BIOLOGICAL OVERVIEW

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. It remains unknown how this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology. Drosophila Rho-kinase (Rok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Rok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain (Mizuno, 1999 and Winter, 2001), and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton (Winter, 2001).

The rok transcript is ubiquitous in all stages of embryogenesis and in imaginal discs. Drok² mutants die before developing into wandering 3^rd instar larvae. To investigate the function of rok in the morphogenesis of adult tissues, somatic Drok² clones generated by mitotic recombination were examined. Homozygous Drok² clones in the eye are of similar size as their wild-type siblings (twin spots), indicating no gross defects in proliferation or survival of Drok² mutant cells. However, about 50% of ommatidia exhibit an increase or decrease in photoreceptor numbers. A change of photoreceptor number has also been reported in eye clones homozygous for a hypomorphic RhoA allele. Of those ommatidia with the correct number of photoreceptors, 60% were misrotated, whereas in control clones all ommatidia were properly rotated. These phenotypes resemble those of planar cell polarity (PCP) mutants such as frizzled, dishevelled, and RhoA, suggesting that rok may also participate in the signaling cascade controlling PCP. Unlike fz and dsh, however, very few ommatidia showed aberrant chirality (Winter, 2001).

The function of rok in the wing was determined, where PCP is exhibited at a single cell level. In wild-type wings, each cell produces a single, distally oriented wing hair, while in Drok² adult wing clones, greater than 70% of the cells produce multiple hairs. In contrast to most known PCP genes, including fz and dsh, which exhibit both stereotypical orientation and multiple hair defects, most hairs within Drok² clones maintain a largely distal orientation (Winter, 2001).

To investigate the nature of the rok phenotype during the development of wing cell polarity, phalloidin was used to examine the F-actin distribution in Drok² clones during prehair initiation. Mutant clones were positively marked using the MARCM strategy. In control clones or in heterozygous tissue neighboring Drok² mutant clones, each wing cell generates a single F-actin bundle from its distal vertex, extending toward its distal neighbor. However, in Drok² clones, a majority of the cells generate more than one F-actin bundle. The F-actin-based prehairs always initiate at the cell periphery. Notably, the majority of the prehairs maintain a roughly distal orientation (in the case of multiple hairs, the vector average remains distal). The similarities between the pupal and adult clonal phenotypes indicate that the multiple hair phenotype of adult Drok² clones is likely the result of failure to restrict F-actin bundle assembly to a single site during prehair formation. The multiple hair/actin phenotype is seen only in mutant cells and never in neighboring heterozygous or wild-type cells, indicating that rok acts in a cell-autonomous manner (Winter, 2001).

The similarity of the Drok² clonal phenotype in the wing to aspects of the phenotypes of fz, dsh, and RhoA led to the hypothesis that Rok may act downstream of Fz/Dsh. To assess the genetic interactions among these genes, the multiple hair phenotype was quantitated in a defined region: the ventral surface of the proximal-anterior region of the wing. Use was made of the dsh¹ allele, which is defective for PCP function without affecting Wg signaling. In dsh¹ hemizygous males, an average of 16.8 cells with multiple hairs (12% of the cells) are present in this region. When Rok is overexpressed via a tubP-Drok transgene in the dsh¹ hemizygous background, the average number of cells exhibiting multiple hairs is reduced by more than 7-fold to 2.3 per wing region. tubP-Drok expression in a wild-type genetic background does not give rise to any obvious phenotypes. Suppression of the dsh¹ multiple hair phenotype by tubP-Drok expression could also be seen when F-actin-based prehairs were visualized in the pupal wing. In contrast to the suppression of the dsh¹ phenotype by overexpression of Rok, reduction of rok dosage by 50% (assuming that Drok² is null) results in a 2.5-fold increase in the number of multiple hair cells (Winter, 2001).

Genetic interactions between rok and fz were examined in a different assay. The proper level of Fz/Dsh signaling is critical for the generation of wild-type PCP, since both overexpression and loss of function of these genes result in polarity defects in the eye and the wing. Overexpression of Fz 30 hr after puparium formation (APF) produces primarily a multiple hair phenotype that is suppressed by dsh¹ heterozygosity. Similarly, reducing the wild-type copy number of RhoA and rok by half suppresses the phenotype by 2- to 2.5-fold. Taken together, these experiments suggest that Rok functions downstream of Fz/Dsh in restricting the number of F-actin-based prehairs (Winter, 2001).

Rok signaling regulates the phosphorylation of nonmuscle myosin regulatory light chain (MRLC), and hence the activity of myosin II. Does the phosphorylation state of MRLC modify the multiple hair phenotype of dishevelled mutants? Use was made of a series of mutant spaghetti squash (sqh) transgenes (sqh codes for the Drosophila MRLC) with point mutations in the primary (Ser-21) and secondary (Thr-20) phosphorylation sites, changing them either to glutamic acid (phosphomimetic), or to nonphosphorylatable alanine. Can the phosphorylation state of MRLC also modulate Fz/Dsh signaling? An examination was made to determine whether the phosphomimetic and nonphosphorylatable forms of MRLC could directly modify the dsh¹ multiple hair phenotype. Introducing one copy of sqhE20E21 reduces the number of multiple hair cells in dsh¹ mutants by 5-fold. sqhE21, or sqhA20E21, also suppresses the dsh¹ phenotype by more than 2-fold. In contrast, introduction of sqhA21 into the dsh¹ background enhances the multiple hair phenotype. The involvement of MRLC in the Fz/Dsh pathway was also examined using the Fz-overexpression assay. Reducing the wild-type sqh gene dosage from two to one, by introducing a single copy of the sqh^AX3 null allele, results in a 2-fold suppression of the multiple hair phenotype caused by Fz overexpression. These results support the notion that MRLC functions in the PCP pathway to restrict F-actin bundle assembly to a single site (Winter, 2001).

MRLC phosphorylation in response to Rok activation would be predicted to modify the conformation and elevate the catalytic activity of its associated heavy chain, Zipper (Zip). Does Zip also participate in regulating actin distribution/wing hair number in response to Fz/Dsh? Loss of one copy of the zip gene enhances the dsh¹ phenotype by 4.5-fold, consistent with the genetic interaction data between fz/dsh and sqh. These results suggest that myosin II functions positively downstream of Fz/Dsh in regulating actin prehair development (Winter, 2001).

The localization of Zip protein in wing cells further supports its role downstream of Fz/Dsh. At the apical surface of the pupal wing cell, Zip is asymmetrically localized to the distal portion of the cell, where prehair growth initiates. This distal localization appears to coincide, temporally, with prehair initiation. To test whether Zip localization could be modified by Fz/Dsh signaling, Zip distribution at the apical surface was examined in dsh¹ mutants. Instead of being concentrated in the distal region of the cell, Zip is concentrated in the center of the cell, where prehairs form in dsh¹ mutants (Winter, 2001).

Does reduction in myosin II/Zip activity also result in the multihair phenotype? Use was made of the hypomorphic zip⁰²⁹⁵⁷, since zip and sqh null mutations appear to be cell lethal in the wing. As is the case with rok, some homozygous zip⁰²⁹⁵⁷ wing cells possess multiple F-actin prehairs (Winter, 2001).

Tests were performed to see if the gene crinkled (ck) is involved in the Fz/Dsh signaling pathway regulating wing hair number because (1) ck mutant cells in the wing lead to multiple hair and split hair phenotypes, and (2) ck encodes the Drosophila myosin VIIA protein. Mutations in mouse myosinVIIA lead to stereocilia disorganization and the formation of multiple bundles of stereocilia (Winter, 2001 and references therein).

Reduction of ck activity potently suppresses the dsh¹ multiple hair phenotype. This result contrasts with the result that zip¹ enhances the dsh¹ multiple hair phenotype, and suggests that the two myosin heavy chains have opposing effects in regulating prehair assembly (Winter, 2001).

Both myosin heavy chain genes were tested for their ability to interact with the hs-fz induced multiple hair phenotype, and again it was found that they have opposing effects. Surprisingly, loss of one copy of zip slightly but significantly enhances the late hs-fz multiple hair phenotype, while loss of one copy of ck markedly suppresses this phenotype. These results are the reverse of what one would expect based on their interactions with dsh¹, and suggests the possibility that there is a signal from Fz to Ck that is independent of Dsh, or that the multiple hair phenotypes resulting from hypo- or hyper-activity of the Fz/Dsh pathway arise via distinct biochemical mechanisms (Winter, 2001).

To further assess the nature of the relationship between the two myosins, the effect of raising or lowering the activity of MRLC on the ck phenotype was tested. The multiple hair phenotype in animals homozygous for a weak ck mutation is enhanced by one copy of the sqhE20E21 transgene (and hence, a probable increase in myosin II activity), but not by a sqhA20A21 transgene. Taken together, these experiments suggest that a balance between the activities of myosin II and myosin VIIA is important in regulating wing hair number (Winter, 2001).

