BIOLOGICAL OVERVIEW

Rho is a member of the Ras GTPase superfamily; it is a GTP-binding protein that regulates cell shape and motility through modulation of the actin cytoskeleton. A general review of this Rho function and of similar functions for two other Ras GTPase superfamily members, Rac and Cdc42, is provided at the Rac site. This overview will deal with the role of Rho in gastrulation. For general information about Rho activation and inactivation and about the downstream targets of Rho, see the Rho Evolutionary homologs section. In Drosophila, Rho is involved in tissue polarity, and this Rho function is dealt with in the Effects of mutation section.

Rho's role in gastrulation

Ras superfamily proteins function as regulated molecular switches that alternate between active GTP-bound and inactive GDP-bound states. The GTP/GDP balance of these small GTPases is dictated by the net rates of guanine nucleotide exchange and GTP hydrolysis; these rates are tightly controlled by three classes of regulatory molecules: (1) guanine nucleotide exchange factors (GEFs) act as positive regulators that promote the release of GDP and consequent formation of the active GTP-bound state; (2) GTPase activating proteins (GAPs) act as negative regulators that stimulate the intrinsic GTPase activity to cycle them back to the inactive GDP-bound form, and (3) guanine nucleotide dissociation inhibitors (GDIs), associate with GTPases to maintain the existing nucleotide-bound state. Rho, acting through other proteins, mediates actin rearrangements that are likely to be required for the numerous cell shape changes in a developing embryo.

DRhoGEF2 was identified in a genetic screen for Rho signaling pathway components in Drosophila. DRhoGEF2 was found to encode a Rho-specific guanine nucleotide exchange factor (Barrett, 1997). The same gene, DRhoGEF2 was similarly identified in an independent screen carried out by Hacker (1998), a screen designed to characterize the maternal effects of zygotic lethal mutations. The gene was independently cloned by Werner (1997). DRhoGEF2 encodes a protein that contains a PDZ domain near the amino terminus, and a cystine-rich butterfly motif in the central region that is present in isoforms of protein kinase C and the mouse Dbl family oncoprotein Lfc. The C-terminal region of DRhoGEF2 contains an extensive region of homology with two separate protein motifs characteristic of the Dbl family of oncoproteins. The first motif, termed the Dbl homology domain promotes the exchange of guanine nucleotides within Rho family GTPases. The second domain, juxtaposed to the Dbl homology domain and C-terminal to it, is a Pleckstrin homology domain (Hacker, 1998).

Embryos lacking DRhoGEF2 fail to gastrulate due to a defect in cell shape changes required for tissue invagination. Expression of a dominant-negative Rho GTPase in early embryos results in similar defects. Of the DRhoGEF2 homozygotes, 100% die as late embryos or early larvae in the absence of obvious abnormalities. DRhoGEF2 mRNA is expressed uniformly at high levels in the syncitial blastoderm; levels decrease until, by the time gastrulation is initiated, no expression is detected. This apparent maternal contribution of DRhoGEF2 mRNA suggests a likely role for the encoded protein in early embryogenesis. Embryos lacking maternal DRhoGEF2 exhibit normal dorso-ventral and anterior-posterior patterning, as well as mesoderm. However, germband extension and posterior midgut invagination appear to be defective. In addition, the cells of the mesectoderm fail to intercalate at the ventral midline, indicating a defect in ventral furrow formation. In embryos lacking maternal DRhoGEF2, the process of gastrulation is highly disorganized and ventral furrow formation never occurs. In such embryos it appears that random cells within an approximately 20-cell width spanning the ventral midline undergo apical membrane constrictions. There is also a substantially reduced number of addition constrictions in neighboring cells, resulting in a pitted ventral surface. In addition to the defects in ventral furrow formation and invagination of the posterior midgut, DRhoGEF2 mutant embryos are defective in invagination of the anterior midgut, a closely related gastrulation event (Barrett, 1997 and Hacker, 1998).

In addition to the DRhoGEF2 defects, embryos expressing a dominant negative Rho1 exhibit obvious defects in gastrulation. While the furrow in dominant negative Rho1 embryos does form, it fails to extend at the posterior end, resembling the ventral furrow defects in folded gastrulation and concertina mutants. The T-shaped invagination of the anterior midgut does not form normally in these embryos and they also exhibit defects in posterior midgut invagination and germband extension. However, the cephalic furrow forms normally in both DRhoGEF2 and dominant negative Rho1 embryos (Barrett, 1997)

Evidence is also presented that DRhoGEF2 mediates these specific cell shape changes in response to the extracellular ligand, Folded gastrulation. fog was expressed ectopically from a huckebein promoter, which is normally active in a subset of cells at the anterior and posterior ends of the embryo. In all the hkb-fog expressing embryos a characteristic transient depression in the dorsal head region can be seen. The surfaces of cells in this depression exhibit membrane blebbing and constrictions closely resembling those normally seen in cells along the ventral furrow in wild-type embryos. In addition, the nuclei of these cells have migrated from an apical to a basal position. In the absence of DRhoGEF2, ectopic Fog expression fails to induce any detectable cell shape changes, despite equivalent levels of fog transgene expression. Together, these results establish a Rho-mediated signaling pathway that is essential for the major morphogenetic events in Drosophila gastrulation (Barrett, 1997).

The central domain of DRhoGEF2 contains a likely phorbol ester-response motif. The homologous domain in Protein kinase C mediates kinase activation in response to diacylglycerol, which is generate by phospholipase C (PLC). Thus is it possible that the GEF activity of DRhoGEF2 is responsive to diacylglycerol. Since PLC-mediated production of diacylglycerol can be promoted by both receptor tyrosine kinase activation and by activation of receptor-coupled heterotrimeric G proteins, it is possible that the nucleotide-exchange activity of DRhoGEF2 is stimulated by signals transduced by both of these types of receptors. The presence of a PDZ domain in DRhoGEF2, suggests that it may interact with additional signaling proteins. Therefore, it appears that the GEF activity of DRhoGEF2 may be regulated by multiple upstream signals (Barrett, 1997).

It is postulated that Fog acts via the G alpha protein Concertina (see G protein salpha 60A: Evolutionary homologs section for more information about Concertina) to activate DRhoGEF2, thereby promoting Rho1 activation and consequent actin rearrangements. Significantly, the Drosophila G alpha subunit, Concertina, exhibits the strongest sequence similarity to the mammalian Galpha12 and Galpha13 proteins, which mediate the activation of Rho by LPA. Thus, it appears likely that a Rho-mediated signaling pathway linked to heterotrimeric G proteins has been evolutionarily conserved (Barrett, 1997 and references).

The obsevation that DRhoGEF2 is required for the cell shape changes induced by ectopic Fog expression strongly supports the model that a signal from Folded gastrulation via Concertina activates DRhoGEF2 (Barrett, 1997). There is, however, one case of a significant difference between the mutant phenotypes of fog and cta and those of DRhoGEF2: in contrast to DRhoGEF2, fog and cta are not essential for ventral furrow formation. For this reason DRhoGEF2 appears to be activated (at least to some extent) independently of either Fog or Cta. In fact, because of the non-essential function of fog and cta in the mesoderm, a second pathway instructing cells to undergo shape changes has been postulated (Costa, 1994). It is proposed that DRhoGEF2 identifies this pathway as a G-protein-coupled signaling cascade involving the GTPase Rho1. Whether this pathway is also required to transduce additional signals besides that of Fog is presently unclear and this question will require further attention in the future (Hacker, 1998).

Rho's role in dorsal closure

The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. Its role in the developing Drosophila embryonic epidermis during the process of dorsal closure has been investigated. By expressing the dominant negative DRhoA^N19 construct in stripes of epidermal cells, it has been confirmed that Rho function is required for dorsal closure and it is necessary to maintain the integrity of the ventral epidermis. Defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoA^N19 expression. Furthermore, these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoA^N19-expressing cells (Bloor, 2002).

Previous studies on RhoA function during dorsal closure have focused on its role in the formation of the leading edge actomyosin purse-string. This study shows that RhoA is required for proper organization of actin and nonmuscle myosin II throughout the epidermis. In addition, inhibition of RhoA causes misregulation of the JNK transcription activation pathway, loss of DE-cadherin from the cell surface and disruption of the apicolateral distribution of ß_Heavy-spectrin. These changes in cell physiology have differential effects on cell behavior that depend upon the position of the cell within the dorsal-ventral axis. In particular, cell-cell adhesion in the ventral and lateral epidermis is severely compromised, but at the leading edge RhoA^N19-expressing cells form new, ectopic cell-cell adhesions (Bloor, 2002).

Tension generated in the amnioserosa and the leading edge of the lateral epidermis independently contributes to the forces that drive dorsal closure. It has been proposed that nonmuscle myosin II activation generates tension in the leading edge and that this causes a leading edge intracellular actomyosin purse-string to shorten. Signaling downstream of RhoGTPase activates nonmuscle myosin II by modulating the level of myosin regulatory light chain phosphorylation. As such, expression of RhoA^N19 in epidermal stripes might disrupt contraction of the leading edge purse-string. Defects in actin and nonmuscle myosin II organization caused by RhoA^N19 expression are first observed at germband extension, up to 2 hours before purse-string formation. Thus, while actin and nonmuscle myosin II are localized at the leading edge in wild-type tissue, a purse-string structure is never formed in leading edge cells that express RhoA^N19. RhoA^N19 expression therefore effectively cuts the leading edge purse-string at multiple sites. This does not necessarily prevent progression of dorsal closure, confirming previous experiments which demonstrate that the integrity of the leading edge is not required for dorsal closure to continue to completion. It is concluded that small independent regions of leading edge in wild-type epidermal stripes can, in conjunction with contraction of the amnioserosa, migrate dorsally with relative normalcy (Bloor, 2002).

