zipper : Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - zipper

Synonyms - myosin II

Cytological map position - 60E9-F1

Function - myosin heavy chain

Keyword(s) - dorsal closure, cell motility, cytoskeleton, gastrulation, germband extension

Symbol - zip

FlyBase ID:FBgn0287873

Genetic map position - 2-[108]

Classification - nonmuscle myosin

Cellular location - cytoplasmic

NCBI link: Entrez Gene
zip orthologs: Biolitmine
Recent literature
Doerflinger, H., Zimyanin, V. and St Johnston, D. (2022). The Drosophila anterior-posterior axis is polarized by asymmetric myosin activation. Curr Biol 32(2): 374-385. PubMed ID: 34856125
The Drosophila anterior-posterior axis is specified at mid-oogenesis when the Par-1 kinase is recruited to the posterior cortex of the oocyte, where it polarizes the microtubule cytoskeleton to define where the axis determinants, bicoid and oskar mRNAs, localize. This polarity is established in response to an unknown signal from the follicle cells, but how this occurs is unclear. This study shows that the myosin chaperone Unc-45 and non-muscle myosin II (MyoII) are required upstream of Par-1 in polarity establishment. Furthermore, the myosin regulatory light chain (MRLC) is di-phosphorylated at the oocyte posterior in response to the follicle cell signal, inducing longer pulses of myosin contractility at the posterior that may increase cortical tension. Overexpression of MRLC-T21A that cannot be di-phosphorylated or treatment with the myosin light-chain kinase inhibitor ML-7 abolishes Par-1 localization, indicating that the posterior of MRLC di-phosphorylation is essential for both polarity establishment and maintenance. Thus, asymmetric myosin activation polarizes the anterior-posterior axis by recruiting and maintaining Par-1 at the posterior cortex. This raises an intriguing parallel with anterior-posterior axis formation in C. elegans, where MyoII also acts upstream of the PAR proteins to establish polarity, but to localize the anterior PAR proteins rather than Par-1.
Losick, V. P. and Duhaime, L. G. (2021). The endocycle restores tissue tension in the Drosophila abdomen post wound repair. Cell Rep 37(2): 109827. PubMed ID: 34644579
Polyploidy frequently arises in response to injury, aging, and disease. Despite its prevalence, major gaps exist in understanding of how polyploid cells alter tissue function. In the adult Drosophila epithelium, wound healing is dependent on the generation of multinucleated polyploid cells resulting in a permanent change in the epithelial architecture. This study shows how the wound-induced polyploid cells affect tissue function by altering epithelial mechanics. The mechanosensor nonmuscle myosin II is activated and upregulated in wound-induced polyploid cells and persists after healing completes. Polyploidy enhances relative epithelial tension, which is dependent on the endocycle and not cell fusion post injury. Remarkably, the enhanced epithelial tension mimics the relative tension of the lateral muscle fibers, which are permanently severed by the injury. As a result, this study found that the wound-induced polyploid cells remodel the epithelium to maintain fly abdominal movements, which may help compensate for lost tissue tension (Losick, 2021).
Chen, W. and He, B. (2022). Actomyosin activity-dependent apical targeting of Rab11 vesicles reinforces apical constriction. J Cell Biol 221(6). PubMed ID: 35404399
During tissue morphogenesis, the changes in cell shape, resulting from cell-generated forces, often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. This study identified an actomyosin-dependent mechanism involving Rab11-mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures ("Rab11 vesicles") by promoting a directional bias in dynein-mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network, and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding.
Lechuga, S., Cartagena-Rivera, A. X., Khan, A., Crawford, B. I., Narayanan, V., Conway, D. E., Lehtimaki, J., Lappalainen, P., Rieder, F., Longworth, M. S. and Ivanov, A. I. (2022). A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair. Faseb j 36(5): e22290. PubMed ID: 35344227
Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. This study found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. These findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.
Selvaggi, L., Ackermann, M., Pasakarnis, L., Brunner, D. and Aegerter, C. M. (2021). Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure. Biophys J. PubMed ID: 34971619
The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. A patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. This tool was used to quantify the in vivo force production of Myosin II waves that are observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing quantitative values on an active Myosin-driven force, this study has elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and the mechanical properties of the dorsal yolk cell surface were characterized.
Selvaggi, L., Ackermann, M., Pasakarnis, L., Brunner, D. and Aegerter, C. M. (2021). Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure. Biophys J. PubMed ID: 34971619
The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. A patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. This tool was used to quantify the in vivo force production of Myosin II waves that are observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing quantitative values on an active Myosin-driven force, this study has elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and the mechanical properties of the dorsal yolk cell surface were characterized.
Guo, H., Swan, M. and He, B. (2022). Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during Drosophila mesoderm invagination. Elife 11. PubMed ID: 35195065
Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, it was find that during Drosophila mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that Drosophila mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. It is proposed that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling.
Selvaggi, L., Ackermann, M., Pasakarnis, L., Brunner, D. and Aegerter, C. M. (2022). Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure. Biophys J 121(3): 410-420. PubMed ID: 34971619
The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. A patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. This study used this tool to quantify the in vivo force production of Myosin II waves that were observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing quantitative values on an active Myosin-driven force, this study elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and the mechanical properties of the dorsal yolk cell surface was characterized.
Lv, Z., Zhang, N., Zhang, X., Grosshans, J. and Kong, D. (2022). The Lateral Epidermis Actively Counteracts Pulling by the Amnioserosa During Dorsal Closure. Front Cell Dev Biol 10: 865397. PubMed ID: 35652100
Dorsal closure is a prominent morphogenetic process during Drosophila embryogenesis, which involves two epithelial tissues, that is, the squamous amnioserosa and the columnar lateral epidermis. Non-muscle myosin II-driven constriction in the amnioserosa leads to a decrease in the apical surface area and pulls on the adjacent lateral epidermis, which subsequently moves dorsally. The pull by the amnioserosa becomes obvious in an elongation of the epidermal cells, especially of those in the first row. The contribution of the epidermal cell elongation has remained unclear to dorsal closure. Cell elongation may be a mere passive consequence or an active response to the pulling by the amnioserosa. This study found that the lateral epidermis actively responds. Tensions within tissues and cell junctions were analyzed by laser ablation before and during dorsal closure, the elliptical and dorsal closure stages, respectively. Furthermore, chronic and acute cell contraction were genetically and optochemically induced, respectively. In this way, it was found that tension in the epidermis increased during dorsal closure. A correspondingly increased tension was not observed at individual junctions, however. Junctional tension even decreased during dorsal closure in the epidermis. A strong increase of the microtubule amount was strikingly observed in the epidermis, while non-muscle myosin II increased in both tissues. These data suggest that the epidermis actively antagonizes the pull from the amnioserosa during dorsal closure and the increased microtubules might help the epidermis bear part of the mechanical force.
Ikawa, K., Ishihara, S., Tamori, Y. and Sugimura, K. (2023). Attachment and detachment of cortical myosin regulates cell junction exchange during cell rearrangement in the Drosophila wing epithelium. Curr Biol 33(2): 263-275. PubMed ID: 36543168
Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. By combining live imaging and physical modeling, this study showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, M6 shifts to the adherens junction plane on jub RNAi and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, this study developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions.
Wang, J., Michel, M., Bialas, L., Pierini, G. and Dahmann, C. (2023). Preferential recruitment and stabilization of Myosin II at compartment boundaries in Drosophila. J Cell Sci. PubMed ID: 36718636
The regulation of mechanical tension exerted at cell junctions guides cell behavior during tissue formation and homeostasis. Cell junctions along compartment boundaries, which are lineage restrictions separating cells with different fates and functions within tissues, are characterized by increased mechanical tension compared to cell junctions in the bulk of the tissue. Mechanical tension depends on the actomyosin cytoskeleton, however, the mechanisms by which mechanical tension is locally increased at cell junctions along compartment boundaries remain elusive. This study show that non-muscle Myosin II and F-actin transiently accumulate, and mechanical tension is increased, at cell junctions along the forming anteroposterior compartment boundary in the Drosophila pupal abdominal epidermis. Fluorescence recovery after photobleaching experiments show that Myosin II accumulation correlates with its increased stabilization at these junctions. Moreover, photoconversion indicates that Myosin II is preferentially recruited within cells to junctions along the compartment boundary. These results indicate that the preferential recruitment and stabilization of Myosin II contribute to the initial build-up of mechanical tension at compartment boundaries.
Gustafson, H. J., Claussen, N., De Renzis, S. and Streichan, S. J. (2022). Patterned mechanical feedback establishes a global myosin gradient. Nat Commun 13(1): 7050. PubMed ID: 36396633
Morphogenesis, the coordinated execution of developmental programs that shape embryos, raises many fundamental questions at the interface between physics and biology. In particular, how the dynamics of active cytoskeletal processes are coordinated across the surface of entire embryos to generate global cell flows is poorly understood. Two distinct regulatory principles have been identified: genetic programs and dynamic response to mechanical stimuli. Despite progress, disentangling these two contributions remains challenging. This study combined in toto light sheet microscopy with genetic and optogenetic perturbations of tissue mechanics to examine theoretically predicted dynamic recruitment of non-muscle myosin II to cell junctions during Drosophila embryogenesis. Dynamic recruitment was found to have a long-range impact on global myosin configuration, and the rate of junction deformation sets the rate of myosin recruitment. Mathematical modeling and high frequency analysis reveal myosin fluctuations on junctions around a mean value set by mechanical feedback. The model accounts for the early establishment of the global myosin pattern at 80% fidelity. Taken together these results indicate spatially modulated mechanical feedback as a key regulatory input in the establishment of long-range gradients of cytoskeletal configurations and global tissue flow patterns.
Zhu, H. and B, O. S. (2023). Actomyosin pulsing rescues embryonic tissue folding from disruption by myosin fluctuations. bioRxiv. PubMed ID: 36993262
During early development, myosin II mechanically reshapes and folds embryo tissue. A much-studied example is ventral furrow formation in Drosophila, marking the onset of gastrulation. Furrowing is driven by contraction of actomyosin networks on apical cell surfaces, but how the myosin patterning encodes tissue shape is unclear, and elastic models failed to reproduce essential features of experimental celf contraction profiles. The myosin patterning exhibits substanatial cell-to-cell fluctuations with pulsatile time-dependence, a striking but unexplained feature of morphogenesis in many organisms. In this study, using biophysical modeling wviscous forces were found to offer the principle resistance to actomyosin-driven apical constriction. In consequence, tissue shape is encoded in the direction-dependent curvature of the myosin patterning which orients an anterior-posterior furrow. Tissue contraction is highly sensitive to cell-to-cell myosin fluctuations, explaining furrowing failure in genetically perturbed embryos whose fluctuations are temporally persistent. In wild-type embryos, this catastrophic outcome is averted by pulsatile myosin time-dependence, a time-averaging effect that rescues furrowing. This low pass filter mechanism may underlie the usage of actomyosin pulsing in diverse morphogenetic processes across many organisms.
