfaces in a complex but reproducible pattern that suggests their involvement in junctional conversion and cell alignment. Indeed, depletion of Myosin II or dlg disrupts these processes. These results show that tight spatial regulation of actomyosin contractility is required to produce this high-energy arrangement of cells (Simone, 2010).
The phenomenon of conversion of three-cell junctions to four-cell junctions has been observed in various epithelial tissues, such as during convergent extension (CE), in involuting tracheal placodes and developing wing cells. Thus, junctional conversion is a common element in tissue remodeling. However, inspection of these cases also demonstrates that there are aspects unique to each (Simone, 2010).
During CE and tracheal placode involution, two three-cell junctions are converted to a four-cell junction by the elimination of a specific contact. However, this is not what drives the rearrangements that pattern these tissues. Rather, the resulting four-cell junction resolves further into two three-cell junctions through the growth of a new cell contact orthogonal to the original contact. This orthogonal regrowth is what drives the final cell rearrangements (Simone, 2010).
Three- to four-cell junction conversion also occurs among wing hair cells. This state also resolves, but in a manner distinct from that during CE. The four-cell junction resolves back to two three-cell junctions, but now in a random manner that maximizes cell contacts to assemble a hexagonally packed cell sheet. As in CE and tracheal placode involution, it is the last step, the re-establishment of three-cell junctions, that forms the final pattern (Simone, 2010).
The alignment among the denticle field cells contrasts with these cases. Here, it is the initial shift from three- to four-cell junctions that patterns the field. These cells make denticles, and a columnar alignment would be likely to optimize locomotion. To accomplish this, the underlying epithelial cells adopt a state far from equilibrium, as a polygonal array morphs into a rectilinear array of aligned cells. Junctional conversion is one step to accomplish this. Eventually, cells along the columns tend to re-establish three-cell junctions with cells of neighboring columns, but in a manner that maintains columnar cell alignment. Presumably, once four-cell junctions are established, a secondary interaction, perhaps adhesion, becomes engaged along each column boundary to maintain alignment (Simone, 2010).
A general principle emerging from all these tissues is that the conversion from three- to four-cell junctions is central to patterning epithelia. However, there are key tissue-specific outcomes that often reflect whether and how a final re-establishment of junctions occurs. Thus, although there will be similarities between the mechanisms involved, there will also be significant differences. Currently, the analyses conducted differ in each of these cases, and so the comparisons remain incomplete (Simone, 2010).
There are some commonalities between polarity protein localizations among the above cases. During CE, Myosin II is enriched along shrinking contacts, whereas Baz is enriched along stable contacts. During tracheal placode invagination, Myosin II is enriched along both contacts that shrink and some that do not, but there was no report of Baz localization. This study shows that Baz and Dlg and the molecular motor Myosin II are enriched along specific boundaries during alignment. Crucially, both Dlg and Myosin II functionally assist in alignment. However, as is the case for the tissues described above, two questions remain to be addressed: how are these proteins targeted to specific cellular interfaces, and how do they then mediate junctional conversion (Simone, 2010)?
Considering the selection of interfaces within denticle field cells, perhaps a different polarity complex, potentially engaging in competitive exclusion to set up the enrichments, defines each contact. So far, no candidates have been identifed with compelling patterns. For instance, Crb was enriched generally among denticle field cells, but not at a specific interface, whereas aPKC was subtly enriched but only late in alignment, suggesting a subordinate role, if any. Since Baz defines the short, stable contact, whereas Dlg defines the long, stable contact, a polarity protein might be present that is enriched on the shrinking contact and both Baz and Dlg can be excluded. One candidate is Par1, which phosphorylates and excludes Baz and Dlg from membranes in other contexts. Unfortunately, for many proteins, such as these, it has been difficult to deplete embryos of function without totally disrupting the epithelium (Simone, 2010).
Although it is a compelling idea, competitive exclusion is perhaps not a prime candidate for setting up or maintaining enrichments during alignment, as Baz enrichment was not altered in dlg mutants. Similarly, during CE aPKC and Par6 are not required for Myosin II localization. In fact, during C. elegans gastrulation, PAR-3 and Myosin II actually co-localize to ingressing edges. It has been proposed that Myosin II can be templated by prior F-actin enrichment, but this too is not the case during alignment, where F-actin and Myosin become enriched at the same time (Simone, 2010).