Unlike other characterized PCP mutants that affect both orientation and number of wing hairs, the primary defect in Drok² clones appears to be the presence of multiple hairs per cell, with little or no wing hair orientation defect. This suggested that Rok and what lies downstream are involved in transmitting a subset of the Fz/Dsh signal. Supporting this idea, it was found that tubP-Drok and sqhE20E21 suppress the multiple hair phenotype of dsh¹, but not the hair misorientation phenotype. Additional data supporting this conclusion comes from observing the site of prehair initiation. Prehairs emerge aberrantly from the center of dsh¹ mutant cells, rather than from the distal vertex as seen in wild type cells. Such mispositioning of prehair initiation correlates with the failure to acquire the proper distal orientation. While tubP-Drok expression suppresses multiple prehair formation, it does not affect the site of F-actin initiation in dsh¹. Finally, the hair orientation defect resulting from Fz overexpression (via hs-fz) at 24 hours is suppressed by reducing dsh gene dosage but not that of RhoA, rok, sqh or ck. Taken together, these observations suggest that separate mechanisms allow Fz/Dsh to independently regulate the number and the orientation of prehairs, and that only the former involves Rok signaling (Winter, 2001).

Mammalian and C. elegans Rho-kinase/ROCK have been implicated in regulating actin stress fiber formation in fibroblasts (Leung, 1996; Amano, 1997; Ishizaki, 1997) or changes in hypodermal cell shape during embryonic development (Wissmann, 1997). How Rho-kinase/ROCK is regulated by an extracellular signal in a physiological process is unknown. Drosophila Rho-kinase has been shown in this study to be required for restricting the number of sites of F-actin bundle assembly in response to activation of the Fz/Dsh pathway. Together with the previously established genetic link between Fz/Dsh and RhoA, a molecular pathway from Fz/Dsh to the regulation of actin structure has been established. Proteins that physically mediate the interaction between Fz and Dsh, and between Dsh and RhoA, remain to be identified (Winter, 2001 and references therein).

The data presented in this study suggest that the Rok/myosin II pathway is involved in regulating the number -- but not orientation -- of the wing hair. What then are the components that regulate wing hair orientation? One possibility is that a bifurcation of the pathway occurs at the level of RhoA, with a separate effector pathway regulating wing hair orientation. In the eye, the JNK pathway has been implicated in functioning downstream of RhoA in regulating ommatidial polarity. However, the function of the JNK pathway in the wing has not been described, and a signaling pathway that regulates transcription is unlikely to encode the requisite spatial information necessary for selection of the site of prehair initiation. Therefore, it is likely that a separate signal from or upstream of RhoA may control the selection of the F-actin assembly site, and therefore the orientation of the wing hair (Winter, 2001 and references therein).

Several other genes have been implicated in regulating wing hair number based on their loss-of-function phenotypes, including inturned (in), fuzzy(fy), and multiple wing hair (mwh). These gene products are thought to participate in a distinct branch of the polarity signaling pathway for the following reasons: (1) they each have significant effects on both hair orientation and hair number, they are unlikely to function downstream of Rok; (2) expression of the phosphomimetic Sqh-E20E21, or the nonactivatable Sqh-A20A21, in mwh or in mutant backgrounds does not modify their phenotypes (Winter, 2001); (3) while Rok functions in the eye as well as in the wing, In, Fy, and Mwh have no apparent role in PCP signaling in the eye (Winter, 2001).

The misorientation of ommatidia in Drok² eye clones is also consistent with its functioning in the Fz/Dsh/RhoA pathway. It is conceivable that Rok may act as an effector of RhoA in regulating the process of ommatidial rotation. Alternatively, loss of Rok function could lead to a disruption of the actin cytoskeleton and a consequent loss of the appropriate cell -- cell contacts that support intercellular signaling essential for ommatidial rotation. Although rok mutants exhibit misrotation of ommatidia, they acquire the proper chiral form. Determination of ommatidial rotation may reflect a Rok-dependent subset of PCP signaling in the eye that is, in some way, molecularly analogous to the subset of PCP signaling in the wing, as reflected in the regulation of hair number (Winter, 2001).

Biochemical and cell culture studies have identified many potential substrates for Rho-kinase/ROCK (reviewed in Amano, 2000) The relative contribution of phosphorylation of these different substrates in mediating various functions of Rho-kinase/ROCK is unclear. Strong genetic evidence has been provided that phosphorylation of MRLC at key residues (Ser-21 in Drosophila, equivalent to Ser-19 in mammals) is a critical output for Rok signaling. Phosphomimetic MRLC expression not only suppresses the multiple wing hair phenotype, but also lethality, of Drok² mutants. Biochemical and immunostaining experiments further demonstrate that Rok modulates MRLC phosphorylation in vivo. Previous work has suggested that Rok regulates phosphorylation of MRLC, both by direct phosphorylation and also by phosphorylation and inhibition of the myosin binding subunit of myosin phosphatase (MBS: see Drosophila Myosin binding subunit) (Amano, 2000). However, at present the relative contribution of direct phosphorylation by Rok of MRLC, versus phosphorylation of MBS that indirectly affects MRLC phosphorylation is not known (Winter, 2001).

The direct genetic interactions between dsh¹ and various sqh transgenes further substantiates the notion that regulation of MRLC phosphorylation is not only a major output of Rok signaling, but also of Fz/Dsh signaling to the actin cytoskeleton in regulating hair number (Winter, 2001).

By what mechanism do myosins restrict F-actin bundle formation? In light of the finding that myosin II is concentrated at the site of prehair formation, it seems plausible that myosin II is actively involved in either the recruitment of F-actin to the prehair site, or that it directly participates in the assembly of actin bundles, or both. Studies of mammalian myosin II provide a precedent for a role in the formation of F-actin bundles. Phosphorylation of MRLC promotes a conformational change in myosin II from a folded to an extended state that readily forms multivalent bipolar filaments capable of binding multiple actin filaments. This is thought to result in F-actin bundling and stress fiber formation (Winter, 2001 and references therein).

It appears that in the developing wing, the level of MRLC phosphorylation/myosin II activity must be within an optimal range to establish the formation of a single hair. It is possible that the efficiency of F-actin bundle formation is regulated by MRLC phosphorylation in a manner similar to the control of stress fiber formation. If one further assumes that there are certain bundling substrates present only at limiting concentrations (e.g., F-actin itself), then one would predict that the assembly of one F-actin bundle would reduce the probability of forming a second bundle. When MRLC phosphorylation falls below some threshold level (e.g., in rok mutant cells), the efficiency of primary bundle formation is reduced, and thus the concentration of the limiting substrate remains at sufficient levels to support the assembly of secondary bundles/prehairs. Conversely, if MRLC is hyperphosphorylated (e.g., in Fz-overexpressing cells), the bundling efficiency may increase such that the threshold concentration for bundle formation would be reduced, thereby increasing the probability of assembling multiple bundles/prehairs. Future studies will be required to determine the detailed mechanisms involved (Winter, 2001).

In addition to nonmuscle myosin II, which resembles the myosin II from skeletal muscle, there exists a large class of unconventional myosins that have different properties and potential functions in nonmuscle cells. For instance, several different classes of unconventional myosins are expressed in inner ear epithelium with different subcellular localization. Mutations in three of the unconventional myosins, myosin VI, VIIA, and XV, cause hearing/balancing defects in mice, two of which when mutated in humans result in deafness. Of particular interest in the context of this study is myosin VIIA, mutations of which are responsible for mouse shaker-1 and human Usher's syndrome 1B. Loss-of-function ck (Drosophila Myosin VIIA) mutants exhibit a multiple hair and split wing hair phenotype. ck exhibits strong genetic interactions with components of the signal transduction pathway defined in this study, and has the opposite effects as that of myosin II. The seemingly antagonistic relationship between myosin II and myosinVIIA may suggest a mechanism in which the balance of the activities or stoichiometry of these two myosins is critical for the common process they regulate. For example, myosin II and myosin VIIA may share some common, limiting component(s) required for their activity. Thus, by reducing the myosin VIIA dose, myosin II has a larger share of the common component(s) and thus its activity is upregulated (Winter, 2001 and references therein).

Recent evidence suggests that vertebrate homologs of Dsh and Fz regulate cell morphogenesis using a pathway(s) analogous to that of the Drosophila PCP pathway. The findings described in this study of Rok raise the possibility that the Fz/Dsh cytoskeletal pathway may also be at work in regulating cell polarity in vertebrates, including the stereocilia formation in the inner ear (Winter, 2001).