Gehrels, E. W., Chakrabortty, B., Perrin, M. E., Merkel, M. and Lecuit, T. (2023). Curvature gradient drives polarized tissue flow in the Drosophila embryo. Proc Natl Acad Sci U S A 120(6): e2214205120. PubMed ID: 36724258
Tissue flow during morphogenesis is commonly driven by local constriction of cell cortices, which is caused by the activation of actomyosin contractility. This can lead to long-range flows due to tissue viscosity. However, in the absence of cell-intrinsic polarized forces or polarity in forces external to the tissue, these flows must be symmetric and centered around the region of contraction. Polarized tissue flows have been previously demonstrated to arise from the coupling of such contractile flows to points of increased friction or adhesion to external structures. However, this study shows with experiments and modeling that the onset of polarized tissue flow in early Drosophila morphogenesis occurs independent of adhesion and is instead driven by a geometric coupling of apical actomyosin contractility to tissue curvature. Particularly, the onset of polarized flow is driven by a mismatch between the position of apical myosin activation and the position of peak curvature at the posterior pole of the embryo. This work demonstrates how genetic and geometric information inherited from the mother interact to create polarized flow during embryo morphogenesis.
Jun, Y. W., Lee, S., Ban, B. K., Lee, J. A. and Gao, F. B. (2023). Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis. Autophagy: 1-17. PubMed ID: 36849436
Dysfunction of the endosomal sorting complex required for transport (ESCRT) has been linked to frontotemporal dementia (FTD) due in part to the accumulation of unsealed autophagosomes. However, the mechanisms of ESCRT-mediated membrane closure events on phagophores remain largely unknown. This study found that partial knockdown of non-muscle MYH10/myosin IIB/zip rescues neurodegeneration in both Drosophila and human iPSC-derived cortical neurons expressing FTD-associated mutant CHMP2B, a subunit of ESCRT-III. It was also found that MYH10 binds and recruits several autophagy receptor proteins during autophagosome formation induced by mutant CHMP2B or nutrient starvation. Moreover, MYH10 interacted with ESCRT-III to regulate phagophore closure by recruiting ESCRT-III to damaged mitochondria during PRKN/parkin-mediated mitophagy. Evidently, MYH10 is involved in the initiation of induced but not basal autophagy and also links ESCRT-III to mitophagosome sealing, revealing novel roles of MYH10 in the autophagy pathway and in ESCRT-related FTD pathogenesis.
Nishizawa, K., Lin, S. Z., Chardes, C., Rupprecht, J. F. and Lenne, P. F. (2023). Two-point optical manipulation reveals mechanosensitive remodeling of cell-cell contacts in vivo. Proc Natl Acad Sci U S A 120(13): e2212389120. PubMed ID: 36947511
Biological tissues acquire reproducible shapes during development through dynamic cell behaviors. Most of these behaviors involve the remodeling of cell-cell contacts. During epithelial morphogenesis, contractile actomyosin networks remodel cell-cell contacts by shrinking and extending junctions between lateral cell surfaces. However, actomyosin networks not only generate mechanical stresses but also respond to them, confounding understanding of how mechanical stresses remodel cell-cell contacts. This study developed a two-point optical manipulation method to impose different stress patterns on cell-cell contacts in the early epithelium of the Drosophila embryo. The technique allows production of junction extension and shrinkage through different push and pull manipulations at the edges of junctions. These observations were used to expand classical vertex-based models of tissue mechanics, incorporating negative and positive mechanosensitive feedback depending on the type of remodeling. In particular, this study showed that Myosin-II activity responds to junction strain rate and facilitates full junction shrinkage. Altogether this work provides insight into how stress produces efficient deformation of cell-cell contacts in vivo and identifies unanticipated mechanosensitive features of their remodeling.
Cazzagon, G., Roubinet, C. and Baum, B. (2023). Polarized SCAR and the Arp2/3 complex regulate apical cortical remodeling in asymmetrically dividing neuroblasts. iScience 26(7): 107129. PubMed ID: 37434695
Although the formin-nucleated actomyosin cortex has been shown to drive the changes in cell shape that accompany animal cell division in both symmetric and asymmetric cell divisions, the mitotic role of cortical Arp2/3-nucleated actin networks remain unclear. In this study, using asymmetrically dividing Drosophila neural stem cells as a model system, a pool of membrane protrusions was identified that form at the apical cortex of neuroblasts as they enter mitosis. Strikingly, these apically localized protrusions are enriched in SCAR, and depend on SCAR and Arp2/3 complexes for their formation. Because compromising SCAR or the Arp2/3 complex delays the apical clearance of Myosin II at the onset of anaphase and induces cortical instability at cytokinesis, these data point to a role for an apical branched actin filament network in fine-tuning the actomyosin cortex to enable the precise control of cell shape changes during an asymmetric cell division.
Balaghi, N., Erdemci-Tandogan, G., McFaul, C. and Fernandez-Gonzalez, R. (2023). Myosin waves and a mechanical asymmetry guide the oscillatory migration of Drosophila cardiac progenitors. Dev Cell 58(14): 1299-1313. PubMed ID: 37295436
Heart development begins with the formation of a tube as cardiac progenitors migrate from opposite sides of the embryo. Abnormal cardiac progenitor movements cause congenital heart defects. However, the mechanisms of cell migration during early heart development remain poorly understood. Using quantitative microscopy, this study found that in Drosophila embryos, cardiac progenitors (cardioblasts) migrated through a sequence of forward and backward steps. Cardioblast steps were associated with oscillatory non-muscle myosin II waves that induced periodic shape changes and were necessary for timely heart tube formation. Mathematical modeling predicted that forward cardioblast migration required a stiff boundary at the trailing edge. Consistent with this, a supracellular actin cable was found at the trailing edge of the cardioblasts that limited the amplitude of the backward steps, thus biasing the direction of cell movement. These results indicate that periodic shape changes coupled with a polarized actin cable produce asymmetrical forces that promote cardioblast migration.
Carim, S. C. and Hickson, G. R. X. (2023). The Rho1 GTPase controls anillo-septin assembly to facilitate contractile ring closure during cytokinesis. iScience 26(6): 106903. PubMed ID: 37378349
Animal cell cytokinesis requires activation of the GTPase RhoA (Rho1 in Drosophila), which assembles an F-actin- and myosin II-dependent contractile ring (CR) at the equatorial plasma membrane. CR closure is poorly understood, but involves the multidomain scaffold protein, Anillin. Anillin binds many CR components including F-actin and myosin II (collectively actomyosin), RhoA and the septins. Anillin recruits septins to the CR but the mechanism is unclear. Live imaging of Drosophila S2 cells and HeLa cells revealed that the Anillin N-terminus, which scaffolds actomyosin, cannot recruit septins to the CR. Rather, septin recruitment required the ability of the Anillin C-terminus to bind Rho1-GTP and the presence of the Anillin PH domain, in a sequential mechanism occurring at the plasma membrane, independently of F-actin. Anillin mutations that blocked septin recruitment, but not actomyosin scaffolding, slowed CR closure and disrupted cytokinesis. Thus, CR closure requires coordination of two Rho1-dependent networks: actomyosin and anillo-septin.
Chen, J., Verissimo, A. F., Kull, A. R. and He, B. (2023). Early zygotic gene product Dunk interacts with anillin to regulate Myosin II during Drosophila cleavage. Mol Biol Cell 34(10):ar102 PubMed ID: 37494082
Drosophila melanogaster cellularization is a special form of cleavage that converts syncytial embryos into cellular blastoderms by partitioning the peripherally localized nuclei into individual cells. An early event in cellularization is the recruitment of nonmuscle myosin II ("myosin") to the leading edge of cleavage furrows, where myosin forms an interconnected basal array before reorganizing into individual cytokinetic rings. The initial recruitment and organization of basal myosin are regulated by a cellularization-specific gene, dunk, but the underlying mechanism is unclear. Through a genome-wide yeast two-hybrid screen, this study identified anillin (Scraps in Drosophila), a conserved scaffolding protein in cytokinesis, as the primary binding partner of Dunk. Dunk colocalizes with anillin and regulates its cortical localization during the formation of cleavage furrows, while the localization of Dunk is independent of anillin. Furthermore, Dunk genetically interacts with anillin to regulate the basal myosin array during cellularization. Similar to Dunk, anillin colocalizes with myosin since the very early stage of cellularization and is required for myosin retention at the basal array, before the well-documented function of anillin in regulating cytokinetic ring assembly. Based on these results, it is proposed that Dunk regulates myosin recruitment and spatial organization during early cellularization by interacting with and regulating anillin.
Gaspar, I., Phea, L. J., McClintock, M. A., Heber, S., Bullock, S. L. and Ephrussi, A. (2023). An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport. J Cell Biol 222(7). PubMed ID: 37213090
Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). This study shows that orchestration of oskar RNP transport in the Drosophila germline relies on interplay between two double-stranded RNA-binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). It was found that Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. It was additionally shown that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. These observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity.
Messer, C. L. and McDonald, J. A. (2023). Rap1 promotes epithelial integrity and cell viability in a growing tissue. Dev Biol 501: 1-19. PubMed ID: 37269969
Having intact epithelial tissues is critical for embryonic development and adult homeostasis. How epithelia respond to damaging insults or tissue growth while still maintaining intercellular connections and barrier integrity during development is poorly understood. The conserved small GTPase Rap1 is critical for establishing cell polarity and regulating cadherin-catenin cell junctions. This study identified a new role for Rap1 in maintaining epithelial integrity and tissue shape during Drosophila oogenesis. loss of Rap1 activity disrupted the follicle cell epithelium and the shape of egg chambers during a period of major growth. Rap1 was required for proper E-Cadherin localization in the anterior epithelium and for epithelial cell survival. Both Myo-II and the adherens junction-cytoskeletal linker protein α-Catenin were required for normal egg chamber shape but did not strongly affect cell viability. Blocking the apoptotic cascade failed to rescue the cell shape defects caused by Rap1 inhibition. One consequence of increased cell death caused by Rap1 inhibition was the loss of polar cells and other follicle cells, which later in development led to fewer cells forming a migrating border cell cluster. These results thus indicate dual roles for Rap1 in maintaining epithelia and cell survival in a growing tissue during development.
Klipa, O., El Gammal, M. and Hamaratoglu, F. (2023). Elimination of aberrantly specified cell clones is independent of interfacial Myosin II accumulation. J Cell Sci 136(13). PubMed ID: 37309190
Spatial organization within an organ is essential and needs to be maintained during development. This is largely implemented via compartment boundaries that serve as barriers between distinct cell types. Biased accumulation of junctional non-muscle Myosin II along the interface between differently fated groups of cells contributes to boundary integrity and maintains its shape via increased tension. Using the Drosophila wing imaginal disc, whether interfacial tension driven by accumulation of Myosin is responsible for the elimination of aberrantly specified cells that would otherwise compromise compartment organization was tested. To this end, Myosin II levels were genetically reduced in three different patterns: in both wild-type and misspecified cells, only in misspecified cells, and specifically at the interface between wild-type and aberrantly specified cells. The recognition and elimination of aberrantly specified cells do not strictly rely on tensile forces driven by interfacial Myosin cables. Moreover, apical constriction of misspecified cells and their separation from wild-type neighbours occurred even when Myosin levels were greatly reduced. Thus, it is concluded that the forces that drive elimination of aberrantly specified cells are largely independent of Myosin II accumulation.

