Gene name - zipper
Synonyms - myosin II
Cytological map position - 60E9-F1
Function - myosin heavy chain
Symbol - zip
Genetic map position - 2-
Classification - nonmuscle myosin
Cellular location - cytoplasmic
|Recent literature||Ochoa-Espinosa, A., Harmansa, S., Caussinus, E. and Affolter, M. (2017). Myosin II is not required for Drosophila tracheal branch elongation and cell intercalation. Development 144(16): 2961-2968. PubMed ID: 28811312
The Drosophila tracheal system consists of an interconnected network of monolayered epithelial tubes that ensures oxygen transport in the larval and adult body. During tracheal dorsal branch (DB) development, individual DBs elongate as a cluster of cells, led by tip cells at the front and trailing cells in the rear. Branch elongation is accompanied by extensive cell intercalation and cell lengthening of the trailing stalk cells. Although cell intercalation is governed by Myosin II (MyoII)-dependent forces during tissue elongation in the Drosophila embryo that lead to germ-band extension, it remained unclear whether MyoII plays a similar active role during tracheal branch elongation and intercalation. This study used a nanobody-based approach to selectively knock down MyoII in tracheal cells. The data show that, despite the depletion of MyoII function, tip cell migration and stalk cell intercalation (SCI) proceed at a normal rate. This confirms a model in which DB elongation and SCI in the trachea occur as a consequence of tip cell migration, which produces the necessary forces for the branching process.
|Clement, R., Dehapiot, B., Collinet, C., Lecuit, T. and Lenne, P. F. (2017). Viscoelastic dissipation stabilizes cell shape changes during tissue morphogenesis. Curr Biol 27(20): 3132-3142. PubMed ID: 28988857
Tissue morphogenesis relies on the production of active cellular forces. Understanding how such forces are mechanically converted into cell shape changes is essential to understanding of morphogenesis. This study used myosin II pulsatile activity during Drosophila embryogenesis to study how transient forces generate irreversible cell shape changes. Analyzing the dynamics of junction shortening and elongation resulting from myosin II pulses, this study found that long pulses yield less reversible deformations, typically a signature of dissipative mechanics. This is consistent with a simple viscoelastic description, which was used to model individual shortening and elongation events. The model predicts that dissipation typically occurs on the minute timescale, a timescale commensurate with that of force generation by myosin II pulses. This estimate was tested by applying time-controlled forces on junctions with optical tweezers. Finally, it was shown that actin turnover participates in dissipation, as reducing it pharmacologically increases the reversibility of contractile events. These results argue that active junctional deformation is stabilized by actin-dependent dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation.
|Streichan, S. J., Lefebvre, M. F., Noll, N., Wieschaus, E. F. and Shraiman, B. I. (2018). Global morphogenetic flow is accurately predicted by the spatial distribution of myosin motors. Elife 7. PubMed ID: 29424685
During embryogenesis tissue layers undergo morphogenetic flow rearranging and folding into specific shapes. While developmental biology has identified key genes and local cellular processes, global coordination of tissue remodeling at the organ scale remains unclear. This study combined in toto light-sheet microscopy of the Drosophila embryo with quantitative analysis and physical modeling to relate cellular flow with the patterns of force generation during the gastrulation process. The complex spatio-temporal flow pattern can be predicted from the measured meso-scale myosin density and anisotropy using a simple, effective viscous model of the tissue, achieving close to 90% accuracy with one time dependent and two constant parameters. This analysis uncovers the importance of a) spatial modulation of myosin distribution on the scale of the embryo and b) the non-locality of its effect due to mechanical interaction of cells, demonstrating the need for the global perspective in the study of morphogenetic flow.
|Qin, X., Hannezo, E., Mangeat, T., Liu, C., Majumder, P., Liu, J., Choesmel-Cadamuro, V., McDonald, J. A., Liu, Y., Yi, B. and Wang, X. (2018). A biochemical network controlling basal myosin oscillation. Nat Commun 9(1): 1210. PubMed ID: 29572440
The actomyosin cytoskeleton, a key stress-producing unit in epithelial cells, oscillates spontaneously in a wide variety of systems. Although much of the signal cascade regulating myosin activity has been characterized, the origin of such oscillatory behavior is still unclear. This study shows that basal myosin II oscillation in Drosophila ovarian epithelium is not controlled by actomyosin cortical tension, but instead relies on a biochemical oscillator involving ROCK and myosin phosphatase. Key to this oscillation is a diffusive ROCK flow, linking junctional Rho1 to medial actomyosin cortex, and dynamically maintained by a self-activation loop reliant on ROCK kinase activity. In response to the resulting myosin II recruitment, myosin phosphatase is locally enriched and shuts off ROCK and myosin II signals. Coupling Drosophila genetics, live imaging, modeling, and optogenetics, this study uncovered an intrinsic biochemical oscillator at the core of myosin II regulatory network, shedding light on the spatio-temporal dynamics of force generation.