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

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

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

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

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

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

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

Asymmetrically deployed actomyosin-based contractility generates a boundary between developing leg segments in Drosophila

Classically, it has been assumed that adhesive differences are a primary means of sorting cells to their respective territories. Yet it is becoming clear that no single, simple mechanism is at play. In the few instances studied, an emergent theme along developmental boundaries is the generation of asymmetry in cell mechanical properties. The repertoire of ways in which cells might establish and then put mechanical asymmetry to work is not fully appreciated since only a few boundaries have been molecularly studied. This study characterize one such boundary in the develop leg epithelium of Drosophila. The region of the pretarsus / tarsus is a known gene expression boundary that also exhibits a lineage restriction. This study show that the interface comprising this boundary is strikingly aligned compared to other cell interfaces across the disk. The boundary also exhibits an asymmetry for both Myosin II accumulation as well as one of its activators, Rho Kinase. Furthermore, the enrichment correlates with increased mechanical tension across that interface, and that tension is Rho Kinase-dependent. Lastly, interfering with actomyosin contractility, either by depletion of myosin heavy chain or expression of a phosphomimetic variant of regulatory light chain causes defects in alignment of the interfaces. These data suggest strongly that mechanical asymmetries are key in establishing and maintaining this developmental boundary (Ly, 2017).

A key component comprising a developmental boundary is the special mechanical properties imposed to its interfaces. Insights into these properties have been gained from the few tissues that have been studied, such as rhombomere boundaries in the vertebrate, but especially the study of several boundaries in Drosophila. The latter studies in Drosophila have afforded much higher resolution so far than study of rhombomeres. Still, relatively few boundaries overall have been studied, and that makes it difficult to draw any generalizations for how the underlying mechanics makes the boundary. This paper reports initial studies on the late-arising developmental boundary necessary for leg segmentation. The pretarsal / tarsal boundary was more aligned than the canonical AP compartment boundary. The rail exhibits an asymmetry in actomyosin accumulation as well as one of its activators, Rho Kinase. This is shown to result in increased tension along the boundary, which is important in aligning its interfaces (Ly, 2017).

Polarized actomyosin enrichment leads to increased cell bond tension along the pretarsal / tarsal interfaces. The fold increase of tension compared with the orthogonal rung interfaces is in line with differences observed in several other tissues, such as the Antero-posterior and dorso-ventral compartment boundaries. Here, along the pretarsal / tarsal interface, actomyosin contractility generates a very smooth, arcing boundary. The alignment is significant, as it is even more aligned than the well-studied AP compartment boundary. In itself, this fact strongly suggests that study of the pretarsal / tarsal boundary will complement the information obtained though study of other developmental boundaries (Ly, 2017).

The data revealing enrichment of the Myosin II regulatory light chain as well as Rho Kinase along rail interfaces strongly implicates contractility in alignment, and the degree of mis-alignment observed in zip mutants supports this contention. Furthermore, treatment with a Rho Kinase inhibitor reduced actomyosin enrichment and released tension along the rail, rapidly generating a less aligned state. In addition, since removal of the Rho Kinase inhibitor led to the rapid re-establishment of alignment, the data collectively argue that asymmetric contractility can drive this alignment event. Still, Rho Kinase inhibitors can affect other protein kinases, such as Atypical Protein Kinase (aPKC). Thus, even though a quite selective Rho Kinase inhibitor was used, it is still possible that another kinase also contributes to alignment, perhaps targeting a factor in addition to the myosin regulatory light chain (Ly, 2017).

The expression of a phosphomimetic form of the Myosin II regulatory light chain generated defects along the rail. The precise mechanism involved awaits live-imaging the formation of the aligned interface. Without that capability in this epithelium, it cannot be determined whether the phosphomimetic form of the Myosin II generated defects due to decreased cycling between on and off states along interfaces normally enriched for myosin, or to increased activity along the normally depleted (rung) interfaces. Nevertheless, regulated contractility is certainly important to alignment (Ly, 2017).

Actomyosin enrichment and the resultant increased tension is a theme observed repeatedly along cell interfaces. Interestingly, the outcome of that increase in tension can be quite different in different circumstances. In some cases, tension stabilizes cell interfaces, as has been observed along the parasegment boundary of the embryonic epithelium, as well as the AP and DV compartment boundaries in developing imaginal disk epithelia. While actomyosin enrichment leads to stabilization in those cases, in other instances, enrichment and the associated increased tension drives interface shrinkage. Those shrinkage outcomes are crucial to the directed junctional remodeling events necessary for convergence extension. Similar shrinkage events are also observed in tissues at steady-state. For example, across the epithelial field in the developing wing, junctional shrinkage events maintain the proper geometry of cell packing. Just how actomoysin enrichment and junctional tension can be directed toward two quite diametrically opposed outcomes, shrinkage or stabilization, is unclear at present. This issue will only be resolved by examining more boundaries of each class, and by identifying more components that act along those interfaces (Ly, 2017).

In fact the pretarsal / tarsal boundary described in this study has several features in common with another interface described previously. In the late embryonic epidermis, well-after convergence and extension, a select set of cells within each parasegment organizes into aligned columns. Those aligning cell columns exhibit enrichments similar to those described here along the smooth, arcing pretarsal / tarsal rail. In addition, in both cases the cells that constitute the boundary assume elongate, rectilinear shapes. A comparison of the mechanics underlying these two alignment events could potentially reveal how actomoysin enrichment and junctional tension can be directed toward stabilization (Ly, 2017).

Besides exhibiting alignment, some boundary interfaces, such as the AP and DV compartment boundaries, are resilient to challenges from neighboring cells, whether from cell division or intercalation. The mechanical basis for this is becoming more clear. The pretarsal / tarsal boundary develops a late-acting lineage-restriction, so it is interesting to consider the degree to which increased tension contributes to the restriction. Interestingly, in depleting or manipulating Myosin II activity the pretarsal/tarsal boundary became very irregular. Yet, no evidence was found for 'invasions' from one territory to the other, at least not in these fixed preparations. This suggests that tension is not sufficient for this restriction in the leg. Perhaps like the DV compartment in the wing a combination of mechanical tension, as seen here, plus oriented divisions and cell elongation contribute to boundary integrity. Alternatively, the affinity properties of the pretarsal versus tarsal cells may well contribute to the lineage restriction (Ly, 2017).

Finally, it is noted that the interfaces flanking the rail are also aligned to a significant degree. This differs from the situation observed along the AP compartment boundary where the adjacent interfaces were used as examples of relatively unaligned interfaces. That raises the interesting question of whether the interfaces flanking the rail are actively aligned. For instance, machinery similar to that deployed along the rail might align the -1 and + interfaces. Alternatively, the flanking interfaces might be aligned only passively, as a consequence of the geometry enforced by the rail interface on the other cell interfaces. If there is an active process aligning the flanking interfaces, MyoII would appear to be minimally involved. No significant enrichment of MyoII was observed along the -1 interface compared to the adjacent rung, and although these interfaces retained some tension, the level was much reduced along the -1 and + interfaces compared to the rails (Ly, 2017).

It is not yet known how the polarized enrichments are first established along the pretarsal / tarsal boundary. There is a fairly well-understood gene regulatory hierarchy that establishes the pretarsal and tarsal territories during the mid third instar period of development. The initially rough borders between the two territories are subsequently refined by further cross-regulatory interactions. Thus, it is no surprise that interfering with the transcriptional regulator, C15, can cause defects along the boundary. In addition, among the factors that are genetically regulated by this transcriptional circuitry are Fasciclin II and the leucine rich proteins, Capricious and Tartan (Caps; Trn). However, it is not known how direct that regulation might be. Moreover, neither removing Fasciclin II, nor both Caps and Trn, generated phenotypes that seemed clarifying. This suggests that key factors remain to be defined. A similar limitation extends to the parasegmental, AP and DV boundaries. While the Wingless, Hedgehog and Notch pathways, respectively, have been implicated at those boundaries, the analyses still leave open the possibility that control by each of those pathways is indirect. Unraveling the direct links from cell signaling to the mechanics of tissue boundaries remains an important goal in studying morphogenesis (Ly, 2017).

Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells

Epithelial cells undergo cortical rounding at the onset of mitosis to enable spindle orientation in the plane of the epithelium. In cuboidal epithelia in culture, the adherens junction protein E-cadherin recruits Pins/LGN/GPSM2 and Mud/NuMA to orient the mitotic spindle. In the pseudostratified columnar epithelial cells of Drosophila, septate junctions recruit Mud/NuMA to orient the spindle, while Pins/LGN/GPSM2 is surprisingly dispensable. This study shows that these pseudostratified epithelial cells downregulate E-cadherin as they round up for mitosis. Preventing cortical rounding by inhibiting Rho-kinase-mediated actomyosin contractility blocks downregulation of E-cadherin during mitosis. Mitotic activation of Rho-kinase depends on the RhoGEF ECT2/Pebble and its binding partners RacGAP1/MgcRacGAP/CYK4/Tum and MKLP1/KIF23/ZEN4/Pav. Cell cycle control of these Rho activators is mediated by the Aurora A and B kinases, which act redundantly during mitotic rounding. Thus, in Drosophila pseudostratified epithelia, disruption of adherens junctions during mitosis necessitates planar spindle orientation by septate junctions to maintain epithelial integrity (Aguilar-Aragon, 2020).