Myosins are a functionally divergent group of molecular motors that are involved in various non-muscle cell motile activities. Zipper, hence referred to as Myosin II, or non-muscle myosin, is crucial to the viability of the Drosophila embryo throughout its development. The clearest example of nonmuscle-driven shape change is cytokinesis, in which an actin and myosin-rich contractile ring cleaves the cell during mitosis. Null alleles of zipper coding for the non-muscle heavy chain cause failure of dorsal closure, during which the lateral epidermal cells elongate to cover the dorsal surface of the embryo after germ band retraction. Axon pathfinding and head involution defects also contribute to the embryonic lethality of zipper mutants. Early dorsal closure is normal in these mutants, suggesting that maternal coded Zipper protein is sufficient for early stages of closure. By late closure however, nonmuscle myosin is either improperly localized in or absent from the leading edge of the lateral epithelium. By the time zip mutant embryos arrest during late dorsal closure stages, the leading edge is dramatically disorganized, the lateral epidermis is not fused along the dorsal midline, and the amnioserosa remains exposed. In normal flies, dorsal closure is accompanied by a dramatic cell shape change as the entire epidermal cell sheet thins out to spread over the amnioserosa. As dorsal closure proceeds, more and more cells throughout the epidermis are elongated. This change in epidermal cell shape, driven for the most part by maternal myosin II, is sufficient to account for the spreading of the lateral epithelium over the region occupied by the amnioserosa (Young, 1993).

Nonmuscle myosin II is also required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos. Developing oocytes containing germline mutant spaghetti squash(sqh) clones fail to attain full suze due to a defect in 'dumping,' the rapid phase of cytoplasmic transport in nurse cells. Spaghetti squash is the regulatory light chain of nonmuscle myosin II; along with the Essential light chain, it acts to regulate the function of Myosin II. Two subunits of the regulatory light chain, along with two subunits of the essential light chain, each combine with two subunits of Myosin II to form the functional Myosin II hexamer. spaghetti squash mutant egg chambers show no evidence of ring canal obstruction, and no obvious alteration in the actin network. However, the distribution of myosin II is abnormal. It is thought that the molecular motor responsible for cytoplasmic dumping is supplied largely, if not exclusively by nurse cell myosin II. Regulation of myosin activity is one means by which cytoplasmic transport is controlled during oocyte development. Fertilized eggs developed from spaghetti squash maternal mutant clones begin development by exhibiting an early defect in axial migration of cleavage nuclei toward the posterior pole of the embryo. This is similar to development seen in early cleavage zygotes, in which the actin cytoskeleton is disrupted. Thus both nurse cell dumping and axial migration of the zygotic nuclei require maternally supplied myosin II (Wheatley, 1995).

In order to study the role of myosin II during imaginal disc development, spaghetti squash was put under control of a heat shock promoter and induced at various times in sqh mutants. sqh mutants can be rescued to adulthood by daily induction of sqh from a hsp 70 promoter. When SQH is transiently depleted in larvae, the resulting adult phenotypes demonstrate that SQH is required in a stage-specific fashion for proper development of eye and leg imaginal discs. When SQH is depleted in adult females, oogenesis is reversibly disrupted. Without SQH induction, developing egg chambers display a succession of phenotypes that demonstrate roles for myosin II in morphogenesis of the interfollicular stalks, three morphologically and mechanistically distinct types of follicle cell migration, and completion of nurse cell cytoplasm transport (dumping) (Edwards, 1996).

Normally at stage 9 of oogenesis, approximately eight so-called border cells form at the anterior tip of the egg chamber, delaminate, and migrate past the phalanx of nurse cells until they reach the anterior tip of the oocyte. This migration of border cells between and through the nurse cells is abnormal in sqh mutants. Normal border cell myosin II staining is much brighter than that of the surrounding nurse cells, indicating a presence of myosin II in border cells. Centripetal cells, a second morphologically distinct type of follicle cell, is derived from the most anterior ring of oocyte follicle cells. Centripetal cells normally elongate and plunge inward toward the border cells, which have come to rest at the anterior face of the oocyte. These centripetal cells will eventually cover the anterior of the oocyte and build several specialized structures of the anterior chorion. Each centripetal cell specifically accumulates a bright bar of myosin II staining at the edge of the apical (inner) surface that leads the penetration between the nurse cells and the oocyte (Edwards, 1996).

sqh mutant ovaries also display major defects in migration of centripetal cells. After three days without SQH, centripetal cells fail to elongate. Follicle cells that construct the dorsal appendages also fail to migrate properly as the supply of SQH dwindles and eventually is depleted. A shortage of SQH in migrating cells appears to be the primary cause of the shortened dorsal appendage phenotype found in sqh mutants (Edwards, 1996).

In sqh mutant tissues, Myosin II heavy chain is abnormally localized in punctate structures that do not contain appreciable amounts of filamentous actin or the myosin tail binding protein p127. p127 is coded for by the gene lethal (2) giant larvae tumor suppressor gene. In follicle cells, p127 is normally concentrated at the lateral membranes. In sqh mutant tissue, p127 shows the same pattern, with no punctate cytoplasmic staining. Likewise F-actin is not mislocalized in sqh mutants. Thus sqh mutations cause mislocalization of myosin, but not the other major cytoskeletal proteins (Edwards, 1996).

Control of cell flattening and junctional remodeling during squamous epithelial morphogenesis in Drosophila

Diverse types of epithelial morphogenesis drive development. Similar cytoskeletal and cell adhesion machinery orchestrate these changes, but it is unclear how distinct tissue types are produced. Thus, it is important to define and compare different types of morphogenesis. Cell flattening and elongation were investigated in the amnioserosa, a squamous epithelium formed at Drosophila gastrulation. Amnioserosa cells are initially columnar. Remarkably, they flatten and elongate autonomously by perpendicularly rotating the microtubule cytoskeleton - this is called 'rotary cell elongation'. Apical microtubule protrusion appears to initiate the rotation and microtubule inhibition perturbs the process. F-actin restrains and helps orient the microtubule protrusions. As amnioserosa cells elongate, they maintain their original cell-cell contacts and develop planar polarity. Myosin II localizes to anterior-posterior contacts, while the polarity protein Bazooka (PAR-3) localizes to dorsoventral contacts. Genetic analysis revealed that Myosin II and Bazooka cooperate to properly position adherens junctions. These results identify a specific cellular mechanism of squamous tissue morphogenesis and molecular interactions involved (Pope, 2008).

Amnioserosa tissue morphogenesis involves dramatic cell shape change. Before amnioserosa morphogenesis, cells are columnar with lateral MT bundles in a basket-like array along the apicobasal axis. With amnioserosa morphogenesis, the cells elongate and flatten. Amnioserosa cells could change shape by symmetrically re-positioning cellular contents (full cytoskeleton and/or membrane reorganization) or by perpendicularly rotating cellular components to reorient the long axis of the cell into the plane of tissue extension. To distinguish these possibilities, 3D cell organization was studied over time; it was discovered that the MT array plus the nucleus, centrosomes and ER rotate, apparently as a unit, into the plane of tissue extension. More symmetric adherens junction (AJ) and cortical reorganizations appear to accompany the rotation. MT arrays also reorient to polarize cells during chemotaxis and tissue migration, and to reposition cell contents as occurs during cortical rotation in early Xenopus embryos. These results reveal rotation of the MT cytoskeleton linked to cell shape change and amnioserosa morphogenesis. Similar mechanisms may underlie the development of other squamous epithelial monolayers (Pope, 2008).

Regulated apical MT protrusion appears to initiate amnioserosa rotary cell elongation. MT inhibition perturbs initial elongation, and the process normally begins with MTs protruding into the apical domain, bending perpendicularly and then extending in the axis of cell elongation. Pre-existing lateral MT bundles appear to protrude across the apical domain - they are mainly non-centrosomal and contain older (acetylated) MTs. However, EB1-GFP imaging also revealed bi-directional MT growth across the apical domain. This indicates that the bundles are dynamic, but argues against MT bundle protrusion through polarized individual MT polymerization. Instead, MT bundle protrusion may involve greater net renewal of bundles apically versus basally, motors sliding MTs past MTs in the bundles and/or motors moving bundles along the cell cortex. Distinguishing these models requires further study. As MTs extend apically the actin cytoskeleton appears to inhibit them, as weakening actin leads to excessively long and randomly oriented MT-based protrusions. Actin is normally found around the full apical circumference as amnioserosa cells elongate. By contrast, Myosin II becomes enriched at AP contacts and is gradually lost from the full cell cortex. Thus, different pools of actin may regulate MT protrusion. It is speculated that the gradual overall loss of cortical actin-myosin complexes permits, and may help orient, regulated MT protrusion. Actin also antagonizes cortical MTs in other systems. MT-based primary axons form where cortical actin is weakest. Actin inhibits cortical MT protrusion in neutrophils and Myosin IIA inhibits cortical MTs in mammalian cells. Actin might physically block MT protrusion, but direct or indirect molecular interactions may also be involved (Pope, 2008).

In Drosophila embryos, MT-actin interactions also affect germband cells. At stage 7-8, actin disruption enhances AJ planar polarity at DV contacts. MT disruption suppresses this, suggesting that actin inhibits MT-based AJ positioning in these cells - however, germband cells show minimal shape change with actin disruption at this stage. Remarkably, the same actin disruption causes stage 9-10 germband cells to rotate analogously to early amnioserosa cells. Their apical domains elongate and their lateral regions rotate perpendicularly, becoming exposed to the embryo surface. Implicating MTs in this change, lateral MT bundles run into the extended apical domains and simultaneous MT disruption suppresses the cell shape change. Thus, actin may inhibit apical MTs to regulate tissue structure in many parts of the embryo. This MT inhibition may also involve coordination with AJs, as disrupted germband cells in armm/z mutants also display MTs protruding into extended apical domains (Pope, 2008).

How do MTs elongate the apical domain and how is this linked to the rotation of the full MT cytoskeleton in the amnioserosa? It is proposed that rotary cell elongation occurs in two phases. In phase one, the MT imaging and inhibitor studies indicate that regulated MT protrusion elongates the apical domain. This may involve a combination of physical force, membrane delivery and/or relaxation of actin-myosin contractility. The AJ clustering observed at abnormal apical MT protrusions formed with actin inhibition in both early amnioserosa cells and the later germband suggests that MTs may apply force to AJs. Consistent with this idea, MT inhibition affected both initial amnioserosa cell elongation and later amnioserosa cell-cell interactions. However, amnioserosa cell elongation in armm/z mutants suggests that MTs may not necessarily engage AJs directly. Since Baz localizes apically in early armm/z mutants, and is enriched at DV amnioserosa cell contacts to which MTs rotate in wild type, it is a strong candidate for coordinating these interactions. However, severe early defects in bazm/z mutants would confound analysis of amnioserosa development - this may require conditional mutants. Phase two of rotary cell elongation requires full perpendicular rotation of the MT array, apical and basal membrane growth, and lateral membrane removal. Although it is unclear how full rotation occurs, MT rotation and cortical remodeling may occur in concert. For example, membrane remodeling may explain how amnioserosa cells remain elongated with MT disruption during later development (Pope, 2008).