|Urbano, J. M., Naylor, H. W., Scarpa, E., Muresan, L. and Sanson, B. (2018). Suppression of epithelial folding at actomyosin-enriched compartment boundaries downstream of Wingless signalling in Drosophila. Development 145(8). PubMed ID: 29691225
Epithelial folding shapes embryos and tissues during development. This study investigated the coupling between epithelial folding and actomyosin-enriched compartmental boundaries. The mechanistic relationship between the two is unclear, because actomyosin-enriched boundaries are not necessarily associated with folds. Also, some cases of epithelial folding occur independently of actomyosin contractility. Shallow folds called parasegment grooves that form at boundaries between anterior and posterior compartments in the early Drosophila embryo were investigated. Formation of these folds requires the presence of an actomyosin enrichment along the boundary cell-cell contacts. These enrichments, which require Wingless signalling, increase interfacial tension not only at the level of the adherens junctions but also along the lateral surfaces. Epithelial folding is normally under inhibitory control because different genetic manipulations, including depletion of the Myosin II phosphatase Flapwing, increase the depth of folds at boundaries. Fold depth correlates with the levels of Bazooka (Baz), the Par-3 homologue, along the boundary cell-cell contacts. Moreover, Wingless and Hedgehog signalling have opposite effects on fold depth at the boundary that correlate with changes in Baz planar polarity.
|Dahl-Halvarsson, M., Olive, M., Pokrzywa, M., Ejeskar, K., Palmer, R. H., Uv, A. E. and Tajsharghi, H. (2018). Drosophila model of myosin myopathy rescued by overexpression of a TRIM-protein family member. Proc Natl Acad Sci U S A. PubMed ID: 29946036
Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in MYH7 encoding slow/beta-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the alpha-helical rod domain of MYH7 are mainly associated with Laing distal myopathy. A Drosophila model of Laing distal myopathy was developed by genomic engineering of the Drosophila Mhc locus. Flies expressing only Mhc(K1728del) in indirect flight and jump muscles, and heterozygous Mhc(K1728del) animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of Mhc(K1728del) mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous Mhc(K1728del) larvae also demonstrated structural and functional impairments in heart muscle. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with Mhc(K1728del) were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in Drosophila recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. These observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy.
|Diaz-de-la-Loza, M. D., Ray, R. P., Ganguly, P. S., Alt, S., Davis, J. R., Hoppe, A., Tapon, N., Salbreux, G. and Thompson, B. J. (2018). Apical and basal matrix remodeling control epithelial morphogenesis. Dev Cell 46(1): 23-39.e25. PubMed ID: 29974861
Epithelial tissues can elongate in two dimensions by polarized cell intercalation, oriented cell division, or cell shape change, owing to local or global actomyosin contractile forces acting in the plane of the tissue. In addition, epithelia can undergo morphogenetic change in three dimensions. This study shows that elongation of the wings and legs of Drosophila involves a columnar-to-cuboidal cell shape change that reduces cell height and expands cell width. Remodeling of the apical extracellular matrix by the Stubble protease and basal matrix by MMP1/2 proteases induces wing and leg elongation. Matrix remodeling does not occur in the haltere, a limb that fails to elongate. Limb elongation is made anisotropic by planar polarized Myosin-II, which drives convergent extension along the proximal-distal axis. Subsequently, Myosin-II relocalizes to lateral membranes to accelerate columnar-to-cuboidal transition and isotropic tissue expansion. Thus, matrix remodeling induces dynamic changes in actomyosin contractility to drive epithelial morphogenesis in three dimensions.
|Vanderleest, T. E., Smits, C. M., Xie, Y., Jewett, C. E., Blankenship, J. T. and Loerke, D. (2018). Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during Drosophila germband extension. Elife 7. PubMed ID: 29985789
Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell-cell interfaces, while the function of tricellular vertices has remained unaddressed. This study identifies a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, it was found that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. These results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation.