Adherens junctions have long been thought to be continuously essential for maintaining epithelial form and function. The current findings demonstrate transient loss of adherens junctions during division of pseudostratified epithelial cells, an event that involves adherens junction remodelling during the extensive rounding up of cell shape in mitosis. This study has furthermore shown that loss of adherens junctions is a direct consequence of the increased Rho activity and actomyosin contractility that drives mitotic rounding, which is both necessary and sufficient to regulate the level of junctional E-cadherin in the pseudostratified wing imaginal disc epithelium of Drosophila. These findings are consistent with previous observations that adherens junctions can be removed via E-cadherin endocytosis upon planar polarised Rho activation and actomyosin-driven junctional shrinkage generated during cell-cell rearrangements in the Drosophila embryo and recent optogenetic experiments in human cells. However, the global loss of E-cadherin observed during mitosis of pseudostratified epithelial cells is unprecedented and may be uniquely required by the rapid transformation of these cells from their highly columnar shape to a rounded sphere at mitosis, which involves a rapid increase in apical area and junctional length. This change in cell chape, driven by global actomyosin contractility during mitotic rounding, may both spread out junctions and disrupt cadherin-cadherin contacts between neighbouring cells to favour endocytosis (Aguilar-Aragon, 2020).

Notably, loss of E-cadherin does not seem to occur during mitosis in cuboidal epithelial cells, which undergo a much milder cell shape change during mitotic rounding, such as the Drosophila follicle cell epithelium or in many human cultured epithelial cell lines. Indeed, E-cadherin was reported to play essential roles in planar spindle orientation in cultured human cells. Thus, the pseudostratified epithelia of Drosophila face a unique challenge of orienting the mitotic spindle in the plane of the epithelium without the use of adherens junctions as a cue, which may explain why these cells instead rely on septate junctions, while planar spindle orientation can occur normally in the cuboidal follicle cell epithelium before septate junctions form. In the absence of septate junctions, spindle-orienting factors such as Pins, Mud, Dlg and Scrib localise to lateral membranes, overlapping with E-cadherin, which can directly interact with Scrib in both Drosophila and human cells (Aguilar-Aragon, 2020).

The findings of this study also shed light on the molecular mechanisms linking the cell cycle with control of Rho activation during mitotic rounding. Downstream of the master mitotic kinase Cdk1, a key role for both Aurora A and B kinases (acting redundantly) was identified in initiating mitosis and maintaining cortical rounding, with Aurora B then acting alone to drive furrow formation during cytokinesis. Aurora kinases are known to activate the key cell cycle kinase Plk1/Polo, which can then activate RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl, possibly via the kinesin-like protein MKLP1/KIF23/ZEN4/Pav. One report suggested that Aurora B also acts to directly phosphorylate RacGAP1/MgcRacGAP/CYK4/Tum on S387, but this study found that a CRISPR-knockin mutation of this site (tum_S387A) is homozygous viable and fertile in Drosophila. In addition, Aurora B can also directly phosphorylate MKLP1/KIF23/ZEN4/Pav to oligomerise and activate RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl during cytokinesis, via a mechanism involving plasma membrane association of clustered C1 domains from the RacGAP1/MgcRacGAP/CYK4/Tum protein. Accordingly, it was found that a CRISPR-knockin mutation of this site (pavS734A/S735A) is homozygous lethal in Drosophila, suggesting that Aurora A/B could act via this complex during mitotic rounding. Indeed, MKLP1/KIF23/ZEN4/Pav undergoes cell cycle-dependent re-localisation from the nucleus (in interphase) to the cytoplasm (in mitosis), including a clear localisation to the entire plasma membrane during mitosis and then to the cleavage furrow during cytokinesis. The results show that mitotic activation of the MKLP1/KIF23/ZEN4/Pav binding partner RacGAP1/MgcRacGAP/CYK4/Tum also involves translocation from the nucleus to the cytoplasm, similar to ECT2/Pbl, where all three proteins are then available to bind the entire plasma membrane and generate global Rho activity to drive cortical contractility and loss of adherens junctions during mitotic rounding (Aguilar-Aragon, 2020).

The complex of MKLP1/KIF23/ZEN4/Pav and RacGAP1/MgcRacGAP/CYK4/Tum is often referred to as the 'centralspindlin' complex, due to its association with the spindle midzone at anaphase. However, the use of this term was avoided because association with the spindle midzone is not required for the function of this complex in activating ECT2/Pbl and Rho at the plasma membrane during cytokinesis. Furthermore, the results show that the same complex also functions prior to anaphase or cytokinesis in activating ECT2/Pbl and Rho for cortical rounding and downregulation of adherens junctions from the very onset of mitosis (Aguilar-Aragon, 2020).

Importantly, the function of the three cell cycle-regulated Rho activators discussed in this study - MKLP1/KIF23/ZEN4/Pav, RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl - on the regulation of adherens junctions appears to be dose-dependent and cell shape change-dependent. The sudden re-localisation of these proteins to the cytoplasm at mitosis drives a global increase in Rho activation, cell shape change and loss of junctions. In contrast, the relatively low level of these proteins in the cytoplasm during interphase appears to contribute to the normal maintenance of Rho activity at the junctional actomyosin ring to maintain adherens junctions-at least in mammalian cells. In Drosophila, an interphase role of ECT2/Pbl in contributing to Rho activation and maintenance of adherens junctions is also plausible and should be most easily distinguished from the mitotic role in non-dividing epithelial cells. However, in the ovarian follicle cell epithelium, silencing of ECT2/Pbl by RNAi affected cytokinesis (leading to larger cells) but did not affect overall epithelial architecture after cells arrest their proliferation, suggesting that any interphase function may be obscured by redundancy with other RhoGEFs in this tissue. Note also that, in this cuboidal epithelium, mitotic rounding itself is more subtle and ECT2/Pbl is dispensable for planar spindle orientation and epithelial integrity (Aguilar-Aragon, 2020).

Finally, it is noted that ECT2/Pbl was initially reported to be capable of acting as GEF for another GTPase, Cdc42, in addition to Rho 38. This activity suggested a possible role in regulating the apical Cdc42-Par6-aPKC complex in epithelial polarity and in mitosis. In Drosophila, ECT2/Pbl was found to drive transient apical spreading of the Cdc42-Par6-aPKC complex during mitotic rounding of pupal notum epithelial cells. This study found evidence for a similar function of ECT2/Pbl in activating Cdc42 during interphase in the post-mitotic ovarian follicle cell epithelium, although the loss-of-function phenotype of ECT2/Pbl is obscured due to redundancy with other Cdc42 GEFs such as beta-PIX. Loss of both ECT2/Pbl and beta-PIX reduced the level of the Cdc42-Par6-aPKC complex localising to the apical domain of post-mitotic follicle cells and also reduced apical ZO-1 localisation in human Caco2 intestinal epithelial cells in culture. In contrast, the gain-of-function phenotype caused by ECT2/Pbl overexpression is a neoplastic tumour-like phenotype and clearly involves ectopic spreading of the apical Cdc42-Par6-aPKC complex, in addition to persistent cell rounding in both cuboidal follicle cells and pseudostratified wing epithelial cells (Aguilar-Aragon, 2020).

In conclusion, the findings provide new insights into the cell biology of mitotic rounding, identifying remodelling of adherens junctions as a key event in pseudostratified epithelia, where rounding is extensive, but not cuboidal epithelia, where rounding is more subtle. These results are consistent with the hypothesis that Rho activation and actomyosin contractility can stabilise adherens junctions in the absence of mechanical strain, but that Rho activation can induce E-cadherin endocytosis above a critical strain threshold, be it either junctional shrinkage or expansion, both of which may alter the geometry of the junctional actomyosin ring and disrupt cadherin-cadherin contacts between neighbouring cells to favour endocytic internalisation of E-cadherin. The results also clarify the molecular mechanisms linking cell cycle control machinery, particularly the Aurora A and B kinases, with Rho activation and mitotic rounding. Lastly, this work may have direct relevance to certain human epithelial cancers, such as lung cancer or glioma, where overexpression of ECT2/Pbl has been reported to correlate for poor prognosis, and where the findings suggest it could drive not only disruption of epithelial polarity via activation of Cdc42 or Rac but also loss of adherens junctions via sustained Rho activation to promote tumour progression (Aguilar-Aragon, 2020).