The question that arises is how do epidermal cells expressing RhoA^N19 move dorsally in the absence of a leading edge purse-string? These cells could hitchhike, i.e. they are pulled dorsally by the amnioserosa or dragged along with neighboring wild-type cells. Although spread and disorganized, dorsal RhoA^N19-expressing cells do maintain adhesion with wild-type neighbors and this might then allow passive RhoA^N19-expressing cells to move dorsally with wild-type tissue. This is consistent both with the inverse correlation between integrity of the ventral epidermis and the extent to which dorsal closure proceeds, as well as with observations on the distribution of tension at the embryo surface during dorsal closure. Thus, during the time that the epidermis lateral to the leading edge opposes dorsal closure, ventral failure of epidermal integrity (and hole formation) would release the tensional restraints on the remaining lateral epidermis, allowing it to move dorsally with more success. Similarly, in the absence of this release (i.e. the ventral epidermis retains its integrity and opposes dorsal movement of the epidermis), the leading edge is presumably no longer capable of generating sufficient force to drive dorsal closure to completion (Bloor, 2002).

RhoA^N19 expression causes ectopic activation of the JNK pathway in the lateral epidermis, suggesting that RhoA normally functions to inhibit JNK signaling. JNK activation is antagonized by the protein phosphatase encoded by puc, and in puc mutants JNK signaling is increased at the leading edge and is activated in the lateral epidermis. Thus, JNK signaling in wild-type embryos is not maximal and basal JNK activity in the lateral epidermis is revealed in the absence of either puc or RhoA mediated repression. Interestingly, RhoA^N19 expression does not increase JNK signaling in the leading edge. This difference between the effect of puc mutations and RhoA^N19 expression could be due to ectopic RhoA^N19 suppressing an upstream JNK activator that is itself maximally activated in the leading edge (Bloor, 2002).

Co-expression of RhoA^N19 and GMA, an actin marker in which GFP is fused to the Drosophila Moesin actin-binding domain, demonstrates that, in addition to effects on the actin cytoskeleton, inhibition of RhoA has profound effects on the adhesive properties of epidermal cells. In accordance with this, DE-cadherin is lost from the surface of RhoA^N19-expressing cells. Rho is required for E-cadherin-mediated epithelial cell-cell adhesion in cultured vertebrate cells: in keratinocytes and MDCK cells, blocking Rho function prevents formation of E-cadherin-based junctions and causes preformed junctions to breakdown. This effect is dependent on cell-cell junction maturity; blocking Rho causes E-cadherin to be lost rapidly (within 1 hour) from immature junctions, but E-cadherin can persist for several hours at mature junctions. This differential affect is also observed in this study, since removal of DE-cadherin from the cell surface is not uniform throughout the RhoA^N19-expressing stripe; ventral cells lose surface staining sooner than dorsal cells. This phenomenon most probably reflects regional differences in the maturity of epidermal cell junctions. Cells of the dorsal epidermis form a compact epithelium early in stage 10, while neuroblast delamination in the ventral neurectoderm delays formation of the ventral epidermis proper until well into stage 11, by which time enGAL4- and prdGAL4-driven protein expression is apparent. Alternatively, the differential effect might be due to a dorsoventral gradient in enGAL4- or prdGAL4-driven expression of RhoA^N19. This seems unlikely, since no regional differences in fluorescence are observed when these GAL4 lines are used to drive GMA expression (Bloor, 2002).

If the primary defect associated with the epidermal expression of RhoA^N19 is loss of DE-cadherin, then the phenotypes induced by RhoA^N19 should phenocopy those of shotgun (DE-cadherin) mutants. The defects exhibited by shg embryos are difficult to compare with those shown by embryos expressing RhoA^N19 in epidermal stripes. However, genetic analysis of shg demonstrates that the embryonic dorsal epidermis is less sensitive than the presumptive ventral epidermis to a reduction in DE-cadherin levels; embryos mutant for null shg alleles are missing head and ventral cuticle, while the dorsal cuticle appears unaffected. Thus, as with epidermal expression of RhoA^N19, shg mutants disrupt ventral epidermal integrity and cells undergo apoptosis, while dorsally epidermal cells remain adherent and secrete cuticle. However, there is at least one clear distinction between the genetic reduction of DE-cadherin and the defects induced by expression of RhoA^N19: both null and dominant-negative mutations in shg do not affect epithelial cell polarity, while inhibition of RhoA activity does. It seems likely that this difference reflects the additional function of RhoA in generating cell polarity, possibly through organization of the actin cytoskeleton. It is concluded that the epidermal defects caused by RhoA^N19 expression cannot be explained simply on the basis of loss of DE-cadherin mediated adhesion (Bloor, 2002).

Maintenance of dorsal epithelial integrity in the absence of detectable surface DE-cadherin suggests that a secondary adhesion system must function in the dorsal epidermis. As in vertebrate cells different classical cadherins exhibit cell type dependent sensitivity to Rho inhibition this could involve another member of the cadherin family. The possibility that two cell-cell adhesion systems function in the dorsal epidermis may explain the behavior of RhoA^N19-expressing cells. Embryos that express RhoA^N19 in epidermal stripes differ from shg mutants in the uniformity of DE-cadherin loss from the cell surface. In shg mutants, zygotic DE-cadherin is lost from all cells, while striped expression of RhoA^N19 results in two populations of dorsal epidermal cells -- those with cell surface DE-cadherin and those without. Differential adhesion properties of cell populations are the molecular basis for the classical phenomenon of cell sorting. Thus, the ectopic cell bridges formed by RhoA^N19-expressing cells in these experimental embryos could be due to activation of a cell sorting mechanism between populations of dorsal epidermal cells that associate via different adhesion molecules (Bloor, 2002).

In summary, by expressing the dominant negative RhoA^N19 construct in epidermal stripes in the developing Drosophila embryo, it has been shown that the small GTPase Rho has multiple functions throughout this tissue. It is therefore clear that experiments designed to test the function of Rho family GTPases in the epidermis cannot be interpreted simply in terms of their effect on the leading edge. The challenge ahead lies in dissecting the pathways downstream of Rho and determining how these Rho-dependent processes contribute individually to epidermal function and morphogenesis (Bloor, 2002).

Wash functions downstream of Rho and links linear and branched actin nucleation factors

Wiskott-Aldrich Syndrome (WAS) family proteins are Arp2/3 activators that mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. Wasp and Scar/WAVE, the two founding members of the family, are regulated by the GTPases Cdc42 and Rac, respectively. By contrast, linear actin nucleators, such as Spire and formins, are regulated by the GTPase Rho. A third WAS family member, called Washout (Wash), has Arp2/3-mediated actin nucleation activity. This study shows that Drosophila Wash interacts genetically with Arp2/3, and also functions downstream of Rho1 with Spire and the formin Cappuccino to control actin and microtubule dynamics during Drosophila oogenesis. Wash bundles and crosslinks F-actin and microtubules, is regulated by Rho1, Spire and Arp2/3, and is essential for actin cytoskeleton organization in the egg chamber. These results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism for how cells might coordinately regulate these structures (Liu, 2009).

The actin cytoskeleton consists of linear and branched filament networks required for processes ranging from cell division to migration. How these two networks function and are coordinated is of major interest, as their misregulation results in infertility, immunodeficiency, and tumor metastasis in humans. Linear actin filament networks, required for cytokinesis and filopodia formation, are regulated by nucleators and bundling proteins, which enhance filament formation rates and control filament organization, respectively. Examples include Spire and the formin Cappuccino (Capu), which exhibit both nucleation and bundling activities and are essential for oocyte development during Drosophila oogenesis. Both Spire and Capu are regulated by the GTPase Rho1 of the Rho family of small GTPases, which is upstream of other linear nucleators, such as Diaphanous, and is considered a key regulator of linear filament formation (Liu, 2009).

Branched or dendritic actin filament networks, which are required for phagocytosis and lamellipodia formation, are primarily regulated by the Arp2/3 complex and by nucleation-promoting factors that associate with Arp2/3 and actin monomers to nucleate daughter filaments off of existing mother filaments. Like Spire and Capu, Arp2/3 is essential for Drosophila oogenesis, specifically for maintaining proper nurse cell cyto-architecture and function. One family of Arp2/3 activators, the Wiskott-Aldrich Syndrome (WAS) protein family, has been shown to function downstream of Rho GTPases to mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. WASP and SCAR/WAVE, the two founding subclasses of the family, are activated by the GTPases Cdc42 and Rac, respectively. Two new WAS subclasses, WASH and WHAMM, have recently been reported and have been shown to exhibit Arp2/3-mediated branched nucleation activity. Which GTPases might regulate them, however, is not known (Liu, 2009).