For rotary cell elongation to translate into tissue extension, cell contacts and AJs must be remodeled. Remarkably, amnioserosa cells maintain their neighbor relationships as they elongate, and two contact types develop; highly elongated AP contacts and lesser elongated DV contacts. Each appears to involve unique AJ remodeling. Intriguingly, Myosin II and Baz localize to AP and DV contacts, respectively -- the same reciprocal planar polarized relationship displayed in the germband. In the amnioserosa, Myosin II and Baz synergize to control overall AJ positioning, a regulatory interaction that has not been shown elsewhere (Pope, 2008).

Myosin II and Baz may regulate specific AJ remodeling events occurring at AP and DV contacts, respectively. Amnioserosa cells increase their apical circumference 10-fold, initially doubling the length of their AP cell contacts every 5-10 minutes. Remarkably, AJs localize around the full circumference as this occurs. This contrasts elongating Drosophila follicle cells, which lose AJ continuity, suggesting specific mechanisms for maintaining AJ continuity during amnioserosa morphogenesis. amnioserosa AJs do lose continuity with actin disruption, suggesting a role for actin. More specifically, AJ fragmentation in baz zip double mutants suggests a role for Myosin II. In the neighboring ventral furrow and germband, actin-myosin contractility is coupled to AJs during apical constriction and cell intercalation, respectively. The actin-myosin complexes enriched along amnioserosa AP contacts may also be contractile, but here they may counterbalance MT protrusion. Slowing apical elongation may indirectly allow AJ remodeling. However, Myosin II may also have direct affects on AJs (Pope, 2008).

Baz may regulate distinct AJ re-modeling at DV contacts. It is hypothesized that MT protrusion applies force to the DV contact at the cell 'front', and that cell elongation may also pull the 'rear' contact. Either force could detach AJs and necessitate AJ remodeling. Dynamic looping of DE-CadGFP and ArmCFP was observed at D-V contacts, and BazGFP partly colocalized with these loops. Although further experiments (e.g., photobleaching) are needed to understand this and other amnioserosa AJ remodeling, Baz localization at DV contacts and abnormal AJ aggregation at DV contacts in baz zip double mutants suggests a role for Baz in AJ remodeling at these sites. Baz appears to interact with MTs and Dynein to initially position AJs during Drosophila cellularization, and Baz might re-position AJs at DV amnioserosa contacts in a similar way (Pope, 2008).

In four different cases, cells were observed elongating towards potential sources of pulling forces. First, wild-type amnioserosa cells elongate along the DV axis towards the germband (potential source of DV pulling forces during convergent extension) and the ventral furrow (potential source of DV pulling forces during invagination). Second, amnioserosa cells elongate along the DV axis of bcd nos tsl mutants, in which germband extension fails, but ventral furrow formation occurs. Third, in dl mutants in which the ventral furrow does not form and the amnioserosa forms a ring around the DV axis, amnioserosa cells reoriented along the AP axis towards ectopic contractile furrows. Fourth, in the stage 9-11 wild-type germband, cells artificially induced to flatten and elongate did so in coordinated groups oriented towards contractile regions of the germband. Thus, polarized pulling forces across a tissue may orient rotary cell elongation. In wild-type embryos, these forces may come from germband extension and/or ventral furrow formation. However, Zen must first trigger the amnioserosa cell shape change, while AP patterning may specifically regulate AJ remodeling (Pope, 2008).

Pulsed contractions of an actin-myosin network drive apical constriction

Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. This study shows, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. These results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally (Martin, 2009).

During Drosophila gastrulation, apical constriction of ventral cells facilitates the formation of a ventral furrow and the subsequent internalization of the presumptive mesoderm. Although myosin is known to localize to the apical cortex of constricting ventral furrow cells, it is not known how myosin produces force to drive constriction. Understanding this mechanism requires a quantitative analysis of cell and cytoskeletal dynamics. Methods were developed to reveal and quantify apical cell shape with Spider-GFP, a green fluorescent protein (GFP)-tagged membrane-associated protein that outlines individual cells. Ventral cells were constricted to about 50% of their initial apical area before the onset of invagination and continued to constrict during invagination. Although the average apical area steadily decreased at a rate of about 5 microm2 min-1, individual cells showed transient pulses of rapid constriction that exceeded 10-15 microm2 min-1. During the initial 2 min of constriction, weak constriction pulses were often interrupted by periods of cell stretching. However, at 2 min, constriction pulses increased in magnitude and cell shape seemed to be stabilized between pulses, leading to net constriction. These two phases probably correspond to the 'slow/apical flattening' and 'fast/stochastic' phases that have been described previously. Overall, cells underwent an average of 3.2 ± 1.2 constriction pulses over 6 min, with an average interval of 82.8 ± 48 s between pulses (mean ± s.d., n = 40 cells, 126 pulses). Constriction pulses were mostly asynchronous between adjacent cells. As a consequence, cell apices between constrictions seemed to be pulled by their constricting neighbours. Thus, apical constriction occurs by means of pulses of rapid constriction interrupted by pauses during which cells must stabilize their constricted state before reinitiating constriction (Martin, 2009).

To determine how myosin might generate force during pulsed constrictions, myosin and cell dynamics were simultaneously imaged by using myosin regulatory light chain (spaghetti squash, or squ) fused to mCherry (Myosin-mCherry) and Spider-GFP. Discrete myosin spots and fibres present on the apical cortex formed a network that extended across the tissue. These myosin structures were dynamic, with apical myosin spots repeatedly increasing in intensity and moving together (at about 40 nm s-1) to form larger and more intense myosin structures at the medial apical cortex. This process, which is referred to as myosin coalescence, resulted in bursts of myosin accumulation that were correlated with constriction pulses. The peak rate of myosin coalescence preceded the peak constriction rate by 5-10 s, suggesting that myosin coalescence causes apical constriction. Between myosin coalescence events, myosin structures, including fibres, remained present on the cortex, possibly maintaining cortical tension between constriction pulses. Contrary to the purse-string model, no significant myosin accumulation was seen at cell-cell junctions. To confirm that constriction involved medial myosin coalescence and not contraction of a circumferential purse-string, constriction rate was correlated with myosin intensity at either the medial or junctional regions of the cell. Apical constriction was correlated more significantly with medial myosin, suggesting that, in contrast to the purse-string model, constriction is driven by contractions at the medial apical cortex (Martin, 2009).

Myosin coalescence resembled contraction of a cortical actin-myosin network. Therefore, to determine whether apical constriction is driven by pulsed contractions of the actin-myosin network, the organization of the cortical actin cytoskeleton was examined. In fibroblasts and keratocytes, actin network contraction bundles actin filaments into fibre-like structures. Consistent with this expectation was the identification of an actin filament meshwork underlying the apical cortex in which prominent actin-myosin fibres spanning the apical cortex appeared specifically in constricting cells. An actin-myosin network contraction model would predict that myosin coalescence results from myosin spots exerting traction on each other through the cortical actin network. To test whether myosin coalescence requires an intact actin network, the actin network was disrupted with cytochalasin D (CytoD). Disruption of the actin network with CytoD resulted in apical myosin spots that localized together with actin structures and appeared specifically in ventral cells. Myosin spots in CytoD-injected embryos showed more rapid movement than those in control-injected embryos, suggesting that apical myosin spots in untreated embryos are constrained by the cortical actin network. Although myosin movement was uninhibited in CytoD-treated embryos, myosin spots failed to coalesce and cells failed to constrict. Because myosin coalescence requires an intact actin network, it is proposed that pulses of myosin coalescence represent contractions of the actin-myosin network (Martin, 2009).

Because actin-myosin contractions occurred at the medial apical cortex, it was unclear how the actin-myosin network was coupled to adherens junctions. Therefore E-Cadherin-GFP and Myosin-mCherry were imaged to examine the relationship between myosin and adherens junctions. Before apical constriction, adherens junctions are present about 4 microm below the apical cortex. As apical constriction initiated, these subapical adherens junctions gradually disappeared and adherens junctions simultaneously appeared apically at the same level as myosin. This apical redistribution of adherens junctions occurred at specific sites along cell edges (midway between vertices). As apical constriction initiated, these sites bent inwards. This bending depended on the presence of an intact actin network, which is consistent with contraction of the actin-myosin network generating force to pull junctions. Indeed, myosin spots undergoing coalescence were observed to lead adherens junctions as they transiently bent inwards. Thus, pulsed contraction of the actin-myosin network at the medial cortex seems to pull the cell surface inwards at discrete adherens junction sites, resulting in apical constriction (Martin, 2009).

The transcription factors Twist and Snail regulate the apical constriction of ventral furrow cells. Snail is a transcriptional repressor whose target or targets are currently unknown, whereas Twist enhances snail expression and activates the expression of fog and t48, which are thought to activate the Rho1 GTPase and promote myosin contractility. To examine the mechanism of pulsed apical constriction further, how Twist and Snail regulate myosin dynamics was tested. In contrast to wild-type ventral cells, in which myosin was concentrated on the apical cortex, twist and snail mutants accumulated myosin predominantly at cell junctions, similarly to lateral cells. These ventral cells failed to constrict productively, which supported the cortical actin-myosin network contraction model, rather than the purse-string model, for apical constriction. twist and snail mutants differentially affected the coalescence of the minimal myosin that did localize to the apical cortex. Although myosin coalescence was inhibited in snail mutants, it still occurred in twist mutants, as did pulsed constrictions. This difference was also observed when Snail or Twist activity was knocked down by RNA-mediated interference. However, the magnitude of constriction pulses in twistRNAi embryos was greater than that of twist mutant embryos, suggesting that the low level of Twist activity present in twistRNAi embryos enhances contraction efficiency by activating the expression of snail or other transcriptional targets. Myosin coalescence was inhibited in snail twist double mutants, demonstrating that the pulsed constrictions in twist mutants required snail expression. Thus, the expression of snail, not twist, initiates the actin-myosin network contractions that power constriction pulses (Martin, 2009).

Net apical constriction was inhibited in both snailRNAi and twistRNAi embryos. It was therefore asked why the pulsed contractions that were observed in twistRNAi embryos failed to constrict cells. Using Spider-GFP to visualize cell outlines, it was found that although constriction pulses were inhibited in snailRNAi embryos, constriction pulses still occurred in twistRNAi embryos. However, the constricted state of cells in twistRNAi embryos was not stabilized between pulses, resulting in fluctuations in apical area with little net constriction. This stabilization defect was not due to lower snail activity, because these fluctuations continued when snail expression was driven independently of twist by using the P[sna] transgene. Although the frequency and magnitude of constriction pulses in such embryos were similar to those in control embryos, stretching events were significantly higher in twistRNAi; P[sna] embryos, suggesting a defect in maintaining cortical tension. This defect might result from a failure to establish a dense actin meshwork, because both twist mutants and twistRNAi embryos had a more loosely arranged apical meshwork of actin spots and fibres than constricting wild-type cells did. twist expression therefore stabilizes the constricted state of cells between pulsed contractions (Martin, 2009).

