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).
Mammalian Rho-kinase/ROK alpha, one of the targets of Rho, has been shown to bind to Rho in GTP-bound form and to phosphorylate the myosin light chain (MLC) and the myosin binding subunit (MBS: see Drosophila Myosin binding subunit) of myosin phosphatase, resulting in the activation of myosin. Thus, Rho-kinase/ROK alpha has been suggested to play essential roles in the formation of stress fibers and focal adhesions. A two-hybrid analysis demonstrates that Rok interacts with the GTP-bound form of the Drosophila Rho1 at the conserved Rho-binding site. Rok can phosphorylate MLC and MBS, preferable substrates for bovine Rho-kinase, in vitro. These results suggest that Rok is an effector of Rho1 (Mizuno, 1999).
To test the physical interaction between Rok and various Rho-GTPases, a pull-down assay using GST-GTPase fusion proteins and in vitro translated Rok was used. Rok binds to the constitutively active form of Drosophila RhoA, but not to constitutively active Rac1 or Cdc42. Mutating a key amino acid within the effector-binding domain (T37A) abolishes the interaction with Rok. These results suggest that Rok is an effector specific for Drosophila RhoA (Winter, 2001).
Studies in mammalian cells have identified several downstream substrates for Rho-kinase/ROCK (Amano, 2000). In particular, Rho-kinase regulates the phosphorylation of the nonmuscle myosin regulatory light chain (MRLC) primarily at Ser-19 and secondarily at the adjacent Thr-18 (Amano, 1996; Kimura, 1996). Phosphorylation of MRLC at these sites results in a conformational change that allows myosin II to form filaments and increases its actin-dependent ATPase activity (Winter, 2001).
The amino acid sequence around the phosphorylation site of MRLC is highly conserved between mammalian MRLC and the Drosophila homolog, encoded by spaghetti squash (sqh). Therefore the phosphorylation of the Drosophila MRLC was assessed using an antibody that recognizes mammalian MRLC only when Ser-19 is phosphorylated. Immunoblot analysis shows that this antibody specifically recognizes phosphorylated Sqh in larval extracts -- the single ~20 kDa band in wild-type extract is absent both in extracts of a sqh null, and when wild-type extract is treated with phosphatase. While phosphorylated Sqh is detectable in Drok2 mutant extracts, its level is greatly reduced, whereas the Sqh protein level is not affected in Drok2 mutants. Previous work with bovine Rho-kinase has established that expression of the N-terminal catalytic domain gives rise to a constitutively active kinase (Amano, 1997). Raising the level of Rok activity in vivo by transient expression of the catalytic domain of Rok (Drok-CAT) results in elevated phosphorylation of Sqh as compared to controls in which a kinase-dead form (Drok-CAT-KG) was used. Taken together, these experiments indicate that Rok is required for maintaining the proper level of MRLC phosphorylation in vivo, and that such regulation depends on its kinase activity (Winter, 2001).
The effect of loss of Rok function on MRLC phosphorylation was examined at the cellular level. In wild-type wing cells, phospho-MRLC is enriched at the cortex of the pupal wing cells, whereas in Drok2 mutant cells this perimembrane staining is reduced or absent. Thus, rok is cell autonomously required for maintaining the level of cortical phospho-MRLC in the pupal wing (Winter, 2001).
The next question considered was whether MRLC/Sqh is an effector for Rok in regulating hair number in response to Fz/Dsh signaling. Use was made of a series of mutant sqh transgenes 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. These sqh transgenes are under control of the endogenous promoter and are expressed at levels similar to the native protein. Remarkably, whereas 100% of Drok2 hemizygous animals die before the wandering third instar stage, introducing one copy of a sqh transgene carrying the E20E21 double mutation (mimicking phosphorylation on both sites) results in 4% hemizygous Drok2 survival to adulthood. Likewise, one copy of an analogous transgene expressing SqhE21 also results in Drok2 hemizygotes surviving to adulthood (albeit a lower percentage), with a large fraction surviving to late-stage pupae. No rescue was observed when transgenes expressing the alanine substituted forms (SqhA20A21 or SqhA21) were introduced into the Drok2 background. These observations support the notion that MRLC is a key target (either directly or indirectly) for Rok kinase in vivo, since mimicking its phosphorylation, even in an unregulated fashion, partially rescues Drok2 organismal lethality (Winter, 2001).