This study reports that Drosophila Wash functions downstream of Rho1 and interacts with Spire and Capu to regulate actin and microtubule organization during Drosophila oogenesis. Wash nucleates actin in an Arp2/3-dependent manner, and exhibits F-actin and microtubule bundling and crosslinking activity that is regulated by a pathway involving Rho1, Spire and Arp2/3. Wash genetically interacts with Rho1, Capu, Spire and Arp2/3, and is essential for actin cytoskeleton organization during oogenesis. These results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism of cytoskeletal control through which cells might coordinately regulate linear and branched architectures (Liu, 2009).

It has been suggested that Rho1 regulates the timing of ooplasmic streaming by regulating the MT/microfilament crosslinking that occurs at the oocyte cortex. In this model, crosslinking antagonizes the formation of the dynamic subcortical MT arrays that are required for ooplasm streaming, but does not require the actin-nucleation activity of these proteins. The current model depends on the presence of SpirC and the cortical localization of Rho1, Capu, the Spire isoforms, and now Wash during late-stage oocytes. Support for this model comes from a recent study demonstrating that chickadee, encoding fly Profilin, is required for the formation of cortical actin bundles in the oocyte, and that Capu and Spire anchor the minus ends of MTs to a scaffold made from these cortical actin bundles. These results suggest dual or multifaceted biochemical roles for these proteins in regulating developmental processes. Consistent with this concept, non-actin-nucleating roles for other formins (i.e. actin severing/depolymerization, MT stabilization, signaling, and transcriptional regulation) are beginning to be reported (Liu, 2009).

St Johnston and colleagues have recently proposed an alternative model in which Capu and Spire are required to organize an isotropic mesh of actin filaments in the oocyte cytoplasm that suppresses the motility of kinesin, a plus-end directed MT motor protein that is required for ooplasmic streaming (Dahlgaard, 2007). Their model was formulated with the assumptions that the SpirC isoform does not exist, that spir^RP is a null allele, and that the cortical localization of Capu and Spire is lost in late-stage oocytes. This study found these assumptions not to be the case. mRNA and protein evidence is provided for the existence of the SpirC isoform. The existence of SpirC is also supported by ESTs from the Drosophila Genome Project. The spir^RP allele affects only the SpirA and SpirD isoforms; it does not affect the SpirC isoform because this isoform has a unique 5' end. Ectopic SpirC expression would not be expected to rescue spir^RP because it is already being expressed. The cortical localization of Capu and the Spire proteins during the late stages is masked by intense yolk auto-fluorescence in the green channel when using live imaging of GFP fusions, but can be observed by fixing, by antibody staining, or by the use of ChFP ('cherry' fusion protein). In addition, a subsequent study has shown that kinesin is not required for this cytoskeletal reorganization, suggesting that Capu and Spire might not act as indirect kinesin regulators, but as direct modulators of the MT cytoskeleton). One possibility is that Capu and Spire are bundling and crosslinking MTs to Profilin-dependent F-actin at the oocyte cortex, as has been demonstrated in vitro (Liu, 2009).

Since the discovery of Arp2/3 activators and other actin-nucleation promoting factors, much of the work examining the functions of these proteins has been focused on the properties of their nucleation activities. Recent studies, however, have begun reporting novel biochemical activities for actin nucleators, including MT stabilization activity by mammalian Diaphanous, filopodia inhibition by WAVE/Arp2/3, and F-actin and MT bundling and crosslinking by Spire and Capu (Rosales-Nieves, 2006). Consistent with this, not all disease-associated WASP mutations are predicted to affect its actin-nucleation activity (Notarangelo, 2008). The current results contribute to this growing list of actin nucleators with significant non-nucleation activities, since this study shows that Wash is both an Arp2/3 activator and a crosslinker/bundler of F-actin and microtubules. What is unique about Wash, however, is that its combination of biochemical activities suggests that it is an important intermediary molecule functioning at the intersection of linear and branched actin architectures, with Spire, Rho and Arp2/3 acting as the factors that direct these dual functions of Wash. Based on these findings, the following model is proposed for Wash function in the context of Drosophila oogenesis. In the nucleation pathway, upstream signals and factors, possibly Rho, induce Wash activation, which acts with Arp2/3 to promote branched filament formation and cytoskeletal integrity in nurse cells. In the crosslinking/bundling pathway, Wash bundles and crosslinks filaments of actin and MTs, under the control of Rho and SpirD, to maintain cortical bundle stability in the oocyte and to prevent premature ooplasmic streaming. Together with Capu and Spire (Rosales-Nieves, 2006), Wash maintains the correct timing of ooplasmic streaming by preventing the formation of the microtubule tracks required for motor proteins to drive cytoplasmic flow. The dual functions of Wash might also be regulated spatially by Arp2/3 and depend on the availability or concentration of Arp2/3. Since the nucleation activity of Wash is Arp2/3 dependent, Wash-mediated actin nucleation might require some threshold concentration of locally available Arp2/3; for example, at the ring canals. Spatiotemporal regulation is also possible through the changing levels of Arp2/3 during oogenesis. Arp2/3, for example, might transiently accumulate at the oocyte cortex during the onset of streaming to disrupt Wash bundling activity (Liu, 2009).

These findings contribute to previous studies examining the functions of Wasp and Scar in Drosophila, and together describe a spectrum of phenotypes that illustrate the multiple functions exerted by WAS family members in development. Scar has been shown to be required for axon development, egg chamber structure, adult eye morphology and myoblast fusion; Wasp has been demonstrated to be required for Notch-mediated cell-fate decisions, rhabdomere microvilli formation, bristle development and myoblast fusion; and Wash is required for pupal development and oogenesis, as described in this study. Mutants in various subunits of Arp2/3 have also been described, offering additional insight into how Wash, Wasp and Scar shape the cytoskeleton during development. Interestingly, the spectrum of Arp2/3 mutant phenotypes reported does not completely overlap with all of the phenotypes associated with these WAS family mutants. This might be because Arp2/3 has not been examined in all of the processes in which WAS members play a role, or it might be an indication that WAS members have additional, Arp2/3-independent functions, which is the case for Wash. The current observations support the idea that these and other actin nucleators, such as Capu and Spire, are required at different times or locations during development, and are thus tightly regulated spatiotemporally by Rho GTPases and other factors (Liu, 2009).

The data indicate that Wash acts as a downstream effector of Rho. Indeed, Rho is shown to regulates the bundling/crosslinking activity of Wash through the relief of SpirD inhibition. However, Rho does not enhance the ability of Wash to induce Arp2/3-mediated actin nucleation, raising the question of how or whether Rho might regulate the Arp2/3-associated functions of Wash. Interestingly, the results are consistent with studies examining the Cdc42 regulation of Wasp in Drosophila, which conclude that Cdc42 activation of Wasp is not required for Wasp function in myoblast fusion or bristle development. Although Wasp exhibits a strong and specific interaction with active Cdc42^GTP in vitro, these studies provide strong evidence that, at least for the subset of developmental processes examined, Wasp is not regulated upstream by Cdc42^GTP. As previously noted, Drosophila Wasp differs from mammalian homologs in that it is not auto-inhibited; Cdc42, therefore, might not be required for the activation of its actin nucleation-promoting functions. This might also be the case for Wash, as it too appears to be constitutively active, and might act as a downstream effector of Rho only where its bundling/crosslinking activities are concerned. The data, however, do not rule out the possibility that the nucleation activity of Wash is regulated by a complex in vivo. In fact, recent reports have shown that two proteins originally associated with Scar regulation, Abi and Kette, control Wasp function in Drosophila as well. It remains to be determined whether Abi and Kette also regulate Wash function, and whether Rho might play a role in mediating these interactions (Liu, 2009).

Wash requires Arp2/3 for actin nucleation, but, interestingly, this association appears to disrupt the ability of Wash to bundle and crosslink F-actin and microtubules, as a loss of F-actin/MT bundling favored branching actin filaments. This suggests that Arp2/3 might act as a molecular switch that shifts Wash function from bundling to nucleation and, in terms of cytoskeletal remodeling, supports the hypothesis that Arp2/3 regulates the balance between linear and branched actin architectures in the cell. This is predicated on the assumption that the Wash bundling/crosslinking and nucleation-inducing activities are mutually exclusive, and would represent a previously uncharacterized function of Arp2/3. However, scenarios cannot be ruled out in which nucleation and bundling might coexist. F-actin bundling might be preserved if the branched-actin structures created by Wash and Arp2/3 in vitro are bundled by Wash in parallel (form angled, branching bundles rather than the tortuous bundles observed under non-Arp2/3 conditions), or if filaments emanating from vertices are clamped together by Wash at the branching point to form angled bundles that branch from these vertices. An example of this latter case has been reported in a recent study examining the concerted actions of N-Wasp and Hsp90 to nucleate branched actin filaments (via N-Wasp activation of Arp2/3) and clamp the angled filaments to form a linear bundle (mediated by Hsp90). Wash therefore, in having both nucleation and bundling activities, might perform both functions simultaneously in the presence of Arp2/3. At the very least, Arp2/3 abolishes the ability of Wash to bundle MTs and crosslink them to actin, and so might contribute to regulating crosstalk between the actin and microtubule cytoskeletons. Further studies examining the molecular interactions of WAS family members and Arp2/3 will be invaluable for understanding the full range of cytoskeletal regulation in the cell (Liu, 2009).