Thus, a 'ratchet' model is proposed for apical constriction, in which phases of actin-myosin network contraction and stabilization are repeated to constrict the cell apex incrementally. In contrast to the purse-string model, it was found that apical constriction is correlated with pulses of actin-myosin network contraction that occur on the apical cortex. Pulsed cortical contractions could allow dynamic rearrangements of the actin network to optimize force generation as cells change shape. Because contractions are asynchronous, cells must resist pulling forces from adjacent cells between contractions. A cortical actin-myosin meshwork seems to provide the cortical tension necessary to stabilize apical cell shape and promote net constriction. The transcription factors Snail and Twist are critical for the contraction and stabilization phases of constriction, respectively. Thus, Snail and Twist activities are temporally coordinated to drive productive apical constriction. Despite the dynamic nature of the contractions in individual cells, the behaviour of the system at the tissue level is continuous, in a similar manner to convergent extension in Xenopus. Pulsed contraction may therefore represent a conserved cellular mechanism that drives precise tissue-level behaviour (Martin, 2009).

Nonmuscle myosin II is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis

Metazoan development involves a myriad of dynamic cellular processes that require cytoskeletal function. Nonmuscle myosin II plays essential roles in embryonic development; however, knowledge of its role in post-embryonic development, even in model organisms such as Drosophila, is only recently being revealed. In this study, truncation alleles were generated and enable the conditional perturbation, in a graded fashion, of nonmuscle myosin II function. During wing development they demonstrate novel roles for nonmuscle myosin II, including in adhesion between the dorsal and ventral wing epithelial sheets; in the formation of a single actin-based wing hair from the distal vertex of each cell; in forming unbranched wing hairs; and in the correct positioning of veins and crossveins. Many of these phenotypes overlap with those observed when clonal mosaic analysis was performed in the wing using loss of function alleles. Additional requirements for nonmuscle myosin II are in the correct formation of other actin-based cellular protrusions (microchaetae and macrochaetae). Genetic interaction studies were confirmed and extended to show that nonmuscle myosin II and an unconventional myosin, encoded by crinkled (ck/MyoVIIA), act antagonistically in multiple processes necessary for wing development. Lastly, it was demonstrated that truncation alleles can perturb nonmuscle myosin II function via two distinct mechanisms -- by titrating light chains away from endogenous heavy chains or by recruiting endogenous heavy chains into intracellular aggregates. By allowing myosin II function to be perturbed in a controlled manner, these novel tools enable the elucidation of post-embryonic roles for nonmuscle myosin II during targeted stages of fly development (Franke, 2010).

The array of phenotypes caused by the directed expression of an allelic series of myosin II truncation constructs shows a variety of new roles for zip/MyoII in wing morphogenesis and confirms expected roles. Perturbing zip/MyoII function resulted in: ectopic and/or expanded vein and crossveins, the formation of multiple wing hairs instead of a single hair, the branching of individual wing hairs and the loss of adhesion between the dorsal and ventral wing epithelial cell sheets. An expected role of myosin II in cytokinesis was very likely the cause of reduced cell proliferation. Truncation allele expression in the thorax indicates these new roles for myosin II are not restricted to wing morphogenesis and likely extend to the correct morphogenesis of actin-based protrusions (hairs or setae; bristles or microchaetae and macrochaetae) throughout the fly (Franke, 2010).

During wing development, expression of these dominant negative truncation alleles cause wing blister phenotypes, which are also caused by mutations in integrin genes. Nonmuscle myosin II is known to participate in integrin-based adhesion of individual migrating cells to extracellular matrix and ablation of nonmuscle myosin IIA heavy chain caused cell-cell adhesion defects in early mouse embryogenesis. Nonmuscle myosin II also functions in the correct lateral arrangement and regulation of cells within a single epithelia. In Drosophila embryonic myofibril formation, correct nonmuscle myosin II localization requires a PS2 integrin. This study extends the role of nonmuscle myosin II in cell adhesion to include an essential role in the apical adhesion of two different epithelial cell sheets to one another. The loss of zip/MyoII function in one of the two wing epithelia sheets was sufficient to abolish adhesion, indicating that zip/MyoII function is necessary in both cell sheets. An accumulation of zip/MyoII at the dorsal-ventral compartment boundary in wing imaginal discs has been reported and may be important for myosin's role in the adhesion between these two cell sheets. Understanding how zip/MyoII functions and coordinates with known adhesion molecules in cell sheet adhesion is an important area for further investigation (Franke, 2010).

Multiple wing hair phenotypes were most easily quantified when GFP-zip/MyoII-Rod(ΔNterm58) was expressed. Like numerous other proteins, zip/MyoII appears to have a direct role in the production of a single hair from the distal vertex of each wing cell. Branching phenotypes were observed in wing hairs, setae and bristles (micro- and macrochaetae) when GFP-zip/MyoII-Rod(ΔNterm58), zip/MyoII-Rod or GFP-zip/MyoII-Neck-Rod was expressed. Branching phenotypes are not generally observed in planar cell polarity mutants and show that zip/MyoII plays a distinct, yet important role in the correct morphology of different actin-based cellular protrusions. Branching of individual hairs or bristles has been characterized in furry and tricornered mutants and can also result from drug treatments (cytochalasin D or latrunculin A) that affect the actin cytoskeleton. As bristles and wing hairs use different assembly strategies of actin bundles to generate their morphology, the simplest explanation of these results is that zip/MyoII plays an early role in these processes. Consistent with these findings, zip/MyoII mutants have defects in shaping and positioning of actin-based protrusions in the embryonic epidermis. Proteins known to contribute to planar polarity (e.g., the proximal and distal proteins Flamingo, Frizzled, Dishelveled, Diego, Strabismus, also known as Van Gogh, and Prickle) all appear to function upstream of the actin cytoskeleton and there are no reports of a direct physical interactions between these proteins and the actin cytoskeleton. As a consequence it is suspected that zip/MyoII functions downstream of planar polarity patterning. How it coordinates with proteins known to be involved in hair morphogenesis will require further investigation (Franke, 2010).

Ectopic and/or expanded vein and crossvein wing patterning phenotypes were observed with the expression of each truncation allele and in the absence of other phenotypes indicating that vein and crossvein positioning defects result directly from zip/MyoII perturbation. The most obvious defects observed were in the positioning of the posterior crossvein, resulting in both ectopic crossveins as well as expanded crossvein tissue. While several loci have been identified that affect vein and crossvein patterning, only a few give rise to ectopic or expanded tissue. Mutant forms of the Dachsous and fat protocadherins have been shown to shift the relative position of the anterior and posterior crossveins with respect to one another but do not cause ectopic tissue. Thus these findings suggest a potentially novel role for zip/MyoII in tissue patterning (Franke, 2010).

The truncation alleles developed in this study will be useful for the analysis of myosin function elsewhere in development. Indeed the current studies show roles for zip/MyoII in movements that contribute to other regions of the adult fly epidermis al well (Franke, 2010).

This study found that full-length zip/MyoII is required for correct function and localization in Drosophila, consistent with findings in S. pombe, which showed that truncations of its myosin heavy chains are not capable of rescue. This contrasts findings in S. cerevisiae where the tail region of myosin II can functionally substitute for full-length protein. The dependence of GFP-zip/MyoII-Neck-Rod on full-length zip/MyoII for localization could occur through two, not necessarily mutually exclusive, mechanisms. First, an individual GFP-zip/MyoII-Neck-Rod protein could heterodimerize with an endogenous, wild-type zip/MyoII heavy chain. Second, GFP-zip/MyoII-Neck-Rod proteins could form homodimers, which associate with endogenous zip/MyoII homodimers, and assemble into bipolar filaments. The results indicate that GFP-zip/MyoII-Neck-Rod heavy chains predominantly form homodimers (Franke, 2010).

The observation that zip/MyoII-HMM(ΔCterm407)-GFP does not induce a multiple wing hair or branching phenotype may provide insights into how zip/MyoII contributes to these processes. One possibility is that zip/MyoII rod-induced aggregates, which recruit endogenous zip/MyoII, may also recruit proteins important for the establishment of PCP thereby altering their subcellular localization. Immunostaining imaginal discs for known PCP proteins that express rod-containing zip/MyoII fragments could help address this (Franke, 2010).

The results demonstrate that nonmuscle myosin II function can be dominantly perturbed by two distinct mechanisms. Previous findings in D. discoidium, yeast, tissue culture cells and Drosophila have demonstrated that myosin II function can be perturbed by expression of truncation constructs. Expression of GFP-zip/MyoII-Rod(ΔNterm58) and zip/MyoII-Rod resulted in the formation of very large intracellular aggregates. Aggregation is consistent with in vitro findings that the light meromyosin region (LMM) is insoluble at physiological ionic strength. Consistent with this study, it has been shown that the formation of myosin II rod aggregates in D. discoidium were intracellular and could contain endogenous, full-length myosin II. Thus, expression of the rod domain recruits full-length nonmuscle myosin II into intracellular aggregates, thereby depleting the total cellular pool of functional nonmuscle myosin II (Franke, 2010).

The second mechanism results in the titration of free light chains from endogenous, full-length nonmuscle myosin II thereby depleting the total cellular pool of nonmuscle myosin II that can be regulated through light chains. A construct capable of function through both mechanisms (one containing both the tail and neck domains) is expected to be the most potent nonmuscle myosin II dominant negative. The results are consistent with this -- GFP-zip/MyoII-Neck-Rod consistently generated the most severe phenotypes. Moreover, these observations suggest that both mechanisms function to simply titrate endogenous, wild-type zip/MyoII heavy chain and are therefore comparable to loss of function alleles (Franke, 2010).

The C-terminal and N-terminal antisera enable semi-quantitative analysis of the expression of a truncation allele to endogenous zip/MyoII. Western blot analysis of whole 3rd instar larvae may be misleading as it does not provide a cellular context for comparing expression levels. Comparing the relative amount of fluorescence between a control region (endogenous zip/MyoII) and an experimental region (endogenous zip/MyoII and rod truncation allele; the current results likely provides a more accurate means for comparing expression -- expression of GFP-zip/MyoII-Neck-Rod is approximately two to three times that of zip/MyoII (Franke, 2010).

That GFP-zip/MyoII expression caused a mild wing phenotype is likely the consequence of heavy chain overexpression without comparable light chain expression. When GFP-zip/MyoII was placed in a heterozygous zipper background the penetrance of phenotypes decreased. The most parsimonious explanation is that the total amount of heavy chain (endogenous plus transgene) was reduced due to the heterozygous background, which helped alleviate the imbalance of heavy and light chains (Franke, 2010).

Having distinct truncation alleles that perturb zip/MyoII function to different extents enables one to screen for enhancers and suppressors of zip/MyoII in desired processes. Expressing of GFP-zip/MyoII-Rod(ΔNterm58) identified both genetic enhancers and suppressors of the multiple wing hair phenotype with components of the PCP pathway (Franke, 2010).

