InteractiveFly: GeneBrief

cysts: Biological Overview | References

Gene name - cysts

Synonyms - Dp114RhoGEF

Cytological map position - 37E4-37E5

Function - signaling

Keywords - RhoGEF that maintains tissue polarity and integrity - together with RhoGEF4 plays roles to couple cytokinesis with de novo junction formation - acts via Rho1, participating in the neighbor mechanosensing response, promoting daughter-daughter cell membrane juxtaposition in preparation to de novo junction formation - Crumbs and its complex partner Cysts support myosin and apical constriction to ensure robust ingression dynamics - recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3 - required for cell intercalation in the extending ectoderm, where it activates Rho1 specifically at junctions - localization is restricted to adherens junctions and is under Gbeta13F/Ggamma1 control - Myosin VI promotes recruitment of the heterotrimeric Galpha12 protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA

Symbol - cyst

FlyBase ID: FBgn0032796

Genetic map position - chr2L:19,502,335-19,508,764

NCBI classification - PH_ARHGEF2_18_like: rho guanine nucleotide exchange factor

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

Cysts orthologs: Biolitmine
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).

It is now well established that cytokinesis and, more generally, cell division entail a mechanosensing response in the neighbors. Despite such conserved mechanical responses and the proposed roles of RhoGTPases in mechanosensing, the nature of the RhoGEF associated with the response to cytokinesis ring contraction has remained unknown. This work establishes that the RhoGEF Cysts modulates the response to endogenous mechanical forces generated by ring contraction in the neighboring cells, thereby enhancing daughter cell membrane juxtaposition. It has been previously reported that ring contractile forces are sensed by the local decrease of Ecad (Pinheiro, 2017). Since that study found that reducing the level of Ecad is sufficient to promote Cysts accumulation, Cysts response to mechanical force can be directly caused by the decrease in Ecad during ring contraction. In addition, MyoII flows and accumulation can also promote Rho local activation, and Cysts recruitment at the rim of the cytokinesis ring depends on Rok. Thus, the Cysts response to mechanical force can be also indirectly caused by the Rok and MyoII accumulation due to the Ecad decrease. Interestingly, whereas previous studies in cell culture showed that the Cysts ortholog p114RhoGEF is recruited at sites of Ecad accumulation, this study uncovered an additional mechanism of Cysts localization promoted by local Ecad decrease due to mechanical forces. To further understand how cells respond to mechanical forces associated with a local Ecad decrease, it will be relevant to explore how Cysts is recruited to sites of lower Ecad and how Rok-dependent contractility feedbacks on Cysts dynamics. Notably, at least two distinct Ecad dynamics have been observed during epithelial cytokinesis in vertebrates. In the avian embryo at stage EGK-X, cell division is concomitant to a local depletion of basal Ecad and an accumulation of MyoII and F-actin, whereas in Xenopus, ring contraction triggers a well-known force-sensing response associated with the accumulation of Ecad and Vinculin. Building on the findings on Cysts and the ones on p114RhoGEF in vitro (Acharya, 2018), one could hypothesize that Cysts/p114RhoGEF represents a common platform for the response to mechanical force during cytokinesis, thus calling for the investigation of the function of RhoGTPases in cells neighboring the dividing cells in vertebrates. This study found that in response to cytokinesis forces, cysts and rho1 mutants showed a similar decrease in MyoII accumulation but that the rho1 phenotype on junction juxtaposition was stronger. Cysts is therefore a major regulator of Rho1 to regulate MyoII accumulation, but the data also suggest that Rho1 plays an additional Cysts-independent function in the control of membrane juxtaposition during cytokinesis. Lastly, Cysts is involved in AJ integrity, cell-cell rearrangements, and cell ingression in interphasic cells in the early Drosophila embryo (de las Bayonas, 2019; Silver, 2019; Simoes, 2022) which are processes entailing the production of mechanical forces and the remodeling of the actomyosin cytoskeleton. One could therefore envision that cytoskeleton and junction dynamics might also depend on the Cysts mechanosensing activity in these developmental processes (di Pietro, 2023).