In motile cells the actin cytoskeleton can be represented as a dynamic sum of two general geometries - strands or bundles of linear actin filaments, and broad dendritic networks of branched filaments. The mechanisms by which these two networks are remodeled and coordinated are areas of intense investigation and are important for understanding how processes such as lamellipodia and filopodia formation occur. It is intriguing to note that, in the latter case, the biochemical properties of Wash suggest that it might play a role in the convergent extension model of filopodia formation, whereby uncapped actin filaments nucleated from a dendritic branched-actin array are captured at the cell periphery and bundled to form long extensions (Mattila, 2008). Wash, as both an Arp2/3 activator and an F-actin bundling protein, is in an ideal position in which to carry out both the nucleation and the bundling functions, and might thus be an important regulator of filopodia formation alongside previously discovered molecules (Mattila, 2008). The presence of Spire and Arp2/3 at the dendritic bed and active Rho at the cell membrane could form two zones of differential activity to switch Wash function from nucleation to bundling and crosslinking. This form of spatial regulation is analogous to how Rho, Cdc42 and Rac define regions of differential activity during wound healing and cell adhesion. Further investigation into the role of Wash in filopodia and lamellipodia formation will be important, as these protrusions play essential roles in wound healing, substrate adhesion and neurite outgrowth (Liu, 2009).

In humans, the misregulation of WAS members results in disorders such as Wiskott-Aldrich Syndrome, and cancer metastasis. As a new member of the WAS family, human WASH appears to also be clinically relevant. WASH has been reported to be overexpressed in a breast cancer cell line and might, like the overexpression of N-WASP and Scar/WAVEs, contribute to metastasis (Leirdal, 2004). Moreover, the subtelomeric location of human WASH places it at high risk for deletion and rearrangement, as subtelomeres are hotspots of meiotic interchromosomal sequence transfers. The data presented in this study demonstrate that Wash is essential for development in Drosophila, and suggest that Wash might function in actin organization in other contexts. Further work will be required to understand how Wash and other WAS family members coordinate linear- and branched-actin networks during oogenesis and other cellular processes, and how the misregulation of these processes results in disease (Liu, 2009).

Rho GTPase controls Drosophila salivary gland lumen size through regulation of the actin cytoskeleton and Moesin

Generation and maintenance of proper lumen size is important for tubular organ function. This study reports on a novel role for the Drosophila Rho1 GTPase in control of salivary gland lumen size through regulation of cell rearrangement, apical domain elongation and cell shape change. Rho1 controls cell rearrangement and apical domain elongation by promoting actin polymerization and regulating F-actin distribution at the apical and basolateral membranes through Rho kinase. Loss of Rho1 results in reduction of F-actin at the basolateral membrane and enrichment of apical F-actin, the latter accompanied by enrichment of apical phosphorylated Moesin. Reducing cofilin levels in Rho1 mutant salivary gland cells restores proper distribution of F-actin and phosphorylated Moesin and rescues the cell rearrangement and apical domain elongation defects of Rho1 mutant glands. In support of a role for Rho1-dependent actin polymerization in regulation of gland lumen size, loss of profilin (Chickadee) phenocopies the Rho1 lumen size defects to a large extent. Ribbon, a BTB domain-containing transcription factor, functions with Rho1 in limiting apical phosphorylated Moesin for apical domain elongation. These studies reveal a novel mechanism for controlling salivary gland lumen size, namely through Rho1-dependent actin polymerization and distribution and downregulation of apical phosphorylated Moesin (Xu, 2011).

Rho1 acts both in salivary gland cells and in the surrounding mesoderm to maintain apical polarity during gland invagination and to mediate cell shape change during gland migration. This study demonstrates a novel role for Rho1 in controlling salivary gland lumen size through regulation of actin polymerization and distribution and regulation of Moesin activity. By analyzing Rho1 alleles for which salivary gland cells invaginated and formed a gland, it was shown that zygotic loss of function of Rho1 results in shortening and widening of the gland lumen, which is accompanied by defects in cell shape change and cell rearrangement and failure of apical domains to elongate along the Pr-Di axis of the gland. These effects of Rho1 are mediated through Rok; inhibition of Rok completely phenocopies loss of Rho1 in these cellular events. Based on these studies, a model is proposed for Rho1 control of salivary gland lumen size, in particular lumen width, which is determined by cell rearrangement and apical domain elongation. Rho1 and Rok, through inhibition of cofilin, regulate cell rearrangement and apical domain elongation by promoting actin polymerization to localize F-actin at the basolateral membrane and by limiting the apical accumulation of F-actin. In parallel to its role in actin polymerization and distribution, Rho1 acts independently of Rok to limit apical p-Moe with Rib by an unknown mechanism and this function of Rho1 is specific for apical domain elongation. The data on cofilin (Twinstar) are consistent with those in cultured HeLa cells that showed that mammalian ROCK can inhibit cofilin activity indirectly through LIMK-mediated phosphorylation of cofilin (Xu, 2011).

Although manipulating Moe activity through gland-specific expression of Moe^T559D was sufficient to completely phenocopy the Rho1 lumen defects, including cell rearrangement, it did so without disrupting actin polymerization or distribution. This is likely to be due to activated Moe strengthening the link between the actin cytoskeleton and the apical plasma membrane (without affecting levels of apical F-actin), which would increase apical membrane stiffness and remove the ability of gland cells to rearrange. Indeed, Moesin has been shown to control cortical rigidity during mitosis of cultured Drosophila S2R+ cells. Thus, Rho1 regulates cell rearrangement and apical domain elongation by controlling the actin cytoskeleton and Moesin activity through distinct mechanisms (Xu, 2011).

The observation that chic mutant glands phenocopy Rho1 mutant glands to a large extent, suggests that Rho1 control of salivary gland lumen size is mainly dependent on a requirement for Rho1 in actin polymerization. However, as the chic and Rho1 gland lumen phenotypes are not identical, with chic mutant glands lacking the apical accumulation of F-actin and p-Moe observed in Rho1 mutant glands, Rho1 probably has an additional function in limiting accumulation of F-actin and p-Moe at the apical membrane. This function of Rho1, at least for limiting apical F-actin, might partly involve Rab5- or Shi-mediated endosome trafficking, because inhibition of Rab5 alone or Shi alone led to accumulation of F-actin at the apical membrane. Although Rab5^DN- or Shi^DN-expressing salivary gland cells were enriched with apical F-actin, lumen size was not affected. This could be due to Rab5^DN and Shi^DN affecting a pool of apical F-actin distinct from that affected by Rho1 and/or because Rab5^DN-expressing gland cells retain basolateral F-actin and the ratio of apical to basolateral F-actin is not altered sufficiently to cause lumen size defects. In Rho1^1B mutant gland cells, some early endosomes were not coated with F-actin. Actin is known to contribute to multiple steps of the endocytic pathway, including movement of endocytic vesicles through the cytoplasm and their transport to late endosomes and lysosomes. One possible mechanism by which Rho1 normally limits apical accumulation of F-actin is by promoting its removal from the apical membrane and accumulation on endocytic vesicles (Xu, 2011).

Currently, it is not know how Rho1 limits accumulation of apical p-Moe. Membrane localization and activity of Moesin can be regulated via a number of mechanisms, such as its phosphorylation on a conserved Threonine residue, binding to phosphatidylinositol-(4,5)bisphosphate [PtdIns(4,5)P2] and association with components of the sub-membrane cytoskeleton, such as Crb. Studies in cultured mammalian cells have demonstrated that Rho signaling activates Moe either through phosphorylation of Moe by ROCK or through ROCK-mediated inhibition of myosin phosphatase, which is known to dephosphorylate p-Moe. Although it is possible that Drosophila Rho1 positively regulates Moe activity by one or more of these mechanisms, this study shows that in the developing salivary glands Rho1 in fact negatively regulates Moe activity. In rib mutant embryos, in which p-Moe is enriched apically, salivary gland and tracheal cells showed decreased staining for Rab11 GTPase, which localizes to the apical recycling endosomes and to secretory vesicles destined for the apical membrane. Thus, Rho1, like Rib might limit apical p-Moe through its membrane transport (Xu, 2011).

In Drosophila imaginal disc epithelia, Moe negatively regulates Rho1 activity to maintain epithelial integrity and to promote cell survival. These studies demonstrating that in the developing salivary gland Rho1 antagonizes Moe activity by limiting its localization at the apical membrane, shed novel insight into the functional relationship between Rho1 and Moe. It is possible that in a dynamic epithelium, such as the developing salivary gland, Rho1 contributes to the precise spatial and temporal regulation of Moe activity to fine-tune selective changes in apical domain shape. By contrast, in the imaginal disc epithelium, Rho1 regulation of Moe might not be necessary and, instead, Moe regulation of Rho1 activity is required to maintain epithelial integrity and cell survival. Thus, Rho and Moe can antagonize each other's activities depending on the type of epithelia or cellular event (Xu, 2011).

Rescue studies with Rho1^WT demonstrate that Rho1 functions predominantly in the salivary gland cells to control apical domain elongation and cell rearrangement. Interestingly, expression of Rho1^WT in the mesoderm with twi-GAL4 has no effect on cell rearrangement and has little effect on apical domain elongation and lumen size, whereas it has been shown that Rho1^WT expression in the mesoderm significantly rescues the gland migration defect of Rho1^1B mutant embryos. This suggests that gland migration and lumen size control are regulated by distinct mechanisms. In support of this conclusion, embryos mutant for multiple edematous wings, encoding the αPS1 integrin subunit, which have defects in gland migration, show no defects in gland lumen width. Identifying the distinct and overlapping mechanisms by which salivary gland lumen width and length are controlled will help to elucidate the mechanisms by which lumen size is controlled in tubular organs (Xu, 2011).

Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6

Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell-cell junctions. This study shows that collective invasion by cancer cells depends on decreasing actomyosin contractility at sites of cell-cell contact. When actomyosin is not downregulated at cell-cell contacts, migrating cells lose cohesion. A molecular mechanism is provided for this downregulation. Depletion of discoidin domain receptor 1 (DDR1) blocks collective cancer-cell invasion in a range of two-dimensional, three-dimensional and 'organotypic' models. DDR1 coordinates the Par3/Par6 cell-polarity complex through its carboxy terminus, binding PDZ domains in Par3 and Par6. The DDR1-Par3/Par6 complex controls the localization of RhoE to cell-cell contacts, where it antagonizes ROCK-driven actomyosin contractility. Depletion of DDR1, Par3, Par6 or RhoE leads to increased actomyosin contactility at cell-cell contacts, a loss of cell-cell cohesion and defective collective cell invasion (Hidalgo-Carcedo, 2011).

Collective movement requires the coordination of actomyosin organization between cells. Actomyosin contractility is high around the edge of the cell cluster and low between cells. At the margin of the group, actomyosin is organized in a supracellular structure analogous to the 'purse-string' observed in epithelial wound closure. Both the elevated actomyosin levels observed around the edges of groups of invading cancer cells and 'purse-string' wound closure are dependent on Cdc42. Force is transmitted between cells through cell-cell contacts near the edge of the group. However, if force is applied uniformly around the cell margin, the cell junctions become compromised and the coordination of movement between neighbouring cells fails. Consistent with this, contact inhibition of locomotion and cell-cell repulsion are associated with increased Rho-driven actomyosin contraction function after cell-cell contact. Therefore a mechanism is required to decrease actomyosin contractility at sites of cell-cell contact. DDR1 acts in a new non-collagen-binding capacity at cell-cell contacts. The localization of DDR1 to cell-cell contacts requires E-cadherin. Once localized at cell-cell contacts, DDR1 helps to recruit Par3 and Par6; these molecules are required for efficient collective invasion. Cell polarity regulators are required for optimal migration in two-dimensional scratch/wound assays, which have some aspects of a collective nature. Moreover, Par3 and Par6 are required for collective migration of border cells in the Drosophila embryo41. The DDR1-Par3/Par6 complex then controls the localization of RhoE. RhoE may be localized through the intermediary p190ARhoGAP, which can bind both Par6 and RhoE. Consistent with this, depletion of p190ARhoGAP gave a similar phenotype to that after DDR1 or Par3/Par6 depletion. Both RhoE and p190ARhoGAP can antagonize Rho-ROCK-mediated regulation of actomyosin. The Par3-dependent suppression of actomyosin that was observe reciprocates the suppression of Par3 function by the actomyosin regulator ROCK. It is likely that the reciprocal nature of these negative interactions serves to segregate Par3 and actomyosin robustly. The DDR1-dependent mechanism that is describe most probably acts together with other proteins to decrease Rho-ROCK function at cell-cell contacts, such as p120 catenin-dependent mechanisms. This analysis has not yet allowed determination of all the components of DDR1 complexes at cell-cell contacts (Hidalgo-Carcedo, 2011).

Various regulators of cell polarity become misregulated in cancer; this has been linked to increased metastasis. It is believed that disruption of DDR1-dependent Par3 localization to cell-cell contacts might be expected to favour blood-borne metastasis. The data do not exclude a positive role for DDR1 in metastasis as a collagen receptor. Indeed, it was found that interference with DDR1 function in metastatic MTLn3 cells decreased their ability to colonize lung tissue. DDR1 expression may therefore not correlate simply with metastatic ability, but it is important to consider whether it is acting in a cell-matrix or cell-cell adhesion context: in the former it may promote single-cell cancer invasion and processes such as lung colonization; in the latter it may only promote more local and lymphatic invasion and hinder haematogenous metastasis. It is likely that DDR1 engages in different molecular complexes depending on whether it is involved in cell-cell interactions or cell-matrix interactions. For example, the data suggest that DDR1 does not associate with myosin IIa at cell-cell contacts but it has been reported to associate with myosin IIa in other contexts (Hidalgo-Carcedo, 2011).

This study described a mechanism that is required to decrease actomyosin contractility at sites of cell-cell contact. DDR1 acts in a new non-collagen-binding capacity at cell-cell contacts. DDR1 helps to recruit Par3 and Par6; this complex then controls the localization of RhoE, which can antagonize Rho-ROCK-mediated regulation of actomyosin. Thus, DDR1 functions at cell-cell contacts to keep actomyosin activity at low levels. Without this decrease in actomyosin activity, cell cohesion cannot be maintained during collective cell migration (Hidalgo-Carcedo, 2011).

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).

Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis

During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. This study used optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. Precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal-ventral or anterior-posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes (Izquierdo, 2018).

The results presented in this study show that localized activation of Rho signaling at the apical surface of cells, which are otherwise not programmed to invaginate, is sufficient to cause tissue invagination and to recapitulate major cell- and tissue-level behaviors associated with endogenous invagination processes. Mechanisms other than apical constriction control a variety of different forms of invaginations during animal development. The current results do not challenge this view, rather, they argue that if considering a monolayer of epithelial cells, apical constriction is sufficient to fold it into a U-shape invagination. However, apical constriction is not sufficient to drive closure of an invagination into a tube-like structure, as seen for example during ventral furrow formation. Additional pushing forces exerted by lateral non-invaginating cells and/or loss of myosin II from the basal surface and basal relaxation might be required to complete this step (Izquierdo, 2018).

At the tissue-level, the contractile behavior of individual cells depends on the geometry of photo-activation. While a square box results in isotropic apical constrictions, a rectangular shape causes cells to constrict preferentially along the minor axis of the photo-activated area and to elongate along the major axis. This anisotropic contractile behavior resembles the one of ventral furrow cells, which are also organized in a rectangular pattern and constrict preferentially along the short axis of the tissue. Anisotropy in ventral furrow cells is not genetically determined but arises as a consequence of tissue geometrical and mechanical constraints. Consistent with these studies, the increase in the degree of anisotropic constriction as a function of the rectangularity of the photo-activated area can be explained if considering that it is mechanically less favorable to shrink cells along the major axis of a rectangle than along the short axis. Indeed, the former deformation requires the endpoints of the constricting tissue to move farther, and thus a larger deformation of neighboring tissues along that axis (Izquierdo, 2018).

The results also reveal an interesting correlation between pulsatile constrictions and tissue invagination. During endogenous morphogenetic processes, two different pulsatile behaviors have been described. One is based on cycles of myosin II accumulation at the medio-apical plane of the cell, during the contraction phase, and dissolution during the relaxation phase. This type of pulsatile behavior has been first described during dorsal closure in Drosophila and it is not linked to tissue invagination. Another type of pulsatile behavior is based on an incremental accumulation of myosin II at each contraction, which is followed by a stabilization period of cell shape without an intervening relaxation phase (ratcheted contractions). Ratcheted contractions require the radial polarization of Rho signaling components. However, in certain mutant conditions that interfere with the establishment of radial polarity, contractions become non-ratcheted with myosin II miss-localizing at three-ways junctional vertices. The optogenetic-induced pulsatile contractions described in this study display a non-ratchet behavior, and similarly to dorsal closure contractions, are characterized by myosin II medio-apical accumulation and dissolution in phase with contraction and relaxation of the apical surface. However, differently from dorsal closure, optogenetic-induced pulsatile contractions display a higher degree of synchrony with photo-activated cells constricting and relaxing in concert. This difference could be explained if considering that a light pulse provides a coherent and synchronous input, while activation of signaling in a developing tissue might be more subject to cell-to-cell variability. Lack of tissue internalization upon induction of pulsatile constrictions is likely due to the absence of a stabilization phase after constriction of the apical surface, which might result in a dissipation of the forces that are normally needed to build tension and drive invagination. Consistently, continuous administration of light induced synchronous contractile behavior and invagination, mimicking the activity of signaling molecules such as Fog whose function is to control the transition from stochastic to collective contractile behavior during ventral furrow invagination. Pulsatile behavior could be elicited either by a discontinuous administration of light, or by continuous illumination at a lower laser power, or by a single pulse at a higher laser power. These results are interpreted to suggest that pulsations can be induced by the stimulation of a Rho-dependent mechano-chemical oscillatory system up to a certain threshold, above which cells constrict without pulsing. Stimulation of Rho signaling above a certain threshold could override, for example, the activity of a RhoGAP, which is required to control the normal spatio-temporal dynamics of Rho GDP/GTP cycling. In agreement with this proposal, pulsatile constrictions during ventral furrow invagination require the activity of a specific RhoGAP. However, while ventral cells pulse with a mean period of ~80 s, optogenetic-induced pulsations display a mean period of ~150 s, a limit probably imposed by the reversion kinetics of the CRY2/CIB1 system in the dark (Izquierdo, 2018).

In conclusion, these data illustrate the utility of applying concepts of synthetic biology (e.g., precise orthogonal control over signaling pathways, guided cell behavior) to the field of tissue morphogenesis and in particular of how the nascent field of synthetic morphogenesis can help defining the minimum set of requirements sufficient to drive tissue remodeling. The data argue that while normally tissue differentiation and tissue shape are intimately linked, it is possible to direct tissue shape without interfering with complex layers of gene regulatory network and tissue differentiation programs. This might have important implications also for tissue engineering, where it might be desirable to shape any given tissue of interest without changing its fate (Izquierdo, 2018).