Previously, zip/MyoII was shown to genetically interact with dsh. The current results extend previous findings to include interactions with other PCP pathway genes (Fz and ck/MyoVIIA). The genetic interaction with Fz suggests that in addition to having a direct role in the production of a single wing hair, zip/MyoII may also participate in wing hair polarity. The genetic studies with ck/MyoVIIA show that it acts antagonistically to zip/MyoII with respect to the multiple wing hair phenotype and to other processes in wing morphogenesis resulting in more severe wing phenotypes (e.g., wing blisters). A role for myosin VIIs in cell adhesion has been demonstrated in different organisms. The mechanism(s) by which these heavy chains function in these different processes will require further investigation (Franke, 2010).

Each truncation allele fulfilled criteria of being specific zip/MyoII dominant-negatives: each caused phenotypes in a dose-dependent manner and these phenotypes were overlapping among the different truncation alleles. The allelic series of myosin II truncation constructs generated allow nonmuscle myosin II function to be variably perturbed in a cell, tissue and temporally specific, and therefore, conditional manner. Thus, these tools now make it possible to interrogate zip/MyoII function during any desired time interval or developmental process in Drosophila (Franke, 2010).

Dynamic myosin phosphorylation regulates contractile pulses and tissue integrity during epithelial morphogenesis

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).

Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension

Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. This study shows that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation (Simoes, 2014).

Rho-kinase is an essential regulator of actomyosin contractility, but the mechanisms that generate Rho-kinase asymmetry to produce spatially regulated forces during development are not well understood. This study shows that Rho GTPase signaling is required for the planar polarized localization of Rho-kinase and myosin II during Drosophila axis elongation. Direct interaction between Rho and Rho-kinase recruits Rho-kinase to adherens junctions but is not sufficient for full Rho-kinase planar polarity, suggesting that other mechanisms amplify the effects of Rho signaling. This study provides evidence that the actin-binding protein Shroom regulates Rho-kinase localization and planar polarized actomyosin contractility to promote sustained cell rearrangements during axis elongation. Shroom is present in a planar polarized distribution at adherens junctions in intercalating cells, consistent with a direct and localized function. Shroom planar polarity requires Rho activity, indicating that Shroom is an effector of Rho signaling. In Shroom mutants, Rho-kinase and myosin II junctional localization and planar polarity initiate normally but fail to be amplified and maintained during axis elongation. Consequently, planar polarized contractile forces and multicellular rosette rearrangements are reduced in Shroom mutants, resulting in decreased convergent extension. These results support a role for Shroom in regulating planar polarized actomyosin contractility and junctional remodeling during convergent extension, expanding the morphogenetic functions of this highly conserved protein beyond its known role in apical constriction (Simoes, 2014).

The data support a model in which Rho GTPase and Shroom have distinct functions in regulating Rho-kinase localization and planar polarized myosin contractility during convergent extension. Rho GTPase recruits Rho-kinase to adherens junctions and initiates planar polarity, and Shroom plays a modulatory role in enhancing and maintaining planar polarized myosin contractility downstream of Rho signaling. Rho GTPase binds to Rho-kinase and could regulate its localization directly. Rho does not bind to Shroom but may regulate Shroom planar polarity indirectly through its effect on the actin cytoskeleton. Rho-kinase, usually viewed as a downstream effector of Shroom, feeds back to maintain Shroom planar polarity and its own planar polarized localization. Rho-kinase could directly phosphorylate Shroom to reinforce planar cell polarity. Alternatively, Rho-kinase could promote Shroom localization through remodeling of the actin cytoskeleton, as the Shroom actin-binding domain is necessary and sufficient for targeting to planar junctions, and Rho-kinase can phosphorylate known regulators of actin (Simoes, 2014).

These findings may be relevant to neural tube development in vertebrates, which involves a combination of apical constriction, polarized junctional remodeling, and cell shape changes. Shroom3 is required for neural tube closure in the mouse, frog, and chick, and disrupting the interaction between Shroom and Rho-kinase reduces the number of rosettes in the chick neural plate. Unlike mutants that have disrupted rosette-based movements caused by defects in cell adhesion, the defects in Shroom mutants are likely a result of reduced myosin II activity. Rosette behaviors in Drosophila predominate midway through elongation at stage 8, coinciding with the stage when myosin becomes mislocalized in Shroom mutants. A failure to reinforce actomyosin contractility during elongation in Shroom mutants could selectively disrupt later-onset, higher-order cell rearrangements, with no effect on local neighbor exchange events that are more frequent at earlier stages. Alternatively, rosette formation may require more force, as rosettes form through the contraction of multicellular actomyosin cables that are under a higher level of tension and accumulate more myosin. In Shroom mutants, defects in myosin junctional localization may prevent contractile forces from reaching the levels necessary to produce rosette-based convergent extension movements. It will be interesting to explore whether planar polarized Shroom activity plays a general role in promoting junctional remodeling and enhancing mechanical force generation in processes that require strong actomyosin contractility during development (Simoes, 2014).

Rho GTPase signaling is an excellent candidate to break planar symmetry, as a small fraction of active Rho protein can trigger rapid and dramatic changes in the actin cytoskeleton. In one model, a subtle increase in Rho activity at AP cell boundaries could provide an instructive cue, guiding planar cell polarity by recruiting Rho-kinase, modifying the actin cytoskeleton, and facilitating the cortical association of the Rho-kinase regulator Shroom. Alternatively, Rho could regulate Rho-kinase planar polarity indirectly through its role in promoting Rho-kinase apical localization. Although it is challenging to visualize a small and highly dynamic population of active Rho protein in vivo, several findings support the idea that localized Rho activity could play an instructive role in planar polarity. First, myosin planar polarity and directional cell rearrangements occur normally at early stages in Shroom mutants, suggesting that other signals are able to generate localized myosin activity. The partial planar asymmetry of a fragment containing the RB domain of Rho-kinase, which is predicted to interact with the active pool of Rho GTPase, suggests that Rho could contribute to this asymmetry. Second, Rho is required for the planar polarized localization of Shroom, raising the possibility that Rho signaling could provide an essential source of Shroom asymmetry. Third, the upstream Rho activator RhoGEF2 in Drosophila and PDZ-RhoGEF in the chick display a subtle planar asymmetry during epithelial bending and elongation. Multiple activators and inhibitors of Rho could act together to generate a spatially localized pattern of Rho activity, as is the case for apical constriction. Notably, although Rho GTPase activity is necessary to establish Rho-kinase and myosin planar polarity, it is not sufficient to maintain their activity at high enough levels to allow sustained force generation and rosette rearrangements in Shroom mutants. It is proposed that Rho promotes the recruitment of Shroom as part of a positive feed-forward mechanism that reinforces planar polarized actomyosin contractility during convergent extension (Simoes, 2014).

Planar polarized cell rearrangements require the active maintenance of cell polarity in large populations of dynamically moving cells. This study shows that Shroom and Rho GTPase signaling play distinct roles in the establishment and maintenance of polarized actomyosin contractility during convergent extension. The upstream spatial cues that localize actomyosin contractility to specific planar cellular domains are not known. An asymmetry in the organization of the actin cytoskeleton is the earliest evidence of planar polarity in the Drosophila embryo. Distinct actin-binding domains in different Shroom isoforms have been proposed to target Shroom protein and its effectors to different regions of the cell. Moreover, the actin-binding domain is critical for Shroom planar polarity. These findings support the idea that an asymmetry in the actin cytoskeleton is an essential spatial input that regulates the localization of Shroom, the contractile machinery, and ultimately the forces that control cell rearrangement and tissue structure. The upstream spatial cues that generate these asymmetries could involve an asymmetry in Rho signaling, perhaps through the local activation of upstream signaling proteins that regulate Rho GTPase activity. Alternatively, the critical event in the establishment of planar cell polarity could be a Rho-independent reorganization of the actin cytoskeleton that biases the activity of Shroom, Rho-kinase, and myosin, which in turn modify the cytoskeleton to allow robust and sustained cell polarization. Elucidation of the upstream spatial cues that regulate actomyosin localization and dynamics will provide insight into the mechanisms that direct polarized cell behavior (Simoes, 2014).

The tricellular junction protein Sidekick regulates vertex dynamics to promote bicellular junction extension

Remodeling of cell-cell junctions drives cell intercalation that causes tissue movement during morphogenesis through the shortening and growth of bicellular junctions. The growth of new junctions is essential for continuing and then completing cellular dynamics and tissue shape sculpting; however, the mechanism underlying junction growth remains obscure. This study investigated Drosophila genitalia rotation where continuous cell intercalation occurs to show that myosin II accumulating at the vertices of a new junction is required for the junction growth. This myosin II accumulation requires the adhesive transmembrane protein Sidekick (Sdk), which localizes to the adherens junctions (AJs) of tricellular contacts (tAJs). Sdk also localizes to and blocks the accumulation of E-Cadherin at newly formed growing junctions, which maintains the growth rate. It is proposed that Sdk facilitates tAJ movement by mediating myosin II-driven contraction and altering the adhesive properties at the tAJs, leading to cell-cell junction extension during persistent junction remodeling (Uechi, 2019).

To generate tissue shapes, cell collectives show various dynamics, such as cell division and cell deformation. Among them, cell intercalation is a multicellular behavior in which cells change their position through the remodeling of cell-cell contacts, leading to the directional elongation and expansion of tissues across species. Especially in epithelia, this cell-cell junction remodeling involves the shortening and loss of bicellular junctions and the subsequent growth of bicellular junctions in a new direction. Junction shortening initiates tissue dynamics and is driven in a conserved manner by contractile forces generated by actomyosin (actin and non-muscle myosin II complex) associating with the cadherin-catenin core complex, including E-Cadherin, β-Catenin, and other related adherens junction (AJ) components, at the AJs of shortening junctions. Junction growth is also essential for continuing and then completing cellular dynamics and tissue shape sculpting. Several studies using flies have suggested that myosin II has a role in junction growth during developmental events. In the germ band, medial pulses of myosin II in the cells surrounding junctions and toward the posterior ectoderm regulate junction growth during cell intercalation-driven convergent extension [germ band extension (GBE)]. A similar contribution of myosin II pulses in the surrounding cells to junction extension is also observed in the apical cell oscillation of amnioserosa cells during dorsal closure. In developing wing epithelia, a decrease in myosin II levels at newly formed junctions facilitates junction growth to organize the epithelial cellular pattern. However, despite its importance, the mechanisms underlying junction growth remain unclear, in contrast to junction shortening (Uechi, 2019).