Moreover, the multiple hair defect resulting from rok loss of function is almost completely suppressed by the presence of the sqhE20E21 transgene in the rescued adults. Taken together with the modulation of MRLC phosphorylation by Rok, these results demonstrate that the regulation of MRLC phosphorylation is a principal function of Rok in regulating F-actin prehair number (Winter, 2001).
Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference has identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. RhoGAP inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by RhoGAP. Local regulation of RhoGAP could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of RhoGAP by integrin and Src, both implicated in neural plasticity (Billuart, 2001).
Drosophila Rho-kinase (Drok), an effector of RhoA, is essential for transducing signals to the actin cytoskeleton in wing cells (Winter, 2001). Since the effector domain mutant analysis of RhoA suggests that a cytoskeletal pathway is important for axon retraction, tests were performed to see if the Drok pathway is involved. Carboxy-terminal truncation of mammalian Rho-kinase/ROCK results in its constitutive activation. Expression of an analogous activated form (Drok-CAT; Winter, 2001) in MB neurons led to truncated dorsal lobes similar to the phenotypes of p190 RNAi and weak RhoA activation. A presumptive kinase-dead point mutation (Drok-CAT.KG; Winter, 2001) has no effect, indicating that Drok signaling is dependent on its kinase activity. Developmental studies indicate that the Drok-CAT phenotypes also results from axon retraction, as does the p190 RNAi phenotype (Billuart, 2001).
Neuroblast clones homozygous for Drok2 (Winter, 2001) do not show apparent defects in cell proliferation, because the adult clones contain dorsal axon lobes contributed by later born neurons. Close examination of Drok2 neuroblast clones reveals that 10 of 17 contain at least one axon that extends significantly further than the heterozygous neurons within the same MB. Although this phenotype is subtle, it is not seen in 19 control clones, the parental chromosome for the Drok2 mutant, nor in many other genotypes studied. Thus, it is concluded that Drok is required to limit dorsal axon extension (Billuart, 2001).
Biochemical and genetic evidence indicates that a key output for Drok signaling in vivo is the regulation of phosphorylation of myosin regulatory light chain (MRLC) encoded by spaghetti squash (sqh) (Winter, 2001). To test if endogenous MRLC is part of the axon retraction pathway regulated by p190, genetic interaction experiments were performed by reducing the dose of endogenous sqh in the context of the p190 dsRNA expression. Marked suppression of the phenotype was observed in flies heterozygous for a null mutation of sqh (sqhAX3). In contrast, expression of a phosphomimetic mutant, Sqh-E20E21, markedly enhanced the p190 phenotype, whereas analogous expression of a nonphosphorylable form (Sqh-A21) had no effect. Further, truncation of the medial lobe was frequently observed when Sqh-E20E21 was expressed with the intermediate p190 RNAi line. This is evident from the FasII staining, showing that the medial ß axons (strongly FasII positive) only extend a fraction of the length of the medial lobe. This phenotype was only observed in the strongest p190 RNAi lines, never in the intermediate line alone. Taken together, these results strongly suggest that Drok and phosphorylation of Drosophila MRLC participate in mediating axon retraction as a result of p190 inactivation (Billuart, 2001).
Drosophila Rho-kinase associates with the GTP-bound Drosophila Rho1 and can phosphorylate the vertebrate MRLC and myosin binding subunit (MBS). Rho-kinase is involved in the establishment of planar polarity in adult structures such as the compound eye and wing. Drosophila Myosin-binding subunit (MBS) has been characterized to elucidate the functions of myosin phosphatase in morphogenesis, revealing that MBS functions in dorsal closure and that it acts antagonistically to the Rho signaling cascade and its effector Rho-kinase (Mizuno, 2002).