Gilgamesh (Gish)/CK1gamma regulates tissue homeostasis and aging in adult Drosophila midgut

Adult tissues and organs rely on resident stem cells to generate new cells that replenish damaged cells. To maintain homeostasis, stem cell activity needs to be tightly controlled throughout the adult life. This study shows that the membrane-associated kinase Gilgamesh (Gish)/CK1gamma maintains Drosophila adult midgut homeostasis by restricting JNK pathway activity and that Gish is essential for intestinal stem cell (ISC) maintenance under stress conditions. Inactivation of Gish resulted in aberrant JNK pathway activation and excessive production of multiple cytokines and growth factors that drive ISC overproliferation. Mechanistically, Gish restricts JNK activation by phosphorylating and destabilizing a small GTPase, Rho1. Interestingly, this study found that Gish expression is down-regulated in aging guts and that increasing Gish activity in aging guts can restore tissue homeostasis. Hence, this study identifies Gish/CK1gamma as a novel regulator of Rho1 and gatekeeper of tissue homeostasis whose activity is compromised in aging guts (Li, 2020).

Adult stem cells are essential for tissue homeostasis and regeneration, and their activity needs to be tightly controlled in order to maintain the normal cellular architecture and physiological function of adult organs. The JNK pathway plays a pivotal role in the Drosophila midgut homeostasis and injury response. In addition, JNK pathway activity is elevated in aging guts, leading to aberrant stem cell proliferation and loss of tissue homeostasis. How the JNK pathway is kept in check during gut homeostasis and aging is not well understood. This study provides both genetic and biochemical evidence that the membrane-associated kinase Gish/CK1γ restricts JNK pathway activity by phosphorylating and destabilizing Rho1, an upstream activator of the JNK pathway. This regulatory pathway is critical for the maintenance of midgut homeostasis as loss of Gish results in elevated JNK pathway activity and ISC proliferation. Interestingly, Gish expression in the midgut declines with aging, which may contribute to the elevated JNK pathway activity and ISC overproliferation in old guts. In support of this notion, it was found that transgenic expression of Gish in old guts could restore JNK pathway activity and ISC proliferation to homeostatic levels. How Gish expression is down-regulated in aging guts remains an open question, but it appears to occur at the level of transcription, although the possibility cannot be ruled out that posttranscriptional regulation may also occur. The precise mechanism awaits further investigation (Li, 2020).

Prolonged activation of JNK can induce apoptosis in both Drosophila and mammalian cells. As a negative regulator of JNK pathway activity, Gish synergizes with Puc, another JNK pathway inhibitor, to protect ISCs from undergoing apoptosis; thus, it may play a role in stem cell maintenance under stress condition. The synergistic interaction between Gish and Puc is not restricted to ISCs but also occurs in other tissues. For example, in wing imaginal discs, depletion of Gish only resulted in low levels of apoptosis; however, removal of one copy of puc (puc^E96/+) in Gish-depleted wing discs (MS>GishRNAi, puc^E96/+) resulted in massive cell death, leading to the formation of small wings. The increased apoptosis in MS>GishRNAi, puc^E96/+ appeared to mirror the dramatic activation of the JNK pathway. Hence, the cell- and tissue-protective function of Gish could simply be attributed to its role in suppressing JNK signaling. However, it remains possible that other pathways downstream of Rho1 and/or Gish may contribute to the induction of apoptosis in Gish and Puc double-deleted cells in parallel to heightened JNK pathway activity (Li, 2020).

Although mainly regulated by GTP-GDP cycling, Rho GTPases can also be regulated by posttranslational modifications such as phosphorylation that control their subcellular localization, stability, and complex formation. For example, a previous study showed that Erk2-mediated phosphorylation of RhoA targeted it for ubiquitin/proteasome-mediated degradation in cultured epithelial cells. However, the phosphorylation site(s) on RhoA that regulates its stability remained unidentified, and the physiological role of Erk2-mediated phosphorylation of RhoA was not determined. This study provides both genetic and biochemical evidence that Gish phosphorylates a cluster of sites in the N-terminal region of Rho1, which targets Rho1 for ubiquitination, followed by proteasome-mediated degradation. Gish-mediated down-regulation of Rho1 restricts JNK pathway activity, which is critical for adult Drosophila midgut homeostasis. How Rho1 inhibits JNK pathway remains an open question. A previous study revealed that Rho1 physically interacts with Slpr/JNKKK regardless of its GDP/GTP binding state and that Rho1 promotes Slpr cortical localization. Therefore, it is possible that plasma membrane-associated Rho1 promotes JNK pathway activation by increasing the local concentration of Slpr/JNKKK at the plasma membrane. The precise biochemical mechanism by which Rho1 activates Slpr/JNKKK awaits further investigation (Li, 2020).

Rho GTPases shuttle between plasma membrane and cytoplasm with GTP-bound active forms associated with membrane. Gish/CK1γ belongs to the CK1 family kinases. Unlike other family members that are located mainly in the cytoplasm, Gish/CK1γ is associated with plasma membrane through its C-terminal lipid modification. This study has demonstrated that the function of Gish in restricting ISC overproliferation depended on its kinase activity and membrane association, suggesting that Gish may phosphorylate plasma membrane-associated Rho1 to prevent its aberrant accumulation at the plasma membrane where it can activate the JNK pathway. This may explain why the role of Gish/CK1γ cannot be replaced by other CK1 family members. Since the CK1 phosphorylation sites on Rho1 are also found in all members of mammalian Rho GTPase, it is speculated that CK1γ may play a conserved role in the regulation of Rho GTPase activity. Future study is needed to determine whether CK1γ regulates Rho GTPase activity and whether such regulation plays a role in development, tissue homeostasis, and aging in mammals (Li, 2020).

Coordination of cytoskeletal dynamics and cell behaviour during Drosophila abdominal morphogenesis

During morphogenesis, cells exhibit various behaviours, such as migration and constriction, which need to be coordinated. How this is achieved remains elusive. During morphogenesis of the Drosophila adult abdominal epidermis, larval epithelial cells (LECs) migrate directedly before constricting apically and undergoing apoptosis. This study investigates the mechanisms underlying the transition from migration to constriction. LECs possess a pulsatile apical actomyosin network, and a change in network polarity correlates with behavioural change. Exploring the properties of the contractile network, it was found that cell contractility, as determined by myosin activity, has an impact on the behaviour of the network, as well as on cytoskeletal architecture and cell behaviour. Pulsed contractions occur only in cells with intermediate levels of contractility. Furthermore, increasing levels of the small Rho GTPase Rho1 disrupts pulsing, leading to cells that cycle between two states, characterised by a junctional cortical and an apicomedial actin network. These results highlight that behavioural change relies on tightly controlled cellular contractility. Moreover, constriction can occur without pulsing, raising questions why constricting cells pulse in some contexts but not in others (Companys, 2020).

During development, morphogenetic processes ultimately shape the organism. Such processes are driven by various cell behaviours, e.g. intercalation, division, migration and shape change, all of which need to be coordinated. Little is known about how this coordination is achieved, how cells switch behaviour and how different behaviours occur simultaneously (Companys, 2020).

Cell behaviour is often directional. Planar cell polarity (PCP) directs cell behaviour in the plane of the tissue, coordinating, for example, junctional remodelling, division orientation and migration. Migrating cells have a protruding front and a contracting back, further indicating cytoskeletal polarity. In contrast, apically constricting cells show radial cell polarity (RCP) (Companys, 2020).

Ultimately, cell behaviour depends on the actin cytoskeleton. Cell migration relies on protrusive activity, such as lamellipodia formation. Cell shape changes, including apical constriction, which reduces apical cell area, depend on actomyosin contractility. Increasing evidence suggests that the actin cytoskeleton shows rhythmical activity, such as actin flows during migration and pulsed contractions, e.g. during junctional remodelling, neuroblast ingression, apical constriction, basal constriction and vertebrate neural tube closure. Pulsed contractions are driven by periodic actomyosin contractions, which are thought to lead to cycles of cell area fluctuation, followed by stabilisation of the resulting smaller area ('ratchet model') (Companys, 2020).

Pulsed contractions are regulated by phosphorylation of Myosin II regulatory light chain [MRLC; Spaghetti squash (Sqh) in Drosophila] by Rho kinase (Rok), and its dephosphorylation by Myosin phosphatase. In addition, Rok inhibits the Myosin-binding subunit of the Myosin phosphatase complex (Mbs). Upstream of Rok, the small GTPase Rho1 is involved in regulating actomyosin contractility in many contexts, from rear retraction in migrating cells to pulsed contractions. The activity of Rho1 is regulated by activating guanine nucleotide exchange factors (GEFs) and inhibitory GTPase-activating proteins (GAPs) (Companys, 2020).

To gain insights into the mechanisms underlying the coordination of cell behaviour, larval epithelial cells (LECs) were studied during formation of the adult abdominal epidermis of Drosophila. During this process, the LECs are replaced by the adult histoblasts. Previous work has shown that LECs undergo directed migration followed by a transition to apical constriction, which eventually leads to delamination and apoptosis (Bischoff, 2012). During migration, LECs form crescent-shaped lamellipodia and migrate posteriorly (Bischoff, 2012). LECs can only move if neighbours provide space by either migrating, reducing apical area or undergoing apoptosis (Companys, 2020).