|Manieu, C., Olivares, G. H., Vega-Macaya, F., Valdivia, M. and Olguin, P. (2018). Jitterbug/Filamin and Myosin-II form a complex in tendon cells required to maintain epithelial shape and polarity during musculoskeletal system development. Mech Dev. PubMed ID: 30213743
During musculoskeletal system development, mechanical tension is generated between muscles and tendon-cells. This tension is required for muscle differentiation and is counterbalanced by tendon-cells avoiding tissue deformation. Both, Jbug/Filamin, an actin-meshwork organizing protein, and non-muscle Myosin-II (Myo-II) are required to maintain the shape and cell orientation of the Drosophila notum epithelium during flight muscle attachment to tendon cells. This study shows that halving the genetic dose of Rho kinase (Drok), the main activator of Myosin-II, enhances the epithelial deformation and bristle orientation defects associated with jbug/Filamin knockdown. Drok and activated Myo-II localize at the apical cell junctions, tendon processes and are associated to the myotendinous junction. Further, it was found that Jbug/Filamin co-distribute at tendon cells with activated Myo-II. Finally, it was found that Jbug/Filamin and Myo-II are in the same molecular complex and that the actin-binding domain of Jbug/Filamin is necessary for this interaction. These data together suggest that Jbug/Filamin and Myo-II proteins may act together in tendon cells to balance the tension generated during development of muscles-tendon interaction, maintaining the shape and polarity of the Drosophila notum epithelium.
|Krueger, D., Tardivo, P., Nguyen, C. and De Renzis, S. (2018). Downregulation of basal myosin-II is required for cell shape changes and tissue invagination. EMBO J 37(23). PubMed ID: 30442834
Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin-mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. This study used optogenetics to quantitatively control myosin-II levels at the basal surface of invaginating cells during Drosophila gastrulation. While basal myosin-II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre-invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube-like structure. Collectively, these data demonstrate the requirement of myosin-II polarization and basal relaxation throughout the entire invagination process.
|Das, S., Kumar, P., Verma, A., Maiti, T. K. and Mathew, S. J. (2019). Myosin heavy chain mutations that cause Freeman-Sheldon syndrome lead to muscle structural and functional defects in Drosophila. Dev Biol. PubMed ID: 30826400
Missense mutations in the MYH3 gene encoding myosin heavy chain-embryonic (MyHC-embryonic) have been reported to cause two skeletal muscle contracture syndromes, Freeman Sheldon Syndrome (FSS) and Sheldon Hall Syndrome (SHS). Two residues in MyHC-embryonic that are most frequently mutated, leading to FSS, R672 and T178, are evolutionarily conserved across myosin heavy chains in vertebrates and Drosophila. Transgenic Drosophila were generated expressing myosin heavy chain (Mhc) transgenes with the FSS mutations and the effect of their expression on Drosophila muscle structure and function was characterized. The results indicate that expressing these mutant Mhc transgenes lead to structural abnormalities in the muscle, which increase in severity with age and muscle use. Flies expressing the FSS mutant Mhc transgenes in the muscle exhibit shortening of the inter-Z disc distance of sarcomeres, reduction in the Z-disc width, aberrant deposition of Z-disc proteins, and muscle fiber splitting. The ATPase activity of the three FSS mutant MHC proteins are reduced compared to wild type MHC, with the most severe reduction observed in the T178I mutation. Structurally, the FSS mutations occur close to the ATP binding pocket, disrupting the ATPase activity of the protein. Functionally, expression of the FSS mutant Mhc transgenes in muscle lead to significantly reduced climbing capability in adult flies. Thus, these findings indicate that the FSS contracture syndrome mutations lead to muscle structural defects and functional deficits in Drosophila, possibly mediated by the reduced ATPase activity of the mutant MHC proteins.
|Krueger, D., Quinkler, T., Mortensen, S. A., Sachse, C. and De Renzis, S. (2019). Cross-linker-mediated regulation of actin network organization controls tissue morphogenesis. J Cell Biol. PubMed ID: 31253650
Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. This study analyzed the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. Early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which this study shows to regulate actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis.