Since junctional conversion is a recurring theme, the mechanism by which it occurs is of particular interest. During alignment and tracheal placode invagination, but not during CE, Myosin II is enriched both along contacts that are eliminated and stable contacts. Although Myosin II is required for alignment, just how actomyosin contractility contributes to contact elimination, and how this process is counteracted along Myosin II-enriched stable contacts, are unknown. As during CE, a subtle enrichment on stable junctions is occasionally observed for DE-Cadherin and Armadillo (β-Catenin). With regard to the shrinking contact, one possibility is that Myosin II pulls actin filaments past each other, collapsing the length of the contact, although how this might destabilize Cadherin-based adhesion is unknown. Another possibility is that actomyosin contractility might differentially affect endocytosis and exocytosis. When rates of endocytosis and exocytosis are balanced, interfaces are stably maintained. However, if the equilibrium is shifted towards endocytosis along a certain contact, for instance where contractility is increased, that contact will shrink (Simone, 2010).
It was found that Dlg is enriched along stable Myosin II-enriched contacts and it is hypothesized that Dlg is involved in functionally distinguishing the two types of Myosin II-enriched contact. There are two ways in which Dlg could inhibit Myosin II function where they are co-enriched. First, Dlg might counteract Myosin II activity indirectly by facilitating exocytosis and membrane deposition as it has been suggested to do during cellularization. A bias towards deposition could lead to the apparent stabilization of contacts. Secondly, Dlg might recruit a Myosin II-inhibiting protein, such as Lgl, through one of its PDZ domains (Simone, 2010).
Regardless of how Dlg counteracts Myosin II function, it was surprising that Dlg, a member of the basolateral group of epithelial polarity genes, influences cell-cell relationships apically in the epithelium at this stage. However, recent work has revealed that at about the stage when alignment begins, there is a transfer of basolateral responsibility from the Dlg group to the Yurt/Cora group (Laprise, 2009). That would free Dlg group genes to participate in other polarity events. With this in mind, attempts were made to test whether the basolateral protein Lgl, a negative regulator of Myosin II, would similarly be freed to assist Dlg in alignment. However, lgl germline clones have radically disrupted epithelia and zygotic mutants have persistent maternal contribution (Simone, 2010).
Convergent extension has some contribution from oriented cell divisions and some from junctional conversions. Although oriented cell divisions contribute to CE more in the posterior portion of the embryo, junctional conversions occur for many cells with no spatially reproducible focus for initiation. By contrast, this study found that cell alignment differs between the prospective smooth and denticle fields, and, among denticle field cells, alignment begins along specific boundaries within each parasegment (Simone, 2010).
Among smooth field cells, stretching along the DV axis appears to be the major contributor to alignment. Even though Dlg makes some contribution to alignment here, it does not appear to be enriched on particular interfaces. However, stretching makes a minimal contribution to alignment among denticle field cells. This dramatic distinction between smooth and denticle field cells, coupled to the observation that specific cell columns initiate alignment events, strongly suggests the involvement of positional signals that are limited to, or emanate from, denticle field cells. In addition to starting at the 1/2 and 4/5 boundaries, alignment also initiates from the ventral midline and progresses laterally, eventually covering a third of the ectoderm . These observations raise the possibility that the 1/2 and 4/5 boundaries, that is boundary between cell columns 1 and 2 and the boundary between cell columns 4 and 5, template the other boundaries by propagating a polarity signal from column to column. Candidate signals include Hedgehog signaling across the 1/2 boundary, Notch signaling across the 4/5 boundary and Egfr signaling across either boundary. Differential Egfr activation has been implicated in the conversion of three- to four-cell junctions in the tracheal placode, but how differential Egfr signaling translates into junctional conversion remains unknown (Simone, 2010).
Four-cell junctions have been reported in several tissues. Their appearance during development might be a hallmark of cellular realignments integral to the patterning of that tissue. During development, signaling often occurs across smooth, defined boundaries, such as the DV compartment boundary in wing and segment boundaries in leg. In fact, the tarsus/pretarsus boundary is composed of four-cell junctions that are strikingly reminiscent of those reported in this study (Simone, 2010).