De novo junction formation is inherent to the proliferation of epithelia. In vitro experiments have put forward a fundamental role for Rac in modulating the dynamics of junction formation upon cell-cell contact formation. Previous findings have also underscored a role for Rac activity in cytokinesis regulating both ring constriction and the topology of the daughter-daughter cell interface. This study uncovered that RhoGEF4 promotes the localization of active Rac at the daughter-daughter cell interface to ensure the withdrawal of the neighboring cell membranes. Therefore, RhoGEF4 is a regulator of Rac function in the control of de novo junction length, as well as of the dynamics of the cell-cell arrangements upon cytokinesis. By comparing the role of RhoGEF4 in different tissues, this work also suggests that the initial topology of the junction formed upon cytokinesis can be modulated by the overall dynamics or mechanical properties of epithelial tissues. Lastly, by combining loss of Cysts and RhoGEF4 function, it was establish that the topology of de novo junctions formed upon cell division is synergistically regulated both by membrane juxtaposition during ring constriction and by membrane withdrawal upon midbody formation. Altogether, this analyses of Cysts and RhoGEF4 functions provide a far better understanding of the mechanisms of Rho and Rac regulation during epithelial cell division, and they illustrate how their distinct activations in the dividing and neighboring cells couple cytokinesis and junction formation (di Pietro, 2023).

The findings on Cysts and RhoGEF4 also highlight the relevance of screening fluorescently tagged libraries to pinpoint key regulators of small GTPases necessary for cytoskeleton and junctional regulations in vivo. In particular, it is foreseen that family-wide characterization of RhoGEF/GAP localization and dynamics will be instrumental to better understand numerous aspects of the conserved process of cell division in invertebrates and vertebrates. So far, studies on cell division have mainly focused on the roles of two RhoGTPase regulators, ECT2 (Drosophila Pbl) and RACGAP1 (Drosophila Tum). This systematic analysis now uncovered a very diverse set of RhoGEF/GAP localizations during epithelial cell division. Accordingly, the screen suggested several possible avenues to decipher the function of RhoGEF/GAPs in mitotic rounding, polar relaxation, and asymmetric furrowing, as well as in midbody dynamics. It was also observed that several RhoGEF/GAPs display similar localizations. These findings will be instrumental to design double or triple loss-of-function experiments to explore RhoGEF/GAP functions and to complement loss-of-function screens that would overlook specific RhoGEF/GAP functions due to their functional redundancy. In addition, this analysis in two distinct tissues underscores a set of RhoGEF/GAPs with different localization and dynamics. This illustrates the relevance of the in vivo exploration under the control of the endogenous promoters to explore how mitosis and cytoskeleton dynamics are differentially modulated to regulate tissue development or function. More generally, RhoGTPases are central regulators of cell and tissue dynamics in metazoan; it is therefore expected that the library will be a key resource to dissect RhoGTPase spatiotemporal regulation and function in a variety of developmental, homeostatic, and repair contexts in epithelia, as well as in stem cells, migrating cells, and neurons (di Pietro, 2023).

Crumbs complex-directed apical membrane dynamics in epithelial cell ingression

Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cells lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. This study shows that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. These findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer (Simoes, 2022).