Ras acts as a molecular switch between two forms of consolidated memory in Drosophila

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. This study demonstrates that the Ras-->Raf-->rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. The initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. These effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. It is concluded that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM (Noyes, 2020).

Consolidation is a process required for the formation of long-lasting memories. This process of converting memories that are initially sensitive to disruption from a variety of insults to more resilient ones is well conserved and many of its characteristics are shared across species. For example, memory in invertebrates and vertebrates lasts longer following multiple spaced training sessions, undergoes both molecular/cellular and systems consolidation, and can be disrupted by inhibition of protein synthesis (Noyes, 2020).

The fruit fly Drosophila melanogaster forms two distinguishable types of consolidated aversive olfactory memory: 1) anesthesia-resistant memory (ARM), which reportedly decays to negligible levels by 4 d after conditioning, can be generated by a single training session; 2) protein synthesis-dependent long-term memory (PSD-LTM), which shows limited decay, requires spaced training. These two types of consolidated memory are not independent from one another. The formation of ARM impairs either the formation or expression of PSD-LTM. Although circuit mechanisms possibly responsible for this relationship are beginning to be dissected, the molecular requirements in the mushroom body (MB), a brain region critical for the storage and retrieval of PSD-LTM and ARM remain unknown (Noyes, 2020).

The small GTPase Ras85D (Ras) is a Drosophila homolog of the mammalian Ras family genes KRAS, NRAS, and HRAS. Activated Ras proteins act as signaling switches, initiating signaling cascades through multiple downstream effector proteins. Precise induction and regulation of Ras activity is essential for mammalian synaptic plasticity and memory. Although upstream regulators of Ras, like NF1 and DRK, have been explored for their roles in Drosophila learning and memory Ras itself has not been thoroughly examined. A large RNA interference (RNAi) screen identified Ras85D as a memory suppressor gene but did not detail its specific role in memory suppression (Noyes, 2020).

This study reports that Ras activity in the MB acts as a switch between the two forms of consolidated memory, required both for PSD-LTM and inhibition of ARM. Increasing Ras activity dramatically reduced memory expression. This effect was determined to be due to Ras regulation of ARM. Knockdown of Ras enhanced the consolidation of ARM, leading to an overall increase in memory, while Ras knockdown eliminated PSD-LTM following spaced training. Although the effect of Ras on both ARM and PSD-LTM was found to be mediated by Raf, it is independent from the canonical downstream extracellular signal-regulated kinase (ERK). Instead, Ras/Raf-mediated inhibition of rho kinase (ROCK) suppresses ARM and is required for PSD-LTM (Noyes, 2020).

Based on the results, a model in which ARM consolidation is suppressed by a training-induced increase in Ras activity. Raf activity is increased in γ MBn following training, presumably through Ras, but the receptor(s) initiating this signaling are not known. Ras can be regulated through G-coupled protein receptors. It is possible that dopamine or an unknown coneurotransmitter released from dopaminergic neurons (DAn) during training initiates Ras signaling. This would provide a link between MP1 DAn, which are proposed to gate LTM, and Ras. The participation of ROCK in consolidation suggests that PSD-LTM and ARM are modulated by changes in the actin cytoskeleton (36) but does not directly indicate whether these changes occur in the pre- or post-synaptic compartments. Of the several genes known to be required for ARM, Bruchpilot (Brp) is the only one with a well-established, specific subcellular compartmentalization. Brp is localized to presynaptic active zones and is required for normal presynaptic morphology and synaptic transmission, indicating that ARM may result from a form of presynaptic plasticity in the MB. Additionally, the DAn that are required for memory formation innervate MB axons and modulate synaptic strength between MBn and downstream MB output neurons. The results demonstrating that artificial activation of Ras increases axonal pERK in γ MBn is evidence that Ras/Raf signaling participates in axonal signal transduction and is consistent with a previous report highlighting a role for presynaptic Raf activity in γ MBn. ROCK activity in mammalian axons is critical for a number of processes; however, it has not been tested whether ROCK signaling occurs in γ MBn axons (Noyes, 2020).

The hypothesis that ARM inhibits the formation PSD-LTM was based on the observation that spaced training, which generates PSD-LTM, eliminates or precludes ARM. Subsequent research at the systems neuroscience level revealed that two sets of neurons, MP1 DAn and serotonergic projection neurons (SPn), appear to be responsible for the promotion of PSD-LTM through the suppression of ARM. The activity of these neurons is increased during spaced training. This activity reduces ARM, while inhibiting their activity enhances ARM. Blocking the activity of either set of neurons during spaced training does not prevent memory formation but prevents the formation of PSD-LTM. This suggests that without SPn and MP1 DAn activity, ARM occurs by default and is preferentially expressed at the expense of PSD-LTM. Ras fulfills the requirements as the intracellular and molecular switch regulating the inverse relationship between ARM and PSD-LTM. The suppression of ARM and formation of PSD-LTM both require Ras in γ MBn, which are downstream in the circuit from the ARM/PSD-LTM-gating MP1 DAn that synapse directly on to γ MBn (Noyes, 2020).

The mammalian counterpart for ARM, if one exists, is unknown. Protein synthesis-independent ARM has been reported to be measurable up to 4 d after conditioning, while mammalian protein synthesis-independent memory lasts only hours. Despite the lack of a clear and direct mammalian counterpart to ARM, it is becoming apparent that many of the same genes that are involved in ARM also play a role in mammalian memory and plasticity. Ras, Raf, and CDC42 negatively regulate ARM but in mammals are positive regulators of LTM. Conversely, reduced ROCK or dunce, the latter purported to function through the SPn, impair ARM. In mammals, inhibition of ROCK or a mammalian ortholog of dnc (48), PDE4, enhances memory. It seems likely that discovering more genetic regulators of ARM will reveal previously unknown genetic regulators of mammalian memory. Based on the genes and their functions discussed in this paper, it is possible that factors that promote ARM in Drosophila function in memory suppression in mammals (Noyes, 2020).

The effect of ROCK on memory is not restricted to γ MBn. ROCK is also required in α/β MBn for ARM. In this neuron type, the effects of ROCK are not mediated by Ras but through Drk, the Drosophila homolog of Grb2. It is interesting to consider whether the ROCK substrate(s) mediating enhanced ARM in α/β and γ MBn are the same even though the upstream signaling components are distinct. Several ROCK targets have been established as important for normal memory, including cofilin and nonmuscle myosin II (Noyes, 2020).

A recent report indicates that ERK activity in γ MBn slows forgetting. The current results revealing that ERK knockdown reduces memory support this conclusion. However, the former report finds that Raf RNAi expression in γ MBn reduces memory, which is at odds with the finding that Raf RNAi enhances memory. The most likely explanation for this discrepancy is the use of different gal4/UAS-RNAi combinations that produce different levels of gene knockdown. It is interesting to consider that Raf signaling in γ MBn might regulate three forms of memory: consolidated ARM and PSD-LTM through ROCK and labile memory through ERK (Noyes, 2020).

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

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

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

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

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

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

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

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

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

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

A modifier screen identifies regulators of cytoskeletal architecture as mediators of Shroom-dependent changes in tissue morphology

Regulation of cell architecture is critical in the formation of tissues during animal development. The mechanisms that control cell shape must be both dynamic and stable in order to establish and maintain the correct cellular organization. Previous work has identified Shroom family proteins as essential regulators of cell morphology during vertebrate development. Shroom proteins regulate cell architecture by directing the subcellular distribution and activation of Rho-kinase, which results in the localized activation of non-muscle myosin II. Because the Shroom-Rock-myosin II module is conserved in most animal model systems, Drosophila melanogaster was used to further investigate the pathways and components that are required for Shroom to define cell shape and tissue architecture. Using a phenotype-based heterozygous F1 genetic screen for modifiers of Shroom activity, several cytoskeletal and signaling protein were identified that may cooperate with Shroom. Two of these proteins, Enabled and Short stop, are required for ShroomA-induced changes in tissue morphology and are apically enriched in response to Shroom expression. While the recruitment of Ena is necessary, it is not sufficient to redefine cell morphology. Additionally, this requirement for Ena appears to be context dependent, as a variant of Shroom that is apically localized, binds to Rock, but lacks the Ena binding site, is still capable of inducing changes in tissue architecture. These data point to important cellular pathways that may regulate contractility or facilitate Shroom-mediated changes in cell and tissue morphology (Hildebrand, 2021).