A more closely studied example is the actomyosin enrichment along the boundary between the dorsal and ventral cells of the wing imaginal disk. Cells along this boundary form a smooth, lineage-restricted interface and do not intermix. The actomyosin 'fence' was implicated in maintaining this lineage-restricted signaling boundary. Notch signaling across the DV boundary enriches it for F-actin and Myosin II and depletes it for Baz. Although a role for Baz was not tested, when Myosin II contractility is compromised, the integrity of the lineage-restricted boundary is disrupted. Under these conditions, the boundary is jagged and some cells can now mix. This work would now suggest that the normal function of the actomyosin fence is to convert three- to four-cell junctions along the boundary. The failure of this would generate interdigitating cells, resulting in a non-aligned, jagged boundary, which would be the first step to cell mixing. By keeping cells aligned at the boundary, Myosin II activity might prevent them from interdigitating and support compartment integrity (Simone, 2010).
Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum). A subset of the trichome-producing notum cells differentiate as 'tendon cells,' serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin, this study shows that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems. The chas phenotype arises during normal shortening of the IFMs and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechanoresponse of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by one or more interactions with other tissues during development and organogenesis influences the maintenance of its shape and PCP features (Olguí, 2011).
chascon (chas) was identified in a gain-of-function (GOF) screen for genes affecting wing morphogenesis. chas encodes two isoforms containing multiple predicted Src homology and PDZ domain binding sites but no catalytic or conserved protein interaction domains, suggesting an adaptor or scaffold function. Expression of either chas isoform in the posterior compartment of wing discs resulted in defects in cellular polarity, misplaced actin hair formation, and loss of asymmetric Flamingo (Fmi) and Frizzled (Fz) localization, suggesting that Chas can disturb planar cell polarity (PCP) establishment and the localization of core Fz/PCP components. chas GOF also displayed PCP defects in other tissues (e.g., ommatidial under-rotation in the eye). In the notum, Chas expression under pannier-GAL4 (pnrG4) expressed centrally in thorax resulted in orientation and morphologic defects of bristles and trichomes (Olguí, 2011).
To ask whether chas is necessary for PCP and/or morphogenesis, UAS-double stranded RNA (UAS-dsRNA) constructs targeting chasA (chasAiR) were generated, and a public UAS-dsRNA to a common exon (chasABiR) was used. pnrG4-driven chasABiR or chasAiR resulted in notum bristles and trichomes pointing to the midline and multiple trichomes per cell. Epidermal indentations were observed in most anterior notum regions. Because both dsRNAs showed indistinguishable phenotypes, chasABiR was used for subsequent studies. To confirm this, a chas loss-of-function (LOF) allele (chas1) was generated via FLP-FRT deletion method. It lacked both 5' untranslated regions and start codons. chas1 animals were viable and fertile and displayed notal PCP and indentation defects similar to pnrG4>chasABiR; no defects were observed in other tissues in chas1 or chasABiR animals). chas1/Df, tubG4>chasABiR, and chas1 animals displayed similar defects, suggesting that chas1 is a strong LOF or null allele. These LOF conditions exhibited weaker phenotypes than regional dsRNA gene knockdown or chas1 clones, suggesting that differences in chas levels between mutant and adjacent wild-type tissue enhance polarity defects. chas1 defects were rescued by either Chas isoform in clones, confirming chas1 specificity. Furthermore, chas1 clones or regional knockdown influenced nonautonomously the orientation of wild-type cells, similar to fz- clones (Olguí, 2011).
In the notum, Fz/PCP signaling is required early to orient asymmetric divisions of sensory organ precursors (SOPs) and later to polarize cellular trichomes and bristle cells along the body axis. chas LOF did not affect the orientation of asymmetric SOP divisions. Thus, chas appeared to act later, possibly interacting with Fz/PCP signaling during PCP establishment in the notum epidermis. The epistatic relationships between chas and Fz/PCP core members was examined. Strikingly, chas1;fzp21 double mutants displayed a novel phenotype, with bristles and trichomes reoriented toward the anterior. These data suggested that chas and Fz/PCP signaling work in parallel to polarize the notum epidermis (Olguí, 2011).