Drosophila NBs are an outstanding model for scrutinizing the cellular machineries underpinning an EMT-like process with high temporal and spatial resolution. While ingressing, a single NB sequentially loses AJs responding to tensile forces exerted by two pools of actomyosin: a planar polarized pool enriched at anterior-posterior junctions, which disassembles first, and a pulsatile pool at the free apical cortex, which further tugs on shrinking junctions in a ratchet-like manner. However, while actomyosin forces reduce the apical perimeter, it remained unclear how cells lose their apical domain and how polarity regulators contribute to the dynamics of apical domain loss. This study demonstrates that regulation of the Crb complex plays a key role in orchestrating apical domain loss during ingression. During early ingression, cells shrink their apical domain while retaining total levels of Crb, which is crucial for maintaining normal actomyosin in NBs and NICs (neighboring non-ingressing cells) to generate the tension balance required for normal ingression dynamics. During late ingression, Crb is rapidly lost from the apical domain, a process initiated by an interaction between Neur and Sdt, which causes the disassembly of the Crb complex. The loss of the Crb complex then precipitates the concurrent loss of the apical membrane and AJs. A similar regulatory interplay between Crb, Sdt, and Neur was also observed during early neurogenesis in the Drosophila optic lobes. Here, neural stem cells emerge from a wave front in the optic lobes rather than ingress as individual cells. Despite these topological differences, the Crb/Sdt/Neur module appears to be a common cell biological regulator of EMT during early neurogenesis (Simoes, 2022).

Endocytosis and endocytic degradation and recycling are requirements for normal NB ingression dynamics. The apical membrane of neuroepithelial cells is much more active endocytically than the basolateral membrane. Notably, Crb is endocytosed during apical contractions and re-secreted during expansions, suggesting that myosin-driven cell contact contraction promotes endocytosis, consistent with recent data from mammalian cells, whereas expansions allow for enhanced secretion. Blocking endocytosis increases surface levels of Crb and Ecad as expected, and prevents NB ingression, whereas enhancing endocytosis accelerates ingression. Moreover, endocytic trafficking plays a key role in determining ingression speed. Loss of ESCRT complex-mediated degradation appears to enhance apical Crb and slow ingression, whereas loss of Retromer-mediated recycling dramatically reduced surface Crb and accelerated ingression. In fact, Retromer-compromised embryos showed the fastest ingression speed of any condition examined, suggesting that the Retromer not only recycles Crb but also other factors that counteract apical domain loss in NBs. Crb turnover during early ingression maintains a steady Crb surface abundance. During late ingression, Crb endocytosis is enhanced during both apical contraction and expansion as a result of the disruption of the Crb-Sdt interaction by Neur, which likely makes the Crb cytoplasmic tail accessible to the Clathrin adapter AP2. AP2 binds to Crb competitively with Sdt, facilitating the rapid endocytic removal of Crb and the apical membrane (Simoes, 2022).

The relationship between actomyosin contraction, endocytosis, Crb protein levels, and their function in NB ingression illuminates the complexity and robustness of morphogenesis. Disrupting several individual molecular processes, while changing the dynamics of ingression, rarely abrogates ingression entirely. First, although endocytosis is essential for ingression, strong depletion of endocytotic regulator delayed ingression but did not prevent delamination in most cases. Similarly, a dramatic depletion of myosin, which is essential for apical constriction of NBs, extended the ingression period but did not block delamination. Evidence of the mutual dependency of endocytosis and myosin-driven contractions in NBs. Contractions foster endocytosis, whereas endocytosis promotes contractions, a co-dependency that likely finetunes ingression dynamics. Second, maintaining Crb in the apical membrane (through overexpression or expression of a non-endocytosable form of Crb) delayed ingression but did not prevent it in most cases, suggesting that Crb membrane persistence is overcome by other mechanisms such as the Neur-dependent disassembly of the Crb complex. Moreover, failure to resolve the Crb complex in late ingression caused part of the apical membrane to persist as apical plugs. Nevertheless, NB delaminate, detach from apical plugs and animals are viable, suggesting that nervous system development proceeds rather normally. Crb surface abundance is dependent on endocytosis and versicle trafficking, and Crb regulates junctional myosin which contributes to apical contraction. Together, these findings highlight the multilayered regulation of ingression through the co-dependent interactions between apical polarity, vesicle trafficking, and actomyosin contractions (Simoes, 2022).