Studying the LEC cytoskeleton during abdominal morphogenesis, this study showed that LECs possess an apicomedial actomyosin network that undergoes pulsed contractions. The network is planar polarised during migration, undergoing pulsed contractions in the cell back, while the front protrudes. Contractions then re-localise to the cell centre, displaying radial polarity during constriction. Thus, behavioural change correlates with a change in the polarity of the contractile cytoskeletal network. To explore how manipulating actomyosin contractility affects the contractile network, Rho1, Rok and Myosin phosphatase were interfered with. Cellular contractility levels have an impact on the behaviour of the contractile network, with pulsed contractions only occurring in cells with intermediate contractility levels. 'Contractility' is referred to as the ability of a cell to contract its actomyosin network (or increase stress in this network, if cell deformation is resisted), which is ultimately determined by its amount of active myosin II. Interestingly, increasing Rho1 levels interferes with pulsed contractions and leads instead to a cycling of cells between two states characterised by a junctional cortical and an apicomedial actin network. Thus, increasing contractility is sufficient to have an impact on cytoskeletal architecture and, consequently, cell behaviour. Additionally, apical constriction can take place without pulsing, raising questions why constricting cells pulse in some contexts but not in others (Companys, 2020).

A cell's level of contractility determines the behaviour of its actin cytoskeleton, with pulsed contractions depending on intermediate levels of contractility, as well as cytoskeletal architecture and cell behaviour. Moderately increasing contractility causes LECs to cycle between two states, characterised by a junctional cortical and an apicomedial actin network, respectively. Moreover, the data suggest that apical constriction can occur without pulsed contractions, raising questions why constricting cells pulse in some contexts but not in others (Companys, 2020).

During migration, LECs are planar polarised. They protrude at the front and undergo pulsed contractions in the back, where they also create d-v oriented actin bundles. In their junctional cortical network, Sqh::GFP localises preferentially to the a-p junctions, similar to embryonic cells during germband extension. As LECs constrict, their apicomedial network becomes radially polarised, with a central actin focus and radial actin bundles that connect the network to the junctions, resembling a spider's web. In addition, Sqh::GFP is distributed evenly across all junctions. Thus, LEC behavioural change is accompanied by a change in cell polarity from PCP to RCP that underlies a change in cytoskeletal activity as well as an overall reorganisation of cytoskeletal architecture (Companys, 2020).

The polarity of LEC migration depends on PCP signalling, but little is known about the link between PCP and cytoskeletal asymmetry. Cytoskeletal asymmetry could be due to localised activity of small Rho GTPases, with Rho1 controlling pulsed contractions in the cell back and Rac1 promoting lamellipodia formation at the front. Such mutually exclusive localisation has been shown in various cell types. How RCP is established in constricting LECs is still unknown (Companys, 2020).

LECs create very large actin foci when contracting, but their foci period is comparable to other systems that create smaller foci. A possible explanation for the short period of the large foci is that foci in LECs are initiated within a large area in the periphery of the cell and not in a small region from which the contractile event then expands (which would potentially slow down foci formation). Focus formation involves both the recruitment of novel actin and Sqh as well as advection, where already recruited actin and Sqh 'flow' towards the centre of the focus (Companys, 2020).

Interestingly, actin dynamics in LECs involves the formation of distinct regions of contractile activity, depending on LEC behaviour and polarity. Constricting LECs create a single focus in their centre, while migrating LECs generate two alternating foci in their back. This difference cannot easily be explained by a change in cellular polarity. Instead, it could be due to the different shapes of migrating and constricting cells. In migrating cells, which are elongated along the d-v axis, one contractile event might not suffice to constrict the whole apicomedial network. Constricting cells, however, are rounder, which might allow radial actin recruitment over the whole apical area. Alternatively, the two foci could be a consequence of the cytoskeletal architecture of a migrating cell, as seen in keratinocytes, where myosin localises to two areas in the back of the cell, flanking the nucleus (Companys, 2020).

LECs that create actin foci migrate faster than earlier stage LECs, which do not undergo pulsed contractions. However, pulsed contractions are not part of the migratory machinery, as early migrating LECs do not show foci and Rho-CA LECs migrate without pulsatile activity. Thus, occurrence of pulsed contractions in migratory LECs appears to be associated with constriction rather than migration (Companys, 2020).

In contrast to other systems that show pulsed contractions, LECs have a very large apical area, up to 70 μm in diameter. This large area might determine the architecture of the LEC apicomedial network, which consists of persistent actin bundles that connect to the cell cortex, as well as dynamic flows and foci. In large cells, a robust, persistent apicomedial network might be crucial to maintain apical area and transduce contractile forces. In line with these observations, a persistent and a dynamic pool of actin have recently been described in ectodermal cells during germband extension and in amnioserosa cells during dorsal closure in Drosophila, but only the larger amnioserosa cells showed visible actin bundles (Companys, 2020).

Altering levels of contractility in LECs showed that pulsed contractions depend on Rok, Myosin phosphatase and Rho1 activity. In this respect, LECs resemble other cells that undergo pulsed contractions. As shown for Drosophila germband cells and amnioserosa cells, the current results indicate that cytoskeletal network dynamics depends on the level of cell contractility. Only with intermediate, wild-type levels of contractility did LECs show pulsed contractions. Reducing contractility (Rok-RNAi or MbsN300) interfered with pulsed contractions, as did increasing contractility (Rho1-CA or Rok-CAT). In addition to the amount of activated myosin II, contractility is also determined by the availability of G-actin as well as F-actin nucleators and crosslinkers (Companys, 2020).

As well as interfering with pulsed contractions, increasing contractility in LECs had a more far-reaching impact on cytoskeletal architecture and cell behaviour. (1) Over-expression of Rho1-CA resulted in F-actin disappearing apicomedially and localising mostly to the junctional cortex. A similar phenotype has been observed during Drosophila gastrulation, suggesting that Rho1 is involved in determining the ratio of apicomedial versus cortical contractility. (2) While over-expression of Rho1-CA can only activate endogenous Rok, over-expression of Rok-CAT can supply large amounts of activated Rok and thus lead to a stronger and more specific activation of Myosin II. In LECs, this not only resulted in F-actin disappearing apicomedially and localising mostly to the junctional cortex (and thus a loss of pulsed contractions), but also in the formation of cortical actin bundles. In cell culture, activation of myosin has also been shown to create actin bundles during stress fibre formation. (3) Increased junctional cortical contractility in both Rho1-CA and Rok-CAT LECs furthermore induced cell blebbing. Blebbing is driven by strong cortical myosin activation. During Drosophila dorsal closure, activation of Myosin light chain kinase and Mbs led to cell blebbing. (4) In 'strong' phenotypes of both Rok-CAT and Rho-CA pupae, LECs do not generate lamellipodia-like protrusions and do not migrate. Thus, high levels of activated myosin appear to over-ride protrusive activity and force cells to constrict. A comparable hyper-contractile phenotype has been described in CHO.K1 cells, where over-expressing a phosphomimetic form of myosin light chain leads to a loss of cell spreading and migration (Companys, 2020).

Increasing wild-type Rho1 levels (UAS.rho1) proved particularly interesting. Cells appear to cycle between a state in which actin accumulates at the junctional cortex and cells show blebbing, and a state in which the apicomedial network is present but non-pulsatile. This cycling phenotype could be explained by the rhythmical activation and de-activation of Rho1 via the endogenous machinery in a context of increased overall contractility of the network. With increasing levels of activated Rho1 (and thus increased levels of activated myosin), LECs appear to shift their ratio of apicomedial versus junctional cortical actin towards cortical. This is reversed when levels of activated Rho1 (and thus levels of activated myosin) decrease. The current results support this hypothesis, as not only the disappearance of apicomedial actin was observed, but also the beginning of junctional cortical blebbing, which suggests an increase in contractility in the junctional cortical network. These observations highlight the importance of specifically coordinating the junctional cortical and the apicomedial actomyosin networks (Companys, 2020).

Although pulsed contractions are affected by the manipulation of contractility, most LECs in Rho-RNAi, MbsN300, UAS.Rho1, Rho1-CA or Rok-CAT pupae constricted successfully. In all these cases, pulsed contractility of the apicomedial actin network was impaired, but cells still had actin at their junctional cortex. This indicates that contractility at the junctional cortex alone can drive apical constriction. Similar observations have been made in amnioserosa cells and during neural tube formation. The observation that boundary LECs constrict without pulsed contractions and with insignificant cell area fluctuation further supports the notion that the contractile force needed for constriction can be created by the junctional cortical network, acting like a contractile ring during cell extrusion, as suggested for boundary LEC extrusion. Another possibility is that the apicomedial network creates non-pulsatile tension that helps drive constriction. For instance, in boundary LECs, diffuse apical Sqh activity can be observed, which might contribute to apical area reduction (Companys, 2020).

Interestingly, Rok-RNAi LECs labelled with Sqh::GFP showed a phenotype comparable to that of constricting early boundary LECs in controls. This raises the possibility that the change from early boundary cell behaviour to the behaviour of cells undergoing pulsed contractions during later stages of development might be due to an increase of contractility in these cells over time (Companys, 2020).