Tissue architecture is typically defined during specific stages of embryonic development and errors in these processes can result in human disease. One example is formation of the vertebrate neural tube. The neural tube is formed via the concerted effort of many cellular pathways that functionally convert a plate of neural ectoderm into a closed tube. Errors in this process can result in birth defects such as spina bifida, exencephaly, or craniorachischisis. One cellular pathway that controls this process is regulated by the Shroom3 cytoskeletal adaptor protein. Shroom3 controls neural tube morphogenesis via the formation of apically positioned contractile networks of actomyosin and these networks facilitate neural tube closure by inducing apical constriction and the anisotropic contraction of actin filaments. This is accomplished via the modular nature of Shroom3. Shroom3 localizes to the apical compartment of epithelial adherens junctions via a direct interaction with F-actin. This interaction is mediated by the Shroom Domain (SD) 1, a unique actin-binding motif present in most Shroom proteins characterized to date. Shroom3 function is also dependent on Rho-kinase (Rock), such that Shroom3 directly binds to Rock and regulates both its localization and catalytic activity. The interaction between Shroom and Rock has been elucidated at the molecular level and is mediated by the conserved SD2 region of Shroom and a conserved coiled-coil region of Rock. The interaction between Shroom and Rock results in the localized activation of non-muscle myosin II (myosin II) contractility, which provides the mechanical force needed to facilitate neural tube morphogenesis. The regulation of myosin II activity by Rock and other cellular pathways has been well described. Rock modulates myosin II activity in two ways. First, Rock can directly phosphorylate the associated regulatory light chain (RLC), which modulates the actin-associated ATPase activity and the conformation of myosin II. Secondly, Rock negatively regulates the phosphatase that dephosphorylates the RLC, thus preventing the inactivation of myosin II (Hildebrand, 2021 and references therein).

Shroom proteins are required for numerous biological processes and are associated with several human diseases. In mammals, there are three definitive Shroom proteins, Shroom2, Shroom3, and Shroom4, each of which contains an N-terminal PDZ domain, the centrally located SD1, and the C-terminally located SD2. All three proteins can directly interact with F-actin and regulate cell morphology via Rock. In humans, SHROOM2 has been linked to neural tube morphogenesis, colorectal cancer, and medulloblastoma, while in vitro studies indicate it is important for cell migration, vasculogenesis, metastasis, and melanosome biogenesis. SHROOM3 mutations have been implicated in chronic kidney disease, heart morphogenesis, and neural tube closure in humans. Using model organisms or cell culture, Shroom3 has been shown to control neural tube closure, axon growth, intestine architecture, eye morphogenesis, thyroid budding, and kidney development. Finally, SHROOM4 mutations have been associated with X-linked mental defects (Hildebrand, 2021).

The Shroom gene is conserved in Drosophila and encodes multiple protein isoforms that have different subcellular distributions and activities in vivo. The most highly conserved region of Drosophila Shroom is the SD2, the region that binds to Drosophila Rho-kinase (Rok). Drosophila Shroom also contains a divergent SD1 motif and this appears to mediate localization to adherens junctions in polarized epithelia. Consistent with the known activities of mammalian Shroom3, expression of Drosophila Shroom in epithelial cells induces apical constriction in a Rok and myosin II dependent manner. While Shroom3 is essential for mouse and human development, Shroom is not absolutely essential for Drosophila viability, as Shroom null flies can be recovered, albeit with significantly reduced frequency. In Drosophila embryos, Shroom is planarly distributed and works in a complicated network with RhoA, Rok, and myosin II to control convergent extension movements. These elegant studies showing the role of Shroom in regulating directional contractility are supported by observations that Shroom proteins can be polarly distributed in mammalian tissues and cells (Hildebrand, 2021 and references therein).

To better understand the mechanisms that control Shroom-regulated changes in cell and tissue morphology, this study has established tools to perform genetic screens for modifiers of Shroom activity in Drosophila. Shroom gain-of-function phenotypes in the eye and wing can be suppressed or enhanced by known components of the Shroom pathway. Using a candidate approach, several cytoskeletal regulators were identified, including Short stop and Enabled, as participants in Shroom-mediated changes in cell morphology. Shroom regulates the distribution of Ena and this is likely mediated by conserved proline-rich sequences in Shroom and the EVH1 domain of Ena. This study further shows that while Ena is required for the Shroom gain-of-function phenotypes, apical recruitment of Ena is not sufficient to cause changes in cell morphology. Additionally, by using an isoform of Shroom that does not bind Ena, but still engages Rok, this study showed that apical constriction can be modulated by different cellular pathways depending on the context (Hildebrand, 2021).

This study describes a genetic approach to identify cellular pathways that participate in tissue morphogenesis. This method takes advantage of the observation that ectopic Shroom protein can utilize the endogenous contractile machinery within epithelial cells to induce apical constriction and disrupt normal tissue morphology. While this work focuses on candidate genes that encode known regulators of epithelial and tissue architecture, it is predicted these tools can be used to perform unbiased, genome-wide screens to identify novel participants in Shroom-mediated cellular processes. Two different tissues, eye and wing imaginal discs, were used for these studies, and these screens can identify factors that are used in a wide range of tissues and cells to control cell dynamics. This is based on the observations that ShroomA, the isoform most similar to mammalian Shroom3, induces similar cellular phenotypes in both types of imaginal discs, and the phenotypes can be modified in both tissues. A powerful aspect of this screen is that these processes are functionally conserved in vertebrate cells and tissues. Additionally, the simplified nature of the Drosophila genome makes these screens possible. Due to genetic and functional redundancy, it is predicted that the analysis performed in this study would be more complicated using vertebrate or cell culture model systems. Drosophila have single genes for Shroom, Rok, myosin II, and Ena while mammals possess gene families for these factors. In support of this, previous work has shown that both Rock1 and Rock2 must be inhibited to prevent Shroom3-mediated apical constriction in cell culture. This screening approach should allow for the identification of novel genetic interactions in Drosophila that can be further verified in mammalian model systems to define their potential role in human disease (Hildebrand, 2021).

Most of the modifiers identified in this study participate in defining actin or microtubule architecture. Of these, several regulate actin dynamics at the level of polymerization or stability, including Ena, Diaphanous, Chickadee, and Slingshot. Interestingly, three of these proteins can be linked, directly or indirectly, to neural tube formation in mice. It should be noted that several classes of actin regulators did not appear to modify the Shroom phenotypes, including nucleators, binding proteins, or adaptors, suggesting that specific types of actin organization are required for Shroom-induced perturbation of cell architecture. This is further supported by the observation that Tropomyosin was also identified in the screen. Tropomyosin regulates the structure of actin filaments and the binding of other proteins, including myosin II and cofilin, that in turn modulate cell architecture or behavior. It is particularly intriguing to note that Tropomyosin mutations can suppress phenotypes caused by the loss of Flapwing, presumably caused by increased myosin II activity. In addition to the actin cytoskeleton, these studies also support a role for microtubules in Shroom-induced phenotypes. This is consistent with the role of microtubules in apical constriction in Drosophila. Recent evidence indicates that apical-medial microtubules play an important role in ventral furrow invagination and this is mediated by Patronin, a protein known to interact with Shot. These studies show that microtubules stabilize the connection of contractile networks to cell junctions to facilitate tissue morphogenesis. These studies are consistent with the current results in relation to Shroom function and Shot distribution in the wing epithelium. It will be interesting to determine if the identified proteins act upstream or downstream of Shroom. While the data suggest Ena acts downstream of Shroom, proteins such as Tropomyosin could function upstream by regulating the amount of Shroom that can bind to F-actin or downstream by modulating the amount of myosin II that can be recruited or activated by the Shroom-Rok complex. It was surprising that determinants of cell adhesion or polarity, such as cadherins or Par complex proteins, were not identified in this screen. It is possible that these proteins are present in sufficient quantity and reducing the dosage is unable to modify the Shroom overexpression phenotype and thus other genetic approaches will be needed to assess the role of these pathways (Hildebrand, 2021).

The data show that endogenous Shroom protein is expressed in epithelial cells during wing and eye development, suggesting it functions in these tissues under normal circumstances. Shroom null flies that survive to adults do not exhibit significant defects in the eyes or wings, although null embryos do exhibit defects in convergent extension and perhaps this could contribute to the observed reduction in viability. In embryos, Shroom is important for the polarized distribution of contractile myosin II needed for convergent extension. It is possible that Shroom activity in disc epithelial cells is redundant to other pathways that regulate Rok and myosin II and Shroom normally functions to make these pathways more robust or function with higher fidelity. Uncovering these subtle interactions will require additional genetic approaches. The localization of Shroom in the eye and wing disc appears to be highly regulated and is reminiscent of that exhibited by myosin II and phosphorylated Sqh, particularly in the eye imaginal disc. A dramatic increase was observed in Shroom protein in cells that are exiting the morphogenetic furrow and forming the pre-clusters that will give rise to the ommatidia. As the ommatidia form, Shroom expression becomes restricted to the R3/4 cells and eventually is lost from these cells. This distribution is essentially the inverse to that of E-cadherin, which is highest in the radial junctions and lower in the circumferential junctions. This could reflect differences in adhesive interactions between the ommatidia pre-clusters and the inter-ommatidia cells, which facilitates rotation of the ommatidia. This hypothesis is supported by previous studies demonstrating that differential adhesion generates specific cellular organization and compartmentalization in the developing eye. Interestingly, the PCP protein Flamingo is also expressed in R3 and R4 and previous studies have identified interactions between the Shroom3 and PCP pathways in the neural tube. As eye development continues, this study observed Shroom expression in the pigment cells of the pupal retina. In both the imaginal disc and the retina, Shroom distribution is restricted to specific cell junctions, suggesting there are differential adhesive or contractile forces associated with these membranes (Hildebrand, 2021).