Drosophila Rho-kinase physically interacts with Rho1 in the GTP form and phosphorylates the vertebrate MBS in vitro. The sequence at the putative phosphorylation site of vertebrate MBS is well conserved in Drosophila MBS, and tests were performed to see whether DRho-kinase phosphorylates MBS in vitro. The GST-fused DMBS-L was expressed and purified from Escherichia coli, and was found to be phosphorylated by wild-type Drosophila Rho-kinase but not by kinase-dead DRho-kinaseK116A (Mizuno, 2002).
Thr594 may correspond to the major phosphorylation site in vertebrate MBS. The threonine residue was replaced with an alanine, and this recombinant Drosophila MBS was used as a substrate. The level of phosphorylation was significantly reduced, indicating that Thr594 is the major site phosphorylated by Drosophila Rho-kinase. It has been reported that mammalian MBS is phosphorylated at several sites by Rho-kinase (Kawano, 1999), and there presumably are other phosphorylation sites in Drosophila MBS as well (Mizuno, 2002).
Drosophila Rho-kinase is thought to be responsible for the inactivation of myosin phosphatase through phosphorylation of MBS. If this inactivation turns out to have a considerable effect on the levels of phosphorylated MRLC, it can be expected that the phenotypes in the MBS mutant embryos and in the embryos overexpressing DRho-kinase would be similar. When wild-type Rho-kinase was expressed with the arm-GAL4 driver, about 80% of the embryos failed to hatch. A similar result was obtained with the 69B-GAL4 driver that induces the target gene in the ectoderm. Most of the lethal embryos show a dorsal open or dorsal hole phenotype, and the pattern of dorsal hairs is disturbed along the dorsal midline in the remaining embryos as observed in the MBS mutant embryos. Examination of the cell shape and the F-actin distribution reveals the same aberrations as those in the MBS mutant embryos (Mizuno, 2002).
Apical constriction is a cell shape change that promotes epithelial bending. Activation of nonmuscle myosin II (Myo-II) by kinases such as Rho-associated kinase (Rok) is important to generate contractile force during apical constriction. Cycles of Myo-II assembly and disassembly, or pulses, are associated with apical constriction during Drosophila melanogaster gastrulation. It is not understood whether Myo-II phosphoregulation organizes contractile pulses or whether pulses are important for tissue morphogenesis. This study shows that Myo-II pulses are associated with pulses of apical Rok. Mutants that mimic Myo-II light chain phosphorylation or depletion of myosin phosphatase inhibit Myo-II contractile pulses, disrupting both actomyosin coalescence into apical foci and cycles of Myo-II assembly/disassembly. Thus, coupling dynamic Myo-II phosphorylation to upstream signals organizes contractile Myo-II pulses in both space and time. Mutants that mimic Myo-II phosphorylation undergo continuous, rather than incremental, apical constriction. These mutants fail to maintain intercellular actomyosin network connections during tissue invagination, suggesting that Myo-II pulses are required for tissue integrity during morphogenesis (Vasquez, 2014).
Recent studies demonstrated that pulsatile Myo-II contractions drive diverse morphogenetic processes, including Caenorhabditis elegans embryo polarization, Drosophila gastrulation, dorsal closure, germband extension, oocyte elongation, and Xenopus laevis convergent extension. Although Rok, and likely Myo-II activation via Rok phosphorylation, is required for contraction, it was not clear whether Myo-II activation simply regulates cortical Myo-II levels or whether coupling between Myo-II activity and its regulators organizes contractile pulses in space and time. Furthermore, why cells undergo pulsatile, rather than continuous, contraction to drive tissue morphogenesis was unknown. This study was able to answer these questions by visualizing the consequences of uncoupling Myo-II activation from upstream signaling pathways on cell and tissue dynamics (Vasquez, 2014).