That LECs can constrict apically without showing pulsed contractions raises important questions about the role of pulsed contractions in constriction, as also raised by others. LECs begin pulsed contractions while still migrating, at a time when LEC shape changes, and thus tissue remodelling intensifies. Also, for most of morphogenesis, LECs undergo pulsed contractions without apically constricting, merely fluctuating their cell area rhythmically and changing shape . This suggests that pulsed contractions do not drive apical constriction per se. Instead, they might play other roles, such as helping to maintain cell shape and to withstand pushing and pulling forces created by neighbouring cells during morphogenesis. Ultimately, this could help to maintain tissue integrity during morphogenesis. Alternatively, pulsed contractions could cooperate with junctional cortical contractility to create sufficient forces to drive apical constriction more effectively; LEC constriction that is accompanied by pulsed contractions is faster than constriction of boundary LECs without actin foci. To gain further insights into the role of pulsed contractions in LECs, future studies need to consider the interactions between neighbouring LECs to assess the impact of external forces on the behaviour of the apicomedial actomyosin network (Companys, 2020).

This study provides insights into the complexity of the cytoskeleton of a cell during morphogenesis, as well as the importance of the level of cell contractility for the regulation of pulsed contractions, cellular architecture and, consequently, cell behaviour. It also highlights the complex interplay between the junctional cortical and the apicomedial network in changing cell shape and cell area. For LEC migration and the subsequent behavioural transition to constriction, a polarised cytoskeletal network and intermediate contractility levels seem to be crucial. What regulates the transition from migration to constriction is unknown. As LECs transit from PCP to RCP while they change behaviour, studying signals that regulate this change in polarity will be crucial in future studies (Companys, 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).

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).

Systematic analysis of RhoGEF/GAP localizations uncovers regulators of mechanosensing and junction formation during epithelial cell division

Cell proliferation is central to epithelial tissue development, repair, and homeostasis. During cell division, small RhoGTPases control both actomyosin dynamics and cell-cell junction remodeling to faithfully segregate the genome while maintaining tissue polarity and integrity. To decipher the mechanisms of RhoGTPase spatiotemporal regulation during epithelial cell division, this study generated a transgenic fluorescently tagged library for the 48 Drosophila Rho guanine exchange factors (RhoGEFs) and GTPase-activating proteins (GAPs), and their endogenous distributions were systematically characterized by time-lapse microscopy. Therefore, candidate regulators of the interplay between actomyosin and junctional dynamics during epithelial cell division were unveiled. Building on these findings, it was established that the conserved RhoGEF Cysts and RhoGEF4 play sequential and distinct roles to couple cytokinesis with de novo junction formation. During ring contraction, Cysts via Rho1 participates in the neighbor mechanosensing response, promoting daughter-daughter cell membrane juxtaposition in preparation to de novo junction formation. Subsequently and upon midbody formation, RhoGEF4 via Rac acts in the dividing cell to ensure the withdrawal of the neighboring cell membranes, thus controlling de novo junction length and cell-cell arrangements upon cytokinesis. Altogether, these findings delineate how the RhoGTPases Rho and Rac are locally and temporally activated during epithelial cytokinesis, highlighting the RhoGEF/GAP library as a key resource to understand the broad range of biological processes regulated by RhoGTPases (di Pietro, 2023).

In animal cells, cell division entails drastic cell shape changes necessary for the faithful segregation of the duplicated genome into the two daughter cells. These morphological changes include cell rounding required for correct spindle formation and orientation, as well as cytokinesis to separate the cytoplasms of the daughter cells. Studies on single cells and tissues have shown that these cell shape changes are powered by small RhoGTPases that remodel the actomyosin cytoskeleton. Moreover, in multicellular contexts, RhoGTPases are also critical to couple cell shape changes and junction dynamics to control tissue polarity, cohesion, and architecture. Notably, cell division is tightly linked to cell fate specification as well as tissue growth, morphogenesis, and mechanics. Therefore, characterizing the regulation of RhoGTPases and its implication in cytoskeleton and junction dynamics during cell division in tissues is central to understand how cell number and genome integrity are controlled and how tissue architecture and function are established and maintained (di Pietro, 2023).

The small RhoGTPases Rho, Rac, and Cdc42 are key pleiotropic regulators of the actomyosin cytoskeleton and cell junction dynamics. They switch between an active GTP-bound state that binds downstream effectors and an inactive GDP-bound state. These states are primarily regulated by Rho guanine exchange factors (RhoGEFs) that activate RhoGTPases by exchanging GDP for GTP and by RhoGTPase-activating factors (RhoGAPs) that promote GTP hydrolysis to GDP, thereby inactivating RhoGTPases. Individual RhoGEF/GAPs associated with the regulation of cell shape changes, cell division, migration, and polarity have been identified in cultured cells and, to a lesser extent, by targeted RNAi or mutant analyses in multicellular contexts. In addition, by ectopically expressing all human RhoGEF/GAPs in cultured cell lines, a recent study has defined their localizations and biochemical interactomes, enabling a better understanding of single-cell migration. Therefore, the mechanisms mediating the spatiotemporal activation of small RhoGTPases are best understood in individual cells in interphase. However, the spatiotemporal regulation of RhoGTPases remains far less explored during cell division or in tissues, impeding understanding of actomyosin and junction dynamics during interphase and cell division in epithelial tissues (di Pietro, 2023).

During animal cell cytokinesis, RhoGTPases control the pronounced cell deformations associated with cytokinetic ring constriction. Numerous studies have converged to show that the assembly and constriction of the actomyosin cytokinetic ring is powered by the membrane redistribution of the RhoGEF ECT2 (Drosophila pebble, Pbl) and RacGAP1 (Drosophila tumbleweed, Tum) within the dividing cell. Despite these fundamental findings, the role of most RhoGEF/GAPs during cell division remains poorly explored. In addition, in epithelial tissues, several studies have shown that the drastic cytokinesis cell shape changes are coupled with E-cadherin (Ecad) adherens junction (AJ) remodeling and de novo AJ formation. In particular, epithelial cytokinesis shares general features in several vertebrate and invertebrate tissues: (1) de novo AJ formation is coordinated with cytokinesis and relies on mechanosensing processes involving the dividing cell and its neighbors, and (2) the arrangement of the newly formed cell junctions is defined in late cytokinesis, and it is proposed to modulate tissue topology and morphogenesis. Some of these epithelial cytokinetic features are known to be regulated by the Rho and Rac GTPases, but the mechanisms controlling their local and temporal activations remain unknown (di Pietro, 2023).

Toward achieving an integrated view of the spatiotemporal regulation of actomyosin and junction dynamics in proliferative epithelia in vivo, a complete library of fluorescently tagged Drosophila RhoGEF/GAPs was assembled. Then RhoGEF/GAP localizations were systematically analyzed from interphase to cell division in two Drosophila epithelial tissues by time-lapse microscopy. By doing so, this study unraveled a series of putative regulators of epithelial tissue organization, polarity, and dynamics. These results led to a focus on the RhoGEF Cysts and RhoGEF4, and to characterize their respective roles in mechanosensation and AJ formation during epithelial cell division. Altogether, this work advances the understanding of cell division in epithelial tissues and highlights that the RhoGEF/GAP library will be a relevant resource to investigate how actomyosin and junction dynamics are controlled during development, repair, and homeostatic processes (di Pietro, 2023).

By modulating the activity of small RhoGTPases, RhoGEFs and RhoGAPs control cytoskeleton organization and dynamics in a broad range of processes such as cell morphogenesis, division, and migration in all eukaryotes. The functions of RhoGTPases have been extensively and systematically investigated in individual cells. However, multicellularity entails a complex interplay between the cytoskeleton and cell-cell junctions to regulate cell and tissue architecture and dynamics, thus highlighting the relevance of characterizing RhoGEF/GAP function in tissues. Cell division has emerged as a multicellular process since it entails the deformation of the neighboring cells, the remodeling of the dividing and neighbor cell junctions, and de novo junction formation. To better understand how RhoGEFs and RhoGAPs control the cytoskeleton and cell junction dynamics in proliferative epithelial tissues, a family-wide Drosophila transgenic library of fluorescently tagged RhoGEF/GAPs was generated and their distributions were systematically determined during interphase and cell division in two epithelial tissues. This screen revealed multiple uncharacterized RhoGEF/GAP interphasic localizations as well as a complex choreography of RhoGEF/GAPs during cell division have been better defined. Building on this screen, the processes of mechanosensing and junction formation by delineating how the activities of specific RhoGTPases are controlled during epithelial cytokinesis and de novo junction formation. This study has therefore uncovered the first mechanisms of RhoGTPase activation underlying the multicellularity of epithelial cell division (di Pietro, 2023).

GENE STRUCTURE

Bases in 5' UTR - 41

Bases in 3' UTR - 858

PROTEIN STRUCTURE

Amino Acids - 193

Structural Domains

Rho1 is highly related to its mammalian counterpart. Drosophila Rho1 is 86% identical to human rhoA, B and C GTPases at the amino acid level, with the vast majority of the differences occcuring in the C-terminal third of the protein. The Drosophila Rho1 protein also exhibits substantial sequence identity (about 75%) with two yeast rho GTPases that have been reported, Yrho1 and Yrho2, indicating that the rho gene has been well maintained throughout evolution (Hariharan, 1995).

date revised: 1 June 2024

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