Previous studies demonstrated that cell intercalation also contributes to the tissue rotational movement observed for Drosophila male genitalia. The fly genitalia are located at the animal's posterior end, and the male genitalia are surrounded by epithelia known as the A8 segment at the anterior side. At 24 h after puparium formation (APF), the genitalia and the A8 epithelia begin dextral rotation that terminates at around 36 h APF. The rotation consists of an initial 180° movement of the posterior compartment of A8 (A8p) along with the genitalia and a subsequent 180° movement of the anterior component of A8 (A8a), the latter of which starts at around 26 h APF. From 26 h APF in the A8a cells, myosin II accumulates to a greater extent at AJs, forming a right oblique angle with the anterior-posterior (AP) axis than at junctions forming a left oblique angle. This polarized myosin II distribution gives rise to right-biased junction shortening in relation to the AP axis and leads to left-right asymmetric cell intercalation, which is persistently observed during the movement. By combining numerical simulations, it was demonstrated that this repeated junction remodeling in the confined space generates the A8a movement. In this movement, newly formed junctions are sufficiently elongated within a certain time frame to execute the next round of cell intercalation. Incomplete genitalia rotation leads to male sterility (Uechi, 2019).

This study performed time-lapse imaging, developed an optogenetic tool, and analyzed the adhesive protein Sidekick (Sdk), which is known to regulate retinal development in flies and mice and showed that myosin II accumulating at the tricellular contacts (tAJs) of growing junctions is required for bicellular junction growth in A8a cells. Also, Sdk regulates the myosin II and E-Cadherin distributions at the tAJs, thereby maintaining the junction growth rate. These findings suggest that the tAJ is a specialized point promoting cell-cell junction extension (Uechi, 2019).

The process of junction shortening is well characterized and is organized by the contractile forces of actomyosin, which is transmitted to cell-cell contacts via AJ components, such as E-Cadherin. These proteins have important roles in the dynamics of multicellular deformation. Since junction formation and growth are important for the continuation and completion of multicellular dynamics and tissue architecture shaping, it is likely that active mechanisms underlie the extension of cell-cell junctions. Indeed, recent reports suggest that actin and myosin II at the bicellular junctions are involved in the junction extension in cell rearrangement and in cell-shape formation during Drosophila wing and eye development. Polarized medial pulses of myosin II in the cells surrounding junctions regulate junction extension in the Drosophila germ band and amnioserosa. This study used an optogenetic tool that allows for the spatiotemporal inactivation of endogenous myosin II and revealed that myosin II accumulating at the tAJs of newly formed junctions is required for junction growth in the A8a epithelia. This study also demonstrated that the myosin II accumulation and junction growth require the tAJ-localizing protein Sdk. Thus, this report that tAJs are an additional point promoting the extension of bicellular junctions (Uechi, 2019).

Sdk transiently localizes to newly formed junctions as well as tAJs, causing a downregulation of E-Cadherin and a slight increase in intercellular spaces at the AJs of growing junctions, indicative of less tight cell-cell contacts. Recent studies in zebrafish showed that the presence of extracellular spaces and the disassembly of cell-cell contacts contribute to fluidize tissues. During body axis elongation, the extracellular spaces render mesodermal cells fluidized and uncaged and associated with large fluctuations in the lengths of cell-cell contact. Decreases in cell-cell contacts through the destabilization of junctional E-Cadherin, accompanied by an increase in extracellular spaces, induces the fluidization of blastoderm cells and consequently allows blastoderm spreading at the onset of morphogenesis. Analogous to these properties, it is possible that the presence of Sdk at growing junctions confers flexible dynamics to the cell-cell contacts at the level of the AJs of the growing junctions. This study proposes mechanisms of junction growth in which Sdk has dual roles. First, Sdk mediates a driving force of junction growth by anchoring myosin II at tAJs; the contractility of the actomyosin then retracts the membrane of the surrounding cells at tAJs. Second, Sdk assists in the myosin II-driven junction growth by localizing to and decreasing the accumulation of E-Cadherin at the growing junctions and their tAJs; this composition of E-Cadherin and Sdk causes contacts between the vertices of the surrounding cells and the cells forming the growing junction to be less tight. Such adhesion can render the tAJs of growing junctions more sensitive to contractile forces at the vertices of the surrounding cells, supporting the retraction of the membrane of the surrounding cells at tAJs. The latter mechanism is indeed likely to contribute to junction growth since inducing sdk RNAi only in the cell forming the growing junction was sufficient to reduce the junction growth rate, even when the surrounding cells consisted of WT cells (Uechi, 2019).

The precise mechanism by which Sdk blocks the accumulation of E-Cadherin at newly formed junctions is still unclear. While Sdk was already present at growing junctions from the step of four-way vertex resolution, E-Cadherin would be newly recruited to the growing junctions since E-Cadherin is removed from remodeling junctions by endocytosis during junction shortening. A recent study using fluorescence recovery after photobleaching (FRAP) revealed two ways that E-Cadherin is re-distributed to cell-cell junctions, lateral diffusion within the plasma membrane and delivery from the cytoplasm by vesicular trafficking. Since (1) E-Cadherin and Sdk did not interact despite their localization to AJs, (2) showed complementary distributions at newly formed junctions and even at cellular edges in S2 cells where they were ectopically expressed, and (3) changed their distributions when the other protein was depleted, it is possible that there are repelling forces between E-Cadherin and Sdk molecules, which cause them to exclude each other and may delay the diffusion of E-Cadherin from neighboring junctions into newly formed junctions, where Sdk is already enriched. However, this study does not exclude another possibility that Sdk inhibits machineries that deliver E-Cadherin from the cytoplasm, such as blocking their access to growing junctions or biochemically inactivating them (Uechi, 2019).

This study observed the accumulation of myosin II and decreased E-Cadherin levels at the tAJ of growing junctions. These distributions resemble those occurring during new cell-cell contact formation between daughter cells in epithelia. After cytokinesis, myosin II accumulates at the edges of new cell-cell junctions in the neighboring cells of the daughter cells, in response to the local decrease in E-Cadherin levels at these edges, which participates in new cell-cell junction formation. These reports and the current observations suggest a possible common mechanism underlying new cell-cell contact formation among epithelial multicellular behaviors. Although the dynamics and roles of Sdk in cell division are still unclear, an intriguing possibility is that Sdk regulates the dynamics of new cell-cell junctions in concert with myosin II and E-Cadherin not only in the context of cell intercalation but also global epithelial dynamics including cytokinesis (Uechi, 2019).

Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability

Tissue remodelling during Drosophila embryogenesis is notably driven by epithelial cell contractility. This behaviour arises from the Rho1-Rok-induced pulsatile accumulation of non-muscle myosin II pulling on actin filaments of the medioapical cortex. While recent studies have highlighted the mechanisms governing the emergence of Rho1-Rok-myosin II pulsatility, little is known about how F-actin organization influences this process. This study shows that the medioapical cortex consists of two entangled F-actin subpopulations. One exhibits pulsatile dynamics of actin polymerization in a Rho1-dependent manner. The other forms a persistent and homogeneous network independent of Rho1. The formin Frl (also known as Fmnl) has been identified as a critical nucleator of the persistent network, since modulating its level in mutants or by overexpression decreases or increases the network density. Absence of this network yields sparse connectivity affecting the homogeneous force transmission to the cell boundaries. This reduces the propagation range of contractile forces and results in tissue-scale morphogenetic defects (Dehapiot, 2020).

Animal cells can modify their shape to complete complex processes such as cell migration, division or tissue morphogenesis. These behaviours arise from the contractile properties of the actomyosin cortex and its ability to build up tension. Recent advances have shown that cortical contractility can occur in a pulsatile manner by taking the form of local and transient accumulations of myosin II (MyoII). These MyoII pulses underlie a variety of morphogenetic processes, ranging from single-cell polarization to tissue-scale remodelling. Although recent evidence suggest that MyoII pulsatility can spontaneously emerge6, the spatiotemporal pattern of cortical contractility must be controlled to produce reproducible morphogenetic outcomes. In most studied systems, this control is achieved through the conserved RhoA GTPase signalling pathway, which activates MyoII through the regulation of Rho-associated kinase (ROCK; Rok in Drosophila) and MyoII light-chain phosphatase (Dehapiot, 2020).

In addition to MyoII regulation, another key parameter that influences cortical contractility resides in the organization and dynamics of the F-actin network. Typically, the cortex assembles as a thin layer of actin filaments bound to the plasma membrane. The cortical network is both highly plastic and mechanically rigid, conferring to the cells the ability to adapt and exert forces on their environment. These remarkable properties stem from the action of actin-binding proteins (ABPs) regulating the organization and turnover of the network. Actin nucleators, such as the Arp2/3 complex or formins, promote filament polymerization that leads, respectively, to the assembly of highly branched or sparse F-actin networks. These networks can be remodelled by actin bundlers (fascin and plastin) or cross-linkers (filamin and α-actinin), and their filament turnover regulated by profilin, capping proteins or members of the actin-depolymerizing factor and cofilin families. Modulating the dynamic organization of F-actin networks through ABPs can significantly modify how MyoII contractility gives rise to cortical tension (Dehapiot, 2020).

In embryonic Drosophila epithelial cells, MyoII pulses appear in the medioapical (medial) part of the cell and produce sustained or repeated cycles of apical contraction and relaxation. MyoII pulsatility, together with adherens junction (AJ) remodelling, gives rise to a variety of tissue morphogenetic events such as mesoderm-endoderm invagination, convergent extension or tissue dorsal closure (DC). While the mechanisms underlying the emergence of MyoII pulsatility have been widely studied, little is known about how medioapical F-actin supports pulsatile contractility. It has been shown that the spatiotemporal organization of F-actin modifies the viscoelastic properties of the cortex and its ability to propagate tension. Cortical F-actin can also influence MyoII activation by serving as a scaffold for the motor-driven advection of regulators such as Rho1, Rok or Rho GTPase-activating proteins (RhoGAPs), which are required for pulse assembly and disassembly. In this study, focusing on two highly pulsatile tissues, namely the ectodermal cells during germband extension (GBE) and amnioserosa cells during DC, the regulation of the medioapical F-actin network was study and attempts were made to understand how it supports the propagation of contractile forces to the surrounding tissue (Dehapiot, 2020).

While most studies of cortical pulsed contractility have focused on the emergence of MyoII pulsatility, this study examined how cortical F-actin influences this process in embryonic Drosophila epithelial cells. In both ectodermal (GBE) and amnioserosa cells (DC) this study showed that the medioapical cortex consists of two differentially regulated but entangled subpopulations of actin filaments. These two populations share the same subcellular localization but undergo distinct spatiotemporal dynamics. The Rho1-induced pulsatile F-actin, together with MyoII, promotes local cell deformations, while the persistent network ensures homogeneous connectivity between pulses and AJs and hence spatial propagation of deformation (Dehapiot, 2020).

The formin Frl was identified as a critical nucleator that promotes the assembly of the persistent network. This constitutes a previously unappreciated role for this formin, since it has been so far mainly described as participating in lamellipodia and filopodia formation. It would be interesting to know whether, like in other systems, Frl is regulated by Cdc42 or Rac1 to promote the persistent network assembly. Furthermore, it is probable that other formins are involved in this process, since the lack of Frl only partially reduced the network density in ectodermal cells. The formin DAAM would constitute a good candidate, since it cooperates with Frl during axon growth in Drosophila (Dehapiot, 2020).