As in other organs, the first signs of PCP in notal epidermal cells are asymmetric localizations of Fz/PCP core components, evident from 24 hAPF (hours after puparium formation) onward. The localization and levels of Fz and Fmi were not affected in pnrG4>chasABiR animals at 30 hAPF , further supporting the notion that chas and Fz/PCP signaling act in parallel to promote PCP on the notum (Olguí, 2011).
The fat (ft)/dachsous (ds) system controls PCP establishment in parallel to Fz/PCP signaling, so whether chas works through this system was tested. The nota of strong ds combinations (ds38k/dsUA071) or pnrG4-driven ftiR, dsiR, display bristles that are slightly oriented laterally, likely related to their mild thorax cleft and shape and size defects (consistent with one or more roles in tissue growth and cell behavior). Overall, the cleft and shape phenotypes associated with ds/ft LOF or GOF are not altered in chas LOF conditions, nor is the chas LOF phenotype, suggesting that chas and the Fat/Ds system act independently (Olguí, 2011).
chas LOF does not disturb asymmetric localizations of Fz/PCP components but still influences coordinated cell orientations in the notum. In chas LOF, bristle and socket cells reoriented polarity toward the midline between 30 and 32 hAPF, and epithelia displayed local cellular anterior-posterior contractions at the level of anterior dorsocentral macrochaetae. This suggested that chas modulates the epithelial behavior at this stage to maintain PCP (Olguí, 2011).
Most trichome-producing cells of the notum differentiate as “tendon cells,” serving as attachment sites for indirect flight muscles (IFMs). These cells form domains defined by stripe expression, which promotes tendon fate. IFMs start to shorten after 20 hAPF, generating mechanical strain at attachment sites. This is evident in dumpy (dp) mutants, in which notum epithelia are pulled inward, resulting in epidermal indentations similar to chas mutants. Dp, a transmembrane cuticular protein, maintains the tension at muscle attachment sites by providing an anchor for cells to attach to the exoskeleton or by modulating the cuticular matrix composition. Double homozygous chas1;dpov1 animals showed stronger indentations and cell-orientation defects than individual mutants, suggesting that chas is required to modulate mechanical properties of epidermal tendon cells during IFM shortening (Olguí, 2011).
By 30 hAPF, chas expression was detected (via chasNP0733G4-driven CD8-GFP staining) in tendon cells and was absent from socket and bristle cells. This was consistent with the chas LOF phenotype domains, supporting the genetic data showing that chas affects bristle polarity nonautonomously (Olguí, 2011).
Subcellularly, Myc-tagged Chas localized at the apical cortex of tendon cells, colocalizing with E-cadherin at adherens junctions and in tendon cell processes and colocalizing with βPS-integrin at myotendinous junctions, consistent with a role of Chas linking the myotendinous junction and apical cortex (Olguí, 2011).
Next, the notum epithelium, tendon cell processes, and IFMs of live pupae expressing constitutively GFP fused to a fragment of the actin binding protein moesin (sGMCA) and specifically in the notum epithelium were monitored by driving the expression of membrane-tethered RFP (CD8-RFP) with pnrG4. Anterior indentations of pnrG4>chasABiR were detected by 27-28 hAPF and were very obvious by 32 hAPF, coinciding with attachment domains of ventral dorsolongitudinal muscles (DLMs), which are highly contracted at this stage. Accordingly, the strongest bristle orientation defects in chasABiR coincided with attachments of dorsal DLMs. Tendon cell processes connecting DLMs in chasABiR did not elongate at 28-32 hAPF, in contrast to wild-type, suggesting that tendons with reduced Chas levels respond differently to pulling stimuli. At 34-35 hAPF, many cells formed multiple trichomes and oriented toward the midline. This demonstrated that chas LOF defects arise during IFM shortening at DLM attachment domains and associate with elongation defects of epidermal tendon cells. The epithelial contraction of pnrG4>chasABiR/ubi-DE-cadh-GFP coincided with the posterior edges of the most dorsal DLM attachment domains, suggesting that pulling forces generated by muscle shortening alter the shape of these cells (Olguí, 2011).
To confirm that IFMs were causing the cellular strain and defects in chas LOF cells, the strain was eliminated by ablating IFMs genetically via expressing activated Notch (Nintra) in muscle progenitors. In this background, cellular orientation of y, chas1 clones was almost completely rescued or suppressed. These data suggest that, in tendons, Chas balances pulling forces generated by IFM shortening to maintain the shape and PCP of the notal epithelium (Olguí, 2011).