EMT is thought to be initiated by the expression of EMT transcription factors (EMT-TFs) of the Snail, Zeb, or bHLH families that downregulate key adhesion or polarity proteins such as Ecad and Crb. NBs are specified through the combined action of proneural genes that include bHLH proteins of the Achaete-Scute complex, the Snail family protein Worniu, and the SoxB family protein SoxNeuro. However, although genes that encode Ecad and Crb are transcriptionally downregulated in NBs, this repression does not appear relevant for NB ingression. Replacing endogenous Ecad with a transgene expressing Ecad under the control of a ubiquitous promoter had no impact on NB ingression dynamics. This study shows that surface levels of Crb remained high in NBs during early ingression before Crb is rapidly removed by endocytosis during late ingression. This raises the question of how the upregulation of proneural genes in presumptive NBs elicits enhanced actomyosin contractility and endocytic removal of apical membrane and junctions (Simoes, 2022).

One proneural gene target is neur. Neur is found throughout the neuroepithelium participating in Delta-Notch-mediated lateral inhibition to select the NB from an equivalence group of 5-7 cells. Neur upregulation in ingressing NBs is thought to be part of a positive feedback that stabilizes NB fate through persistent asymmetric Delta-Notch signaling. The increase in Neur may also be important for the effective disruption of the Crb complex to destabilize the apical domain. Neur can disrupt the Crb complex across the epithelium but is normally prevented from doing so by Bearded proteins that act as inhibitors of Neur. Increasing Neur concentration may overcome this inhibition in NBs. This raises the possibility that the proneural gene-dependent upregulation of neur contributes to the timing of ingression, consistent with the observation that in Neur-depleted embryos ingression is prolonged. Furthermore, Neur may enhance actomyosin contractility in NBs seen in late ingression as was reported for Neur in the Drosophila mesoderm. It is hypothesized therefore that Neur could be a central regulator of NB selection and ingression, stabilizing NB fate, driving apical membrane constriction through actomyosin contraction, and disrupting the Crb complex to remove apical membrane and junctions. Interestingly, it was also noted that during ingression, the number of alternative isoforms of Sdt is limited to Sdt3, the isoform susceptible to Neur. Hence, NBs appear to develop the molecular competence for apical membrane removal at least in part through rebalancing Sdt splice forms (Simoes, 2022).

The loss of apical-basal polarity is an early event during EMT marked by the loss of epithelial AJs that can trigger expression of EMT-TFs and the disassembly of cell junctions. However, the findings of this study indicate that the loss of apical-basal polarity in NBs is preceded by a period (~20 min; early ingression) of ratcheted apical contractions that reduce the apical area of delaminating cells. The maintenance of normal Crb levels during early ingression is crucial for normal ingression dynamics. Crb stabilizes junctional myosin through its effector, Cyst, which is recruited to the junctional domain by the Crb complex (Silver, 2019). Although the loss of Crb or loss of Cyst causes similar reductions of junctional myosin in the neuroepithelium (Silver, 2019), NB ingression was consistently faster in Cyst-compromised embryos than in controls. In contrast, NBs in Crb-compromised embryos showed much larger variability of ingression speeds, with a small fraction of NBs ingressing rapidly while the majority was slower than controls. Thus, it is likely that Crb makes other contributions to regulating NB ingression in addition to its Cyst-mediated function in supporting junctional actomyosin. Interestingly, the mouse Crb homolog Crb2 is required for myosin organization and ingression during gastrulation. The predominant defect in Crb2-compromised mice appears to be a failure of ingression, which may be similar to the fraction of NBs showing slower than normal ingression seen with the loss of Drosophila Crb. To what extent the differences in cell behavior caused by the loss of Crb and Crb2 depend on the biomechanical specifics of the tissue context or result from differences in molecular pathways in which Crb and Crb2 operate remains to be explored (Simoes, 2022).

Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, this study analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. Cysts was demonstrated to be recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, these findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity (Silver, 2019).