This study also showed that Frl antagonizes apical cell contractility by impairing Rho1 signalling in amnioserosa cells. While this antagonism may depend on cell context, it will be interesting to identify the crosstalk mechanisms operating between Frl and the GTPase. To this end, previous studies report that F-actin can negatively feedback on Rho1 activation. Indeed, it is possible that, like in the Caenorhabditis elegans zygote, some Rho1 inhibitors (for example, RhoGAPs) bind to cortical F-actin in the systems examined in this study. Consequently, modifying the persistent network density, through Frl loss or gain of function, could in turn modulate the levels of apical Rho1 activation. It has also been shown that advection acts as a positive feedback for pulsatility by increasing the local concentration of upstream regulators (for example, Rho1 and Rok) (Dehapiot, 2020).

It will therefore be interesting to study how the persistent network influences this feedback mechanism. The current data also revealed that modulating Frl levels affects epithelial dynamics at the cellular and tissue scales. Although this is probably influenced by the effect of Frl on medial actomyosin pulsatility and/or MyoII junctional density, this study designed a series of analyses to understand how the persistent network may influence pulsed contractility in mechanical terms. It has been suggested that medioapical F-actin acts as a scaffold to transmit contractile forces to AJs and, by extension, to the surrounding tissue. The results revealed that the persistent network does indeed play a key role in this process by promoting the uniform and long-range propagation of contractile forces. A numerical model was desigened to qualitatively recapitulate the experimental measurements and to provide solid evidence that Frl influences epithelial dynamics through the persistent network (Dehapiot, 2020).

Tissue morphogenesis requires interactions between cellular-and tissue-scale deformations, the propagation of which in space and time are little understood. This study showed that differentially regulated subpopulations of actin filaments play a key role in this process by promoting distinctly the emergence and the spatial propagation of cortical deformations. The findings echo previous experimental and theoretical studies demonstrating that the F-actin network, through its cross-linking state, the length of its filaments or its turnover, can mediate the amplitude and the length scale at which cortical stresses propagate. It will be important to unravel how cells tune these properties in different tissues and developmental stages to further understand how mechano-chemical information drives embryo morphogenesis (Dehapiot, 2020).

Drosophila motor neuron boutons remodel through membrane blebbing coupled with muscle contraction

Wired neurons form new presynaptic boutons in response to increased synaptic activity, however the mechanism(s) by which this occurs remains uncertain. Drosophila motor neurons (MNs) have clearly discernible boutons that display robust structural plasticity, being therefore an ideal system in which to study activity-dependent bouton genesis. This study showed that in response to depolarization and in resting conditions, MNs form new boutons by membrane blebbing, a pressure-driven mechanism that occurs in 3-D cell migration, but not previously described to occur in neurons. Accordingly, F-actin is decreased in boutons during outgrowth, and non-muscle myosin-II is dynamically recruited to newly formed boutons. Furthermore, muscle contraction plays a mechanical role, which is hypothesized to promotes bouton addition by increasing MN confinement. Overall, this study identified a mechanism by which established circuits form new boutons allowing their structural expansion and plasticity, using trans-synaptic physical forces as the main driving force (Fernandes, 2023).

Dissection of the mechanisms that regulate activity-dependent bouton formation is critical to understand remodeling strategies required for neuronal growth and wiring. Using live imaging of the Drosophila larval NMJ, a system in which 3-D intercellular and biophysical interactions are preserved, this study showed that bouton formation occurred by membrane blebbing, a mechanism widely used in 3-D cellular migration but, to our knowledge, never reported to be used by neurons to remodel. This study showed that new boutons induced by elevated activity, also called ghost boutons (GB), are bona fide blebs, whose growth does not rely on actin polymerization but is rather pressure driven. Additionally, it was observed that activity-dependent bouton formation was frequently associated with muscle activity and that blocking muscle contraction significantly decreased GB frequency after stimulation. Moreover, the manipulation of synaptic activity and/or muscle contraction resulted in predictable changes in bouton formation in response to stimulation, with a progressive increase in new bouton numbers with higher levels of synaptic activity and/or muscle contraction (Fernandes, 2023).

The results suggest that muscle contraction plays a mechanical role in activity-dependent bouton formation. At the NMJ, the MN is deeply imbedded in the muscle, which by contracting can directly alter neuronal confinement. It is hypothesized that with elevated activity, and in response to a still unclear pre and/or postsynaptic signal to initiate bouton outgrowth, MNs add new boutons by membrane blebbing, and muscle contraction is required to increase neuronal confinement. This confinement results in increased pressure onto the MN membrane, powering bouton outgrowth at places putatively primed for bouton formation. In accordance, it is known that blebs are favored in conditions of high confinement, namely in tissues where cells encounter increased mechanical resistance. Furthermore, cells in compressive 3-D environments naturally go through stiffness gradients, which can polarize the recruitment of molecules required to or that facilitate blebbing in spots of high membrane tension. Likewise, at the NMJ it is possible that regions of higher activity produce more signals for inducing synaptic growth, which is supported by previous studies that showed that synaptic plasticity at the NMJ, including GB formation, requires activity-dependent secretion of postsynaptic signals such as BMP ligands (Maverick and Gbb) and Ca2+-sensitive vesicle regulator Syt-IV, presynaptic Wg and Syt-I mediated NT release and postsynaptic glutamate receptor function. It will be interesting to investigate whether activation of pathways downstream of these factors can converge to determine sites primed for bouton initiation with synaptic activity, by recruiting initiation factors and/or changing cytoskeletal dynamics at these regions (Fernandes, 2023).

Cells migrating in confined environments typically display rounder morphologies and use hydrostatic blebs for movement. Interestingly, it has been shown that neurons from mouse central nervous system (CNS) growing in 3-D matrices, rather than 2-D surfaces, displayed GCs that had an amoeboid-like morphology, characterized by very low adhesive interactions and extensive rounded deformations of the body-wall, challenging the paradigm of the lamellipodia based GC. In the brain, even though there are no muscle compressive forces, synaptic boutons are equally confined and surrounded by other neuronal, astrocytic, or microglial processes. Interestingly, a recent study showed that in the mammalian brain, enlargement of dendritic spines produces mechanical pressure onto boutons thereby enhancing NT release, thus contributing to synaptic strength (a force reported to be comparable to that of muscle contraction). This exciting discovery is in line with the hypothesis that mechanical force directly regulates synaptic function, or as in the case of this study, formation of new boutons. It will be interesting to study how neurons can sense and respond to mechanical signals provided by the cellular environment, and if pathways used for mechanosensing and transduction, likely regulating adhesion and contacts with extracellular partners, are coupled with synaptic transmission machinery during plasticity (Fernandes, 2023).

While the molecular identity of the signal that leads to bouton initiation remains to be discovered, it is well established that blebs nucleate when a small patch of membrane is detached from the actin cortex. This can happen either as a direct consequence of buildup in hydrostatic pressure (that detaches membrane to cortex binding proteins) or by formation of local gaps, resulting from rupture of the cortex in regions of high membrane energy (where accumulation of MyoII helps to weaken the cortex). Our data suggests that, although at the NMJ the two mechanisms may coexist, with elevated activity boutons tend to form mainly because of compressive forces exerted by the muscle. Supporting this, it was found that activity-induced bouton formation was usually fast and correlated with visible muscle contraction, while events with low or no visible muscle contraction were slower and occasional. Additionally, post-stimulation, even though MyoII puncta were observed preceding bouton formation in ~35% of events, boutons were found forming with low MyoII levels and decreasing or inactivation of MyoII did not prevent bouton formation, which suggests that MyoII was not required to propel bouton growth induced by NMJ stimulation (although this result can not directly be extended to a null scenario). Interestingly, MyoII was recruited to ghost boutons in stimulated and unstimulated NMJs, which may explain cases where boutons formed with lessened muscle contractions. The fact that boutons always formed without filamin, which links actin to membrane, further supported that actin polymerization is not required for bouton initiation. Altogether a model is favored in which boutons form in regions of high pressure by detachment of membrane from the actin cortex and that local MyoII activation can facilitate the weaking of the cortex at these sites. Importantly, regardless of the exact signal used to initiate activity-dependent bouton formation, this study showed that growth and stabilization of these boutons requires both actin and MyoII local rearrangements analogous to what is reported for cellular blebs: weakened actomyosin cortex in early-stage boutons and enrichment after expansion (Fernandes, 2023).

This model explains the formation of new synaptic boutons by a physical process-membrane blebbing, which in addition to the more studied transcriptional or biochemical factors, allows fast, on demand expansion of the NMJ. Whether axons are always competent for bleb-dependent bouton expansion or require priming by cytoskeletal regulation remains to be elucidated. Moreover, considering the conservation of presynaptic cytoskeletal components and bouton ultrastructure throughout evolution, it is postulated that this mechanism of synapse remodeling can be present in other organisms, including vertebrates. Overall, the finding that bouton addition at the NMJ occurs as result of a MN-muscle physical interplay highlights the importance of intercellular cooperation during plastic changes. Circuit remodeling as a response to experience requires rapid modulations of bouton number. We speculate that the regulation of confinement by non-neuronal cells (muscle or glia) can be a mechanism widely used by the nervous system to coordinate local activity-dependent structural changes in neurons with its surrounding 3-D cellular microenvironment. Future studies will elucidate whether the understanding of the biochemical and mechanical relations between neurons and their neighboring cells during structural plasticity can help design new strategies to remodel neuronal circuits that have been impaired by neurodevelopmental or neurodegenerative diseases (Fernandes, 2023).


The nonmuscle myosin heavy chain from Drosophila contains an alternatively spliced exon at the 5' end which generates two distinct heavy-chain transcripts: the longer transcript inserts an additional start codon upstream of the primary translation start site and encodes a myosin heavy chain with a 45-residue extension at its amino terminus. The shorter transcript is 3.5 times more abundant than the longer one (Ketchum, 1990).

A second alternative exon (40aa) is close to the nucleotide binding pocket. The position, size and sequence of this exon is conserved in D. simulans and putative alternative exons of different size (7 to 16 aa), but identical positions have been reported for other myosins in many phyla. The functional significance of either alternative splice is not clear (Mansfield, 1996)

Bases in 5' UTR - 96 or 197

Exons - 14

Bases in 3' UTR - 188


Amino Acids - 2057

Structural Domains

The coding sequence of zipper reveals extensive homology with other conventional myosins, especially metazoan nonmuscle and smooth muscle myosin isoforms. Comparisons among available myosin heavy-chain sequences establish that characteristic differences in sequence throughout the length of both the globular myosin head and extended rod-like tail readily distinguish nonmuscle and smooth muscle myosins from striated muscle isoform,s and predict a basis for their functional diversity. The ATP binding region is in the first 200 amino acids. The actin binding region is within 30 amino acids centered on amino acid 670. The myosin tail includes the 1085 residues from leucine-843 to leucine-1927 and is characterized by a strong heptad repeat, common to conventional myosin tails and other alpha-helical coiled-coil proteins. These regions dimerize and fold into the extended rod-like tail of the native Drosophila myosin molecule. The hinge region is in the center of the tail sequence. The terminal 47 amino acids consist of the globular tail region. The tail region contains putative casein kinase II and protein kinase C phosphorylation sites (Ketchum, 1990 and Mansfield, 1996).

zipper : Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised:  1 January 2024

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