This study identified dynamic Myo-II phosphorylation as a key mechanism that regulates contractile pulses. Myo-II pulses are associated with dynamic medioapical Rok foci and myosin phosphatase. In addition, the phosphomimetic sqh-AE and sqh-EE mutants, which exhibited constitutive cytoplasmic Myo-II assembly in vivo, exhibited defects in two properties of contractile pulses. First, phosphomimetic mutants did not initially condense apical Myo-II or F-actin into medioapical foci, resulting in Myo-II accumulation across the apical domain and thus a defect in Myo-II radial cell polarity. Second, phosphomimetic mutants continuously accumulated Myo-II in the apical cortex, lacking clear cycles of Myo-II remodeling that are observed in wild-type embryos. Although the phosphomimetic alleles are predicted to partially activate the Myo-II motor’s ATPase activity compared with normal phosphorylation, the similarity of the Myosin binding subunit (MBS) (see Lee, 2004) knockdown phenotype suggests that the changes in Myo-II organization and dynamics in phosphomimetic mutants reflect defects in the control over Myo-II dynamics rather than a reduction in motor activity. The consequence of persistent Myo-II assembly across the apical surface in phosphomimetic mutants and MBS knockdown is a more continuous, rather than incremental, apical constriction, demonstrating that pulsatile cell shape change results from temporal and spatial regulation of Myo-II activity via a balance between kinase (Rok) and phosphatase (myosin phosphatase) activity (Vasquez, 2014).
Mutants that decrease Myo-II phosphorylation affected contractile pulses in a manner that was distinct from the phosphomimetic alleles. Both the sqh-AA and the sqh-TA mutants exhibited Myo-II assembly into apical foci, potentially mediated by phosphorylation of low levels of endogenous Sqh or phosphorylation of threonine-20, respectively. For the sqh-TA mutant, Myo-II assembly was correlated with constriction, suggesting that Myo-II motor activity is not rate limiting to initiate a contractile pulse. However, Myo-II foci in sqh-TA and sqh-AA mutants were not efficiently remodeled after assembly and coalescence. The persistence of cortical Myo-II foci in sqh-AA and sqh-TA mutants was surprising given that rok mutants and injection of Rok inhibitor reduce cortical localization of Myo-II. One explanation is that high levels of Myo-II activity induce actomyosin turnover and thus could be required to remodel the actomyosin network after contraction. Alternatively, apical recruitment of myosin phosphatase or proteins that negatively regulate Rok could depend on Myo-II phosphorylation or actomyosin contraction. Although future work is needed to address the role of Myo-II motor activity in contractile pulses, the phenotypes of alleles that constitutively reduce phosphorylation further suggest that cycling between high and low phosphorylation states is required for proper Myo-II pulses (Vasquez, 2014).
A model is proposed for contractile pulses in the ventral furrow where, in combination with unknown cortical cues that apically localize Myo-II, local pulses of apical Rok activity within the medioapical cortex polarize Myo-II assembly and coalescence. Rok foci could polarize actomyosin condensation by generating an intracellular gradient of minifilament assembly and tension that results in inward centripetal actomyosin network flow. In addition, local Myo-II activation by Rok foci combined with broader myosin phosphatase activity throughout the apical cytoplasm could generate a gradient of Myo-II turnover that will concentrate Myo-II into medioapical foci. MBS is required to restrict phosphorylated Myo-II to specific cell–cell interfaces during dorsal closure, demonstrating that the balance between Myo-II kinases and phosphatase can generate spatial patterns of Myo-II activation in epithelial cells. Myo-II remodeling after coalescence could result from local decreases in Rok activity and enrichment of apical myosin phosphatase with Myo-II structures. Thus, coupling Myo-II activation to dynamic signals that regulate Myo-II phosphorylation organizes contractile pulses in space and time to drive incremental apical constriction (Vasquez, 2014).
Polarized actomyosin contraction, pulses, and flows generate force and organize the actin cortex in a variety of cellular and developmental. In contrast to the ratchet-like constriction of ventral furrow cells, some cell types undergo extended periods of actomyosin pulsing and area fluctuations without net reduction in area. Furthermore, directional rearrangement of cell contacts, such as during convergent extension in the Drosophila germband, can be achieved through planar polarized accumulation of junctional Rok and Myo-II in conjunction with planar polarized medioapical actomyosin flows. Modulating the spatial and temporal regulation of Myo-II phosphorylation and dephosphorylation provides a possible mechanism to tune contractile dynamics and organization to generate diverse cell shape changes. Consistent with this organizational role, phosphomimetic RLC mutants also disrupt the planar polarized localization of junctional Myo-II in the Drosophila germband. Thus, it will be important to define the principles that control Myo-II activity and dynamics and how tuning Myo-II dynamics impacts force generation and tissue movement (Vasquez, 2014).