The epidermal indentations in chas LOF arise at attachment sites of medial and ventral DLMs, which form more perpendicular angles with the epithelial plane than dorsal DLMs. This characteristic, and time-lapse studies, suggested that the planar component of pulling forces transmitted through oblique attachments of dorsal DLMs impact directionally on the epithelial plane, inducing a change of cellular orientation. The mechanical force, not compensated in chas mutant cells, is transmitted laterally, influencing mechanically the reorientation of neighboring wild-type cells (Olguí, 2011).
Of note, trichomes still localize to posterior cell edges in chas LOF, consistent with Fz/PCP signaling still determining the position of trichome formation. When interfering with both Fz/PCP signaling and chas, the mechanical stress influences polarity more strongly, leading to cellular orientation inverted anteriorly (Olguí, 2011).
To define chas function, genes displaying similar phenotypes were sought within a genome-wide RNA interference screen. Knock down of jitterbug (jbug) phenocopies all aspects of chas LOF. Coexpression of jbugiR and chasABiR (under pnrGal4) showed an enhancement of polarity and indentation defects. Jbug, along with cheerio, encodes the two Drosophila filamin orthologs. Filamins form homodimers that crosslink actin filaments to confer mechanical stability to membranes. They also work as molecular scaffolds linking transmembrane receptors with cytosolic signaling proteins and actin filaments (Olguí, 2011).
Whereas coexpression of chasABiR and jbugiR resulted in stronger defects than each dsRNA separately, coexpression of wild-type JbugL isoform with chasABiR rescued chas LOF phenotypes. In contrast, Chas overexpression did not rescue jbugiR, suggesting that jbug/Filamin acts downstream of chas. Molecularly, Chas and Jbug proteins coimmunoprecipitated and colocalized with actin filaments in S2R+ cells and in tendon cells, suggesting that they participate in a molecular complex associated with actin filaments. These data suggest that chas acts through jbug to maintain the shape and PCP of the notum (Olguí, 2011).
Drosophila embryonic tendon cells contain prominent arrays of F-actin and MyosinII (MyoII), connecting the apical cortex with myotendinous junctions and maintaining the integrity of tendon cells upon stretching. Rheological experiments have shown that actin networks containing FilaminA display enhanced elasticity. Accordingly, chas and jbug could modulate elastic properties of tendon cells either by regulating MyoII activity and/or localization or by contributing in parallel to the formation and/or behavior of apicobasal F-actin networks (Olguí, 2011).
Localization of Zipper (Zip), Drosophila MyoII, and Shortstop (Shot), a plakin that organizes apicobasal microtubule (MT) networks in embryonic tendons, were analyzed. Zip colocalizes partially with F-actin and Shot at tendon processes. chasABiR did not disturb Zip or Shot localization and levels, suggesting that it is not required for F-actin/MyoII arrays or MT/Shot networks (Olguí, 2011).
Zip function was knocked at different pupal development stages via a dominant-negative form of Zip (DN-zip) under the control of pnrGal4 and tubGal80ts(to bypass its cytokinesis role). DN-zip expression from 0 hAPF resulted in bristle orientation defects and epidermal indentations. DN-zip or chasABiR expression at 14 hAPF (18°C) resulted in weak orientation defects. Strikingly, coexpression of chasABiR and DN-zip from 14 hAPF caused strong PCP phenotypes and epidermal indentations. chas LOF phenotypes were not caused by diminished Zip activity, because expression of constitutively active Drosophila myosin regulatory light chain did not rescue chasABiR-associated defects. This suggests that (1) zip/MyoII and chas-jbug/filamin cooperate to confer tendon cells with the ability to adapt to pulling forces generated by IFM shortening, and that (2) the levels and/or activity of filamin and MyoII regulate viscoelastic properties of actin networks that run from myotendinous junctions to the apical cortex and/or the actin arrays present at the apical cortex (Olguí, 2011).