In the wing imaginal disc, expression of Shroom protein was observed in rows of cells that border the anterior half of the wing margin. Consistent with the genetic interactions, a similar expression pattern was observed for both Ena and Shot in these cells. It is currently unclear if the co-expression of Shroom, Ena, and Shot is controlled pre- or post-transcriptionally. It is possible that the expression of Shroom, Ena, and Shot is coordinately regulated in a gene network. Alternatively, the stability or apical localization of these proteins may be interdependent or closely orchestrated. This expression pattern in the anterior wing margin is similar to members of the Irre cell Recognition Module (IRM), including cell surface receptors Roughest, Hibris, and Kirre, which help position the sensory organs. This is particularly interesting in light of the fact that the vertebrate orthologs of these genes, Neph and Nephrin-1, and Shroom3 are all involved in formation of podocytes in the glomerulus of the mammalian kidney. It will be exciting to apply genetic analysis to investigate if these pathways cooperate to regulate tissue morphology (Hildebrand, 2021).

Ena and Shroom show extensive co-expression and colocalization in both the wing and eye imaginal disc, although Ena is more widely expressed than Shroom. In both the wing and eye imaginal disc, Ena is expressed in most cells and is localized primarily in the tricellular junctions with lower expression in the adherens junctions. However, as seen in the wing margin and the morphogenetic furrow, cells that express Shroom protein also exhibit high levels of Ena in the cell junctions. Importantly, reducing the amount of Shroom protein perturbs the localization of Ena in the anterior wing margin. The relationship between Ena, Shroom, Rok, and myosin II in defining cell shape is likely to be complicated. This stems from the observations that these factors could be placed both upstream and downstream of Shroom. For example, it has been previously shown that Shroom distribution to the apical adherens junctions is mediated, at least in part, by direct binding to F-actin. However, it has also been established that RhoA and Rok regulate F-actin architecture to influence Shroom distribution, which then facilitates the polarized distribution of Rok and myosin II. Ena has been shown to have multiple roles in Drosophila development, including axon guidance, collective cell migration, and epithelial morphogenesis. The role Ena plays in Shroom-mediated apical constriction is unclear. The current data suggest that Ena functions downstream of Shroom and is recruited to adherens junctions via an LPPPP-EVH1 interaction. Ena is primarily defined as a modulator of F-actin dynamics that facilitates the formation of long filaments by competing with barbed-end capping and promoting the addition of actin monomers to the barbed end. This activity may be important for providing the substrate for activated myosin II to drive cell contraction. This is consistent with studies in vertebrate cells showing that Diaphanous 1, is also required for contractility in adherens junctions and that this study has also identified Dia as a potential modifier of Shroom activity (Hildebrand, 2021).

Elegant studies from several groups have identified many other signaling pathways that control the distribution of contractile myosin II networks during Drosophila development, including the Fog, PCP, HH, Dpp, EGF, Toll, and integrin signaling pathway. How all these signaling pathways are orchestrated and converge on myosin II at the cellular and tissue level is a fascinating question. It has been shown that the above processes use a variety of methods to regulate the small GTPase RhoA, which activates Rok, including several GTP exchange factors or GTPase Activating Proteins. It should be noted that other GTPases such as Rap1 or CDC42 also regulate apical constriction. This work has shown that Shroom3 may activate Rock independent of RhoA, suggesting that there as mechanisms to bypass small GTPases in the activation of myosin II. It will be informative to utilize this screening approach to further test how these pathways might work with ShroomA to control cell morphology (Hildebrand, 2021).

Filopodia-based contact stimulation of cell migration drives tissue morphogenesis

Cells migrate collectively to form tissues and organs during morphogenesis. Contact inhibition of locomotion (CIL) drives collective migration by inhibiting lamellipodial protrusions at cell-cell contacts and promoting polarization at the leading edge. This study reports a CIL-related collective cell behavior of myotubes that lack lamellipodial protrusions, but instead use filopodia to move as a cohesive cluster in a formin-dependent manner. Genetic, pharmacological and mechanical perturbation analyses were performed to reveal the essential roles of Rac2, Cdc42 and Rho1 in myotube migration. These factors differentially control protrusion dynamics and cell-matrix adhesion formation. Active Rho1 GTPase localizes at retracting free edge filopodia and Rok-dependent actomyosin contractility does not mediate a contraction of protrusions at cell-cell contacts, but likely plays an important role in the constriction of supracellular actin cables. Based on these findings, it is proposed that contact-dependent asymmetry of cell-matrix adhesion drives directional movement, whereas contractile actin cables contribute to the integrity of the migrating cell cluster (Bischoff, 2021).

The ability of cells to migrate as a collective is crucial during tissue morphogenesis and remodeling. The molecular principles of collective cell migration share features with the directed migration of individual cells. The major driving forces in migrating single cells are Rac-mediated protrusions of lamellipodia at the leading edge, formed by Arp2/3 complex-dependent actin filament branching and Rho-dependent actomyosin-driven contraction at the cell rear. Cells can migrate directionally in response to a variety of chemical cues, recognized by cell surface receptors that initiate downstream signaling cascades controlling the activity or recruitment of Rho GTPases. Directional cell locomotion is also controlled by mechanical stimuli such as upon cell-cell contact. A well-known phenomenon is contact inhibition of locomotion (CIL), whereby two colliding cells change direction after coming into contact. Mechanistic evidence has been obtained of how CIL might act in vivo as the driving force to polarize neural crest cells that derived from the margin of the neural tube and disperse by migration during embryogenesis (Bischoff, 2021).

In neural crest cells, CIL involves distinct stages of cell behavior including cell-cell contact, protrusion inhibition, repolarization, contraction, and migration away from the collision. The initial cell-cell contact requires the formation of transient cadherin-mediated cell junctions. Once the cells come in close contact, a disassembly of cell-matrix adhesion near the cell-cell contact and the generation of new cell-matrix adhesions at the free edge occur. Such mechanical crosstalk between N-cadherin-mediated cell-cell adhesions and integrin-dependent cell-matrix adhesions has been recently described in vivo during neural crest cell migration in both Xenopus and zebrafish embryos. However, the loss of cell-matrix adhesions at cell contacts alone is not sufficient to drive CIL. A subsequent repolarization of the cells away from the cell-cell contact and thereby the generation of new cell-matrix adhesions and protrusions at the free edge are required to induce cell migration away from the collision. In neural crest cells, this depends on the polarized activity of the two Rho GTPases, Rac1 and RhoA. A model of CIL has been proposed in which a contact-dependent intracellular Rac1/RhoA gradient is formed that generates an asymmetric force driving directed cell migration. N-cadherin binding triggers a local increase of RhoA and inhibits Rac1 activity at the site of contact. Thus, Rac1-dependent protrusions become biased to the opposite end of the cell-cell contact and cells migrating away from the collision (Bischoff, 2021).

Overall, CIL has been successfully used to explain contact-dependent collective migration of loose clusters of mesenchymal cells such as neural crest cells and hemocytes, but it is still unclear whether mechanisms governing CIL might also contribute to the migratory behavior of cohesive cell clusters or epithelia (Bischoff, 2021).

Using an integrated live-cell imaging and genetic approach, this study identified a CIL-related, contact-dependent migratory behavior of highly cohesive nascent myotubes of the Drosophila testis. Myotubes lack lamellipodial cell protrusions, but instead form numerous large filopodia generated at both N-cadherin-enriched cellular junctions at cell-cell contacts and integrin-dependent cell-matrix sites at their free edge. Filopodia-based myotube migration requires formins and the Rho family small GTPases Rac2, Cdc42, and RhoA, whereas the Arp2/3 complex and its activator, the WAVE regulatory complex (WRC), seem only to contribute to filopodia branching. Rac2 and Cdc42 differentially control not only protrusion dynamics but also cell-matrix adhesion formation. Unlike CIL, RhoA is not activated at cell-cell contacts, but rather gets locally activated along retracting protrusions. Genetic and pharmacological perturbation analysis further revealed an important requirement of Rho/Rok-driven actomyosin contractility in myotube migration (Bischoff, 2021).