Antagonistic interactions between apical and basolateral polarity regulators position AJs at the apico-lateral membrane to form a junctional complex. In turn, AJs are thought to maintain apical-basal polarity through the segregation of the apical and basolateral membrane domains, organization of the cytoskeleton, and direct polarity by acting as signaling centers for polarity complexes. Although a number of Drosophila RhoGEFs and RhoGAPs have been implicated in epithelial polarity and AJ stability, no single RhoGEF or RhoGAP has been found to phenocopy the polarity or junctional defects that are seen in embryos compromised for factors such as Crb, aPKC, or E-cadherin. The current findings suggest that loss of the RhoGEF Cysts causes a polarity phenotype strikingly similar to the loss of core apical polarity proteins. Moreover, this study found that Cyst is recruited to the apico-lateral cortex by the action of polarity proteins and, by activating Rho1, stabilizes AJ-associated actomyosin, which supports junctional and epithelial integrity (Silver, 2019).

In Cysts-compromised embryos, AJ formation is disrupted in early gastrulation, and AJs do not form a circumferential belt. These defects in AJ assembly or stability correlate with reduced and irregular myosin accumulation at the apico-lateral cortex. Given the molecular function of Cysts as a GEF for Rho1, loss of myosin activity is presumably the immediate cause for the defects in AJ formation and the subsequent loss of apicobasal polarity in many epithelial cells. crb-depleted embryos failed to recruit Cysts to apical junctions and showed a similar decline in junctional myosin. Therefore, a major function of the apical Crb polarity complex appears to be the Cysts-mediated support of junctional actomyosin (Silver, 2019).

While many cells in crb or cyst mutants undergo programmed cell death, others retain or recover polarity and form small epithelial cysts, a process seen from mid-embryogenesis (postgastrulation stages) onward. Several polarity proteins such as Crb, Sdt, and Baz are needed for normal epithelial polarization in early embryos but are not essential for polarization in postgastrulation embryos, which explains the ability of some epithelial cells in these mutants to form epithelial cysts with normal polarization. In fact, when programmed cell death is suppressed, cyst formation is shown by all epithelial cells in crb mutants. Formation of epithelial cysts seen in cysts mutant embryos therefore suggests that Cysts is also not essential for epithelial polarity in late embryos. This view is supported by the decline of Cysts protein accumulation at AJs seen in late embryos (Silver, 2019).

Several observations, including the genetic interaction of cysts with genes encoding basolateral polarity proteins, the dependence of the junctional localization of Cysts on the apical polarity proteins Baz and Crb, the physical interactions between Cysts and apical polarity proteins, and the function of Cysts in stabilizing AJs, indicate that Cysts is an integral part of the apical polarity machinery in early Drosophila embryos. A particularly striking finding was the complete suppression of the cysts phenotype by codepletion of the basolateral polarity proteins Scrib or Lgl, seen in double-mutant embryos that showed phenotypes indistinguishable from single scrib or lgl mutants. This mimics previous observations with double mutants of crb or sdt and scrib, lgl or discs large. Moreover, this study found that a reduction of aPKC enhanced Baz mislocalization in Cysts-compromised embryos, suggesting that aPKC cooperates with Cysts and acts upstream or in parallel to Cysts to organize Baz. These findings emphasize that Cysts, similar to Crb and aPKC, is a component of a negative feedback circuit between apical and basolateral regulatory networks that govern epithelial polarity. The dependence of Cysts localization on Crb and Baz suggests that Cysts acts downstream of these two proteins. Once polarized, Cysts appears to maintain polarity and junctional stability through actomyosin remodeling (Silver, 2019).

In vivo structure-function data indicate that the C-terminal region is essential for Cysts activity. Moreover, it was found that the C-terminal region of Cysts can oligomerize, potentially facilitated by the CC domain. It is speculated that clustering of Cyst could enhance its cortical association. The Crb complex protein Patj represents one possible anchor for Cyst clusters at the cortex. Biochemical data show that the Cysts C-terminal region is sufficient for interactions with Patj. Patj has been implicated as a myosin II activator in the embryo. It is proposed therefore that Crb, Patj, and Cyst form a complex that organizes junctional actomyosin. However, as Patj is not essential for embryonic survival, Cysts may interact with additional binding partners within the Crb complex. Another apical binding partner for Cyst is Baz/Par3, which is required for Cyst cortical recruitment, coprecipitates with the Cyst C-terminal region, and coaggregates with Cyst in HeLa cells (Silver, 2019).