The notum epithelium develops in tight association with IFMs. IFMs are directly attached to epithelial 'tendon cells' and generate a pulling force over these cells during PCP establishment. Mechanical properties of tendon cells and their interaction with muscles and the cuticle need to be finely tuned to maintain their shape and polarity. Chas localizes from myotendinous junctions to the apical cortex and is upregulated during their maturation. Its function is likely to regulate filamin activity/or localization to adjust elastic properties of tendon cells during IFM shortening. Because diminished MyoII activity mimics jbug or chas LOFs and tendon process length of chasABiR is not altered, a role of Chas/filamin in regulating elastic properties of F-actin arrays is favored over length adaptation through active polymerization (Olguí, 2011).
Mutations in filamins are associated with human diseases, causing neuronal migration defects, bone and cartilage malformations, myopathies, and vascular defects, among others. The role of filamin in organogenesis and disease progression may be in part related to its structural role modulating the viscoelastic properties of actin filaments. The observations made in this study contribute to the understanding of the role or roles of filamins as regulators of viscoelastic cellular properties during intertissue interactions. How does Chas modulate filamin function? Because Chas does not present any catalytic domains, the idea is favored that it acts as an adaptor for filamin. Thus, lack of chas may affect the composition and/or architecture of tendon actin networks organized by filamin (Olguí, 2011).
In conclusion, this paper proposes that zip/MyoII, chas, and jbug/filamin have complementary roles in the apicobasal mechanical properties of tendon cells, influencing cytoskeletal dynamics at the apical cortex. Thus, alterations in the ability to adjust to tensile forces operating in the apicobasal direction disrupt the epithelial shape and shift the site of trichome formation, their number per cell, and their planar orientation. These data reveal that interactions of two tissues and the ability of cells to adapt mechanically to them during morphogenesis are fundamental to maintain the shape and PCP of an epithelium (Olguí, 2011).
Nuclei are precisely positioned within all cells, and mispositioned nuclei are a hallmark of many muscle diseases. Myonuclear positioning is dependent on Kinesin and Dynein, but interactions between these motor proteins and their mechanisms of action are unclear. This study found that in developing Drosophila muscles, Dynein and Kinesin work together to move nuclei in a single direction by two separate mechanisms that are spatially segregated. First, the two motors work together in a sequential pathway that acts from the cell cortex at the muscle poles. This mechanism requires Kinesin-dependent localization of Dynein to cell cortex near the muscle pole. From this location Dynein can pull microtubule minus-ends and the attached myonuclei toward the muscle pole. Second, the motors exert forces directly on individual nuclei independently of the cortical pathway. However, the activities of the two motors on the nucleus are polarized relative to the direction of myonuclear translocation: Kinesin acts at the leading edge of the nucleus, whereas Dynein acts at the lagging edge of the nucleus. Consistent with the activities of Kinesin and Dynein being polarized on the nucleus, nuclei rarely change direction, and those that do, reorient to maintain the same leading edge. Conversely, nuclei in both Kinesin and Dynein mutant embryos change direction more often and do not maintain the same leading edge when changing directions. These data implicate Kinesin and Dynein in two distinct and independently regulated mechanisms of moving myonuclei, which together maximize the ability of myonuclei to achieve their proper localizations within the constraints imposed by embryonic development (Folker, 2013).
Myonuclei in the developing Drosophila embryo undergo several discrete nuclear movements governed by distinct mechanisms that have been best characterized in the lateral transverse (LT) muscles. At the completion of fusion, all of the myonuclei within the muscles reside in a single cluster near the ventral pole of each LT muscle. Shortly after fusion [stage 14, 10:20-11:20 hours after egg laying (AEL)] this single cluster separates into two clusters -- one ventral and one dorsal. During stage 15 (11:20-13 hours AEL) and stage 16 (13-16 hours AEL) these clusters move toward their respective poles. Finally, during stage 17 (16-20 hours AEL), the myonuclei become evenly spaced throughout the muscle. These different myonuclear movements appear to be mechanistically distinct. For example, embryos that are mutant for the MT-associated protein Ensconsin fail to separate the single post-fusion nuclear cluster into distinct dorsal and ventral clusters. However, Dynein and Kinesin appear to be dispensable for the initial separation of the single post-fusion cluster into two, but are required to move the individual clusters toward the muscle poles. More specifically, Dynein localized to the muscle pole pulls MT minus-ends and attached myonuclei toward the muscle pole. The role of Kinesin is less clear, and whether Dynein and Kinesin drive distinct aspects of myonuclear movement or work within a common pathway is not known (Folker, 2013).