In summary, a model is proposed in which N-cadherin-mediated contact-dependent asymmetry of cell-matrix adhesion acts as a major switch to drive cell movement toward the free space, whereas contractile actin cables contribute to the integrity of the migrating cell cluster (Bischoff, 2021).

The data imply that a contact-dependent migration mechanism acts as a driving force to polarize Drosophila myotubes and to promote their directional movement along the testes. A contact-stimulated migration has been already observed in cultured cells many years ago, but the molecular mechanisms underlying this phenomenon has been never analyzed in more detail. It has been observed that both primary neural crest cells and two neural crest-derived cell lines barely moved when isolated in suspension, but could be stimulated up to 200-fold to migrate following contact with migrating cells. This process might help to ensure the cohesion and coordination of collectively migrating myotubes to form dense muscular sheets in the walls of developing hollow organs. Those muscle fibers that race ahead will immediately cease migration when they lose contact with their neighbors. That is exactly what was observed in the current experiments. After ablation, an isolated myotube awaits restimulation by the other cells of the migrating cluster. Consistently, reduced N-cadherin function promotes single-cell migration toward the free space at the expense of collective directionality. The contact-dependent behavior of myotubes also resembles CIL, a well-characterized phenomenon. CIL regulates the in vivo collective cell migration of mesenchymal cells such as neural crest cells by inhibiting protrusions forming within the cluster at cell-cell edges and by driving actin polymerization at their free edge (Bischoff, 2021).

Different from neural crest cells, myotubes did not migrate as loose cohorts, but maintain cohesiveness (see Comparison between filopodia-based and lamellipodia-based cell migration). In the context of more-adhesive cells, a CIL-related mechanism, termed frustrated CIL has been proposed by which cell-cell junctions can determine the molecular polarity of a collectively migrating epithelial sheet. Evidence has been provided that cell-cell junctions determine the molecular polarity through a network of downstream effectors that independently control Rac activity at the cell-free end and Rho-dependent myosin II light chain activation at cell-cell junctions (Ladoux, 2017; Desai, 2009). At the first glance, myotubes do not show an obvious polarized cell morphology with prominent polarized protrusions. Instead, myotubes form numerous competing protrusions in all directions. However, protrusions pointing to the free space preferentially form more stable cell-matrix adhesions as anchorage sites for forward protrusions, whereas the lifetime of cell-matrix adhesions at cell-cell contacts is decreased. Thus, a contact-dependent asymmetry in matrix adhesion dynamics seems to be important for the directionality of migrating myotubes, a molecular polarity that has been also found in neural crest cells undergoing CIL. Only when one of the adhesions of competing protrusions disassembles, pulling of the cell body toward the competing protrusions might contribute to symmetry breaking and directionality of collective migration (Bischoff, 2021).

Evidence is provided for a differential requirement of the Rho GTPases, Rac2, and Cdc42 in regulating cell-matrix adhesion. cdc42 knockdown cells formed less cohesive clusters and showed a significant increase of cell-matrix adhesion lifetime probably due to a decrease cell-matrix adhesion turnover. In contrast, Rac2 depletion resulted in a prominent loss of cell-matrix adhesions, a phenotype that has already been described in Rac1-/- mouse embryonic fibroblasts. Thus, a model is proposed in which cell-matrix adhesions are downregulated at N-cadherin-dependent cell-cell contacts, a process that requires Cdc42 functions. To finally test whether a contact-dependent reduction of cell-matrix adhesion in filopodia is sufficient to explain the observed collective cell behavior, a simplified simulation model was developed with a few rules governing cell behavior such as protrusive filopodia, matrix adhesion, cell-cell adhesion, and membrane resistance. Unlike comparable computer models, single cells do not possess directional information. A cell's position is defined by the geometric center of all its filopodia, whose emergence/disappearance/elongation causes translation of the centroid, perceived as motion. Upon cell-cell contact, filopodia lose their cell-matrix adhesion and thereby their grip on the ECM, but keep connections through cell-cell adhesions. These adhesions are recognized by both contributing cells to calculate their respective centroids. Using these simple rules, it was possible to model myotube collective migration, provided that cells are positioned in a confined area mimicking the unfolded testis surface. If filopodia disappear directly after contact, cells exhibit a different cell behavior that is very reminiscent of CIL. This simplified model further confirms the observation that local regulation of cell-matrix adhesion suffices to drive collective motility (Bischoff, 2021).

Actomyosin function ensures the integrity of cohesive myotube cluster during migration Myotube migration also requires Rho1 the Drosophila homolog of RhoA. Different from cells undergoing CIL, in migrating myotubes activated Rho1 was not enriched at cell-cell contacts between myotubes, but rather localized as local pulses along retracting filopodial protrusions at free edges. The effects of tensile forces have to be addressed separately in the future, by establishing one of the many existing force measurement techniques such as transition force microscopy or using in vivo FRET-based tensions sensors in this system. Loss of Rok activity, sqh, and zip phenocopies rho1 knock down suggesting that a canonical pathway controls myotube migration in which Rho1 acts through Rok kinase to activate myosin II contractility. This finding supports the notion that in testis myotubes, unlike many other cell types, locally restricted Rho-GTPase regulation outweighs global Rac/Rho regulation along the cell-rear axis to achieve directionality. Previous studies demonstrated that myosin II-dependent contraction is essential for coordinating the CIL response in colliding cells. In myotube migration, Rok-dependent actomyosin contraction seems to be not required to drive the myotube cluster forward, but rather contractile actin cables contribute to the integrity of the migrating cell cluster. Thus, myotube cluster behave more like a collectively migrating monolayered epithelial sheet during gap closure. While myotubes migrate into any given free space, they leave larger gaps within the cell sheet surrounded by prominent circumferential actin cables. Constriction of these supracellular actin cables necessarily might lead to gap closure observed in wild type, but not in cells defective for RhoRok-driven actomyosin contractility (Bischoff, 2021).

Efficient mesenchymal cell migration on two-dimensional surfaces is thought to require the Arp2/3 complex generating lamellipodial branched actin filament networks that serve a major engine to push the leading edge forward (Bischoff, 2021).

Interestingly, epithelial and mesenchymal cells form more filopodia when the Arp2/3 complex is absent. Under these conditions, mesenchymal cells lack lamellipodia and adopt a different mode of migration only using matrix-anchored filopodial protrusions. The data further provide evidence for a filopodia-based cell migration in a physiological context during morphogenesis. This migration mode largely depends on formin as central known actin nucleators generating filopodia. The data also suggest that the Arp2/3 and its activator, the WRC, contribute to a more efficient myotube migration by promoting filopodia branching, and thereby increasing the number of cell-matrix adhesions, thus increased anchorage sites. Overall, filopodia-based migration enables the cell to regulate discrete subunits of membrane protrusions as an answer to the environment. The sum of filopodial protrusions adds up to a net cell locomotion that occurs similarly during lamellipodial migration. Filopodial matrix adhesion complexes not only provide anchorage sites, but also allow cells to directly restructure their microenvironment by membrane-bound matrix proteases. There is indeed increasing clinical evidence suggesting filopodia play a central role in tumor invasion. Similar to invading cancer cells myotubes rather migrate through a 3D microenvironment composed of extracellular matrix restricted by pigment cells from the outside of the testis. Thus, it will be interesting to determine to what extent extracellular matrix restructuring by metalloproteinases is required for myotube migration (Bischoff, 2021).

Taken together, the data suggest that contact-stimulated filopodia-based collective migration of myotubes depends on a CIL-related phenomenon combining features and molecular mechanisms described in mesenchymal and epithelial sheet migration as well. A model is proposed in which contact-dependent asymmetry of cell-matrix adhesion acts as a major switch to drive directional motion toward the free space, whereas contractile actin cables contribute to the integrity of the migrating cell cluster (Bischoff, 2021).

GENE STRUCTURE

cDNA clone length - 6259

Bases in 5' UTR - 1022

Exons - 10

Bases in 3' UTR - 1064

PROTEIN STRUCTURE

Amino Acids - 1390

Structural Domains

The Drosophila homolog of Rho-kinase/ROK alpha, Rok, which has conserved the basic structural feature of Rho-kinase/ROK alpha consisting of the N-terminal kinase, central coiled-coil and C-terminal pleckstrin homology (PH) domains, has been identified (Mizuno, 1999).

Rok structure has been compared with that of rat ROK-beta. Both proteins are composed of a conserved N-terminal domain, a serine/threonine kinase domain, a large coiled-coil domain with an embedded Rho-binding domain, and a C-terminal Plekstrin-homology (PH) domain split by a Cys-rich domain (Winter, 2001).

date revised: 12 December 2022

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