Myo-II phosphomutants resulted in a more continuous apical Myo-II assembly and apical constriction, enabling investigation of the role of pulsation during tissue morphogenesis. Continuous Myo-II assembly and contraction in the sqh-AE mutant resulted in a slower mean rate of apical constriction and thus delayed tissue invagination. This delay suggested that pulsing might be important for the efficiency of apical constriction. However, phosphomimetic mutants might not fully recapitulate the ATPase activity of phosphorylated Myo-II. The sqh-TA mutant, which also perturbs Myo-II remodeling, constricted ventral furrow cells at a rate that is only slightly slower than wild type. In addition, MBS knockdown, which disrupted Myo-II pulses, exhibited a more variable constriction rate, but with a mean rate comparable to control embryos. The current finding is distinct from studies in other cell types where loss of MBS results in excessive phosphorylated Myo-II accumulation and cell invagination. Thus, MBS can regulate Myo-II organization and dynamics without causing a significant increase in apical Myo-II levels. It is concluded that Myo-II pulses are not absolutely required for individual cell apical constriction (Vasquez, 2014).
Although phosphomimetic mutant cells constrict and undergo tissue invagination, the coordination of invagination and the stability of the supracellular actomyosin meshwork were perturbed. Continuous apical constriction was associated with abnormal separation events between Myo-II structures in adjacent cells, resulting in gaps or holes in the supracellular Myo-II meshwork. Thus, continuous Myo-II assembly and a lack of Myo-II dynamics during apical constriction appear to sensitize the tissue to loss of intercellular cytoskeletal integrity during morphogenesis. Although loss of cytoskeletal continuity in phosphomimetic mutants does not block tissue invagination, it is speculated that dynamic Myo-II pulses are important to make tissue invagination robust to changes in tensile stress. One possible function of Myo-II pulses is to attenuate tissue tension or stiffness during morphogenetic movements. Because pulsed Myo-II contractions are staggered between neighboring cells, pulsation could serve as a mechanism to coordinate contractile force generation across the tissue such that intercellular connections are buffered from high levels of tension. Indeed, reducing adherens junction proteins sensitizes the intercellular connections between cytoskeletal networks to tensile forces generated in ventral furrow cells. Alternatively, remodeling of actomyosin networks that occurs during pulses could be required to adapt the cytoskeletal organization such that forces transmitted between cells accommodate the changing pattern of tissue-scale forces during the course of morphogenesis. In either case, the current data suggest that Myo-II pulsing and remodeling are important for collective cell behavior by ensuring proper force transmission between cells in a tissue undergoing morphogenesis (Vasquez, 2014).
A tight spatiotemporal control of actin polymerization is important for many cellular processes that shape cells into a multicellular organism. The formation of unbranched F-actin is induced by several members of the formin family. Drosophila encodes six formin genes, representing six of the seven known mammalian subclasses. Knittrig (Formin homology 2 domain containing ortholog), the Drosophila homolog of mammalian FHOD1, is specifically expressed in the developing central nervous system midline glia, the trachea, the wing and in macrophages. knittrig mutants exhibit mild tracheal defects but survive until late pupal stages and mainly die as pharate adult flies. knittrig mutant macrophages are smaller and show reduced cell spreading and cell migration in in vivo wounding experiments. Rescue experiments further demonstrate a cell-autonomous function of Knittrig in regulating actin dynamics and cell migration. Knittrig localizes at the rear of migrating macrophages in vivo, suggesting a cellular requirement of Knittrig in the retraction of the trailing edge. Supporting this notion, this study found that Knittrig is a target of the Rho-dependent kinase Rok. Co-expression with Rok or expression of an activated form of Knittrig induces actin stress fibers in macrophages and in epithelial tissues. Thus, a model is proposed in which Rok-induced phosphorylation of residues within the basic region mediates the activation of Knittrig in controlling macrophage migration (Lammel, 2014).
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).
The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, Combover (Cmb)/CG10732 was identified as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. These data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh (Fagan, 2014).
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