This study examined the translocation of myonuclei toward the muscle poles during stage 15 (11:20-13 hours AEL); in developing Drosophila muscles, Dynein and Kinesin were found to work together to move nuclei in a single direction. This contrasts with other systems, such as interkinetic nuclear migration in mammalian brains and the C. elegans hypodermis, where Kinesin and Dynein move nuclei in opposing directions. As part of this analysis, it was found that MTs are bidirectionally oriented throughout most of the developing muscle, unlike in these other in vivo contexts, perhaps promoting the cooperation of the two motors. Furthermore, Dynein and Kinesin cooperate to move nuclei by two separate mechanisms that are spatially segregated. First, the two motors work together in a pathway that acts from the cell cortex at the muscle poles. This mechanism requires Kinesin-dependent localization of Dynein to the cell cortex near the muscle pole. At this location, Dynein can pull MT minus-ends and the attached myonuclei toward the muscle pole. Second, the motors exert forces directly on individual nuclei independently of Dynein activity from the cortex, consistent with recent reports in cell culture. However, this study demonstrated that the activities of the two motors on the nucleus are polarized relative to the direction of myonuclear translocation: Kinesin acts at the leading edge of the nucleus, whereas Dynein acts at the lagging edge of the nucleus. Consistent with the activities of Kinesin and Dynein being polarized on the nucleus, nuclei rarely change directions, and those that do, reorient to maintain the same leading edge. Conversely, nuclei in both Kinesin and Dynein mutant embryos change direction more often and do not maintain the same leading edge when changing directions. It is hypothesized that these functions increase myonuclear translocation efficiency in dense in vivo environments and enable myonuclei to reach their proper position within the time constraints of development (Folker, 2013).
Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in MYH7 encoding slow/beta-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the alpha-helical rod domain of MYH7 are mainly associated with Laing distal myopathy. A Drosophila model of Laing distal myopathy was developed by genomic engineering of the Drosophila Mhc locus. Flies expressing only Mhc(K1728del) in indirect flight and jump muscles, and heterozygous Mhc(K1728del) animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of Mhc(K1728del) mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous Mhc(K1728del) larvae also demonstrated structural and functional impairments in heart muscle. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with Mhc(K1728del) were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in Drosophila recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. These observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy (Dahl-Halvarsson, 2018).
Missense mutations in the MYH3 gene encoding myosin heavy chain-embryonic (MyHC-embryonic) have been reported to cause two skeletal muscle contracture syndromes, Freeman Sheldon Syndrome (FSS) and Sheldon Hall Syndrome (SHS). Two residues in MyHC-embryonic that are most frequently mutated, leading to FSS, R672 and T178, are evolutionarily conserved across myosin heavy chains in vertebrates and Drosophila. Transgenic Drosophila were generated expressing myosin heavy chain (Mhc) transgenes with the FSS mutations and the effect of their expression on Drosophila muscle structure and function was characterized. The results indicate that expressing these mutant Mhc transgenes lead to structural abnormalities in the muscle, which increase in severity with age and muscle use. Flies expressing the FSS mutant Mhc transgenes in the muscle exhibit shortening of the inter-Z disc distance of sarcomeres, reduction in the Z-disc width, aberrant deposition of Z-disc proteins, and muscle fiber splitting. The ATPase activity of the three FSS mutant MHC proteins are reduced compared to wild type MHC, with the most severe reduction observed in the T178I mutation. Structurally, the FSS mutations occur close to the ATP binding pocket, disrupting the ATPase activity of the protein. Functionally, expression of the FSS mutant Mhc transgenes in muscle lead to significantly reduced climbing capability in adult flies. Thus, these findings indicate that the FSS contracture syndrome mutations lead to muscle structural defects and functional deficits in Drosophila, possibly mediated by the reduced ATPase activity of the mutant MHC proteins (Das, 2019).
The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. This study used the Drosophila embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, it was shown that engineering human MYH9-related disease mutations into Drosophila myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the Drosophila myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis (Kasza, 2019).
zipper
:
Biological Overview
| Evolutionary Homologs part 1/3
| Evolutionary Homologs part 2/3
| Evolutionary Homologs part 3/3
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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