|Proag, A., Monier, B. and Suzanne, M. (2019). Physical and functional cell-matrix uncoupling in a developing tissue under tension. Development. PubMed ID: 31064785
Tissue mechanics play a crucial role in organ development. They rely on the properties of cells and the extracellular matrix (ECM), but the relative physical contribution of cells and ECM to morphogenesis is poorly understood. This study analyzed the behavior of the peripodial epithelium (PE) of the Drosophila leg disc in the light of the dynamics of its cellular and ECM components. The PE undergoes successive changes during leg development, including elongation, opening and removal to free the leg. During elongation, it was found that the ECM and cell layer are progressively uncoupled. Concomitantly, the tension, mainly borne by the ECM at first, builds up in the cell monolayer. Then, each layer of the peripodial epithelium is removed by an independent mechanism: while the ECM layer withdraws following local proteolysis, cellular monolayer withdrawal is independent of ECM degradation and driven by myosin-II-dependent contraction. These results reveal a surprising physical and functional cell-matrix uncoupling in a monolayer epithelium under tension during development.
|Balaji, R., Weichselberger, V. and Classen, A. K. (2019). Response of Drosophila epithelial cell and tissue shape to external forces in vivo. Development 146(17). PubMed ID: 31399470
How actomyosin generates forces at epithelial adherens junctions has been extensively studied. However, less is known about how a balance between internal and external forces establishes epithelial cell, tissue and organ shape. This study used the Drosophila egg chamber to investigate how contractility at adherens junctions in the follicle epithelium is modulated to accommodate and resist forces arising from the growing germ line. Between stages 6 and 9, adherens junction tension in the post-mitotic epithelium decreases, suggesting that the junctional network relaxes to accommodate germline growth. At that time, a prominent medial Myosin II network coupled to corrugating adherens junctions develops. Local enrichment of medial Myosin II in main body follicle cells resists germline-derived forces, thus constraining apical areas and, consequently, cuboidal cell shapes at stage 9. At the tissue and organ level, local reinforcement of medial junction architecture ensures the timely contact of main body cells with the expanding oocyte and imposes circumferential constraints on the germ line guiding egg elongation. This study provides insight into how adherens junction tension promotes cell and tissue shape transitions while integrating the growth and shape of an internally enclosed structure in vivo.
|Mishra, A. K., Mondo, J. A., Campanale, J. P. and Montell, D. J. (2019). Coordination of protrusion dynamics within and between collectively migrating border cells by myosin II. Mol Biol Cell 30(19): 2490-2502. PubMed ID: 31390285
Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While nonmuscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. This study used genetics, Airyscan microscopy, live imaging, optogenetics, and Forster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. Myosin was found to accumulate transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin colocalize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo.
|Kasza, K. E., Supriyatno, S. and Zallen, J. A. (2019). Cellular defects resulting from disease-related myosin II mutations in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31615886
The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. This study used the Drosophila embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, it was shown that engineering human MYH9-related disease mutations into Drosophila myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the Drosophila myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis.
|Kobb, A. B., Rothenberg, K. E. and Fernandez-Gonzalez, R. (2019). Actin and myosin dynamics are independent during Drosophila embryonic wound repair. Mol Biol Cell: mbcE18110703. PubMed ID: 31553671
Collective cell movements play a central role in embryonic development, tissue repair, and metastatic disease. Cell movements are often coordinated by supracellular networks formed by the cytoskeletal protein actin and the molecular motor non-muscle myosin II. During wound closure in the embryonic epidermis, the cells around the wound migrate collectively into the damaged region. In Drosophila embryos, mechanical tension stabilizes myosin at the wound edge, facilitating the assembly of a supracellular myosin cable around the wound that coordinates cell migration. This study shows that actin is also stabilized at the wound edge. However, loss of tension or myosin activity does not affect the dynamics of actin at the wound margin. Conversely, pharmacological stabilization of actin does not affect myosin levels or dynamics around the wound. Together, these data suggest that actin and myosin regulation during embryonic wound closure in Drosophila are largely independent, thus conferring robustness to the embryonic response to wounds.
|Caldwell, J. T., Mermelstein, D. J., Walker, R. C., Bernstein, S. I. and Huxford, T. (2019). X-ray Crystallographic and Molecular Dynamic Analyses of Drosophila melanogaster Embryonic Muscle Myosin Define Domains Responsible for Isoform-Specific Properties. J Mol Biol. PubMed ID: 31786266
Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. This study reports the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using a system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain were prepared and crystallized. The x-ray crystal structure was solved by molecular replacement, and the resulting model was refined against diffraction data to 2.2 A resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins.
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).
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).
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).
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).
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).
Uechi, H. and Kuranaga, E. (2019). The tricellular junction protein Sidekick regulates vertex dynamics to promote bicellular junction extension. Dev Cell 50(3):327-338. PubMed ID: 31353316
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).
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)
Exons - 14
Bases in 3' UTR - 188
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).
date revised: 15 August 2019
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