A recent independent study also arrived at the conclusion that Cysts activates Rho1 at AJs during germband extension in the Drosophila embryo (de Las Bayonas, 2019). It is further shown that depletion of Cysts acts downstream of a G protein-coupled receptor (GPCR) and the Gβ13F/Gγ1 heterotrimeric G protein in directing cell rearrangements promoting germband extension, and that germband extension is somewhat reduced when Cysts is depleted. Loss of Gγ1 causes an ~20% reduction in Cyst junctional enrichment (de Las Bayonas et al., 2019). These and the current data suggest that the normal junctional recruitment of Cyst requires at least three distinct inputs: interactions with Baz/Par3 and the Crb complex, and heterotrimeric G protein signaling (Silver, 2019).

This study found that Cysts becomes enriched at the apico-lateral cortex after the mesoderm and endoderm have invaginated and the germband starts to elongate. This localization coincides with the assembly of the apical-cortical actomyosin network. Rho-Rho kinase signaling plays a critical role in the activation of myosin II in this process. Structure-function analysis showed that Cyst contains an essential RhoGEF domain as predicted, and the use of Rho activity probes, genetic interactions, and biochemical assays showed that Cysts preferentially targets Rho1. Although the biochemical assay also revealed stimulation of Rac1 activity by Cysts, all other data point to Rho1 as the primary target of Cyst. It is proposed therefore that Cyst activates Rho1 to organize actomyosin at the cortex at a time when AJs assemble into a circumferential belt (stages 6/7). Consistent with this, it was found that Cysts is important for maintaining normal cortical levels of myosin II. A similar loss in junctional myosin was also observed in Crb-compromised embryos in line with the finding that Crb is required for Cysts junctional recruitment. The cysts mutant phenotype suggests that Cysts is the key RhoGEF that activates Rho1 at ectodermal AJs. In contrast, RhoGEF2 functions in the mesoderm and ectoderm, where it becomes apico-cortically enriched and activates Rho1 to recruit myosin II to the apical-medial cortex. Thus, RhoGEF and Cysts act in parallel on Rho1 to orchestrate the balance of cortical and medial actomyosin dynamics (Silver, 2019).

Cysts is the single orthologue of a group of four mammalian RhoGEFs that target RhoA in cell culture . One of the mammalian orthologues (p114RhoGEF) stabilizes tight junctions and AJs through organization of the actin cytoskeleton associated with cellular junctions (Nakajima and Tanoue, 2011; Terry, 2011; Acharya, 2018). p114RhoGEF is recruited to apical junctions through a mechanism involving CRB3A, Ehm2/Lulu2, Par3, Patj, the heterotrimeric G protein Gα12, and the GPCR Sphingosine-1 phosphate receptor 2 (Acharya, 2018). p114RhoGEF requires the polarity regulator Ehm2/Lulu2 (a homologue of Drosophila Yrt) to activate RhoA. In contrast, this study did not detect genetic or biochemical interactions between Cyst and Yrt in Drosophila. Recently, ARHGEF18, the human orthologue of p114RhoGEF, was identified as a gene associated with retinal degeneration, and a fish orthologue is required to maintain epithelial integrity of the retina. ARHGEF18 mutant retinal defects closely resemble those found in patients carrying mutations in the crb homologue CRB1. It is concluded that the function of Cyst and p114RhoGEF/ARHGEF18 in coupling apical polarity proteins and GPCR signaling to junctional Rho activity and actomyosin function is conserved between flies and vertebrates and likely contributes to retinal health in humans, although some of the molecular interactions may have shifted in relative importance (Silver, 2019).

The other mammalian orthologues of Cyst, p190RhoGEF, AKAP-13, and GEF-H1 have not been implicated as regulators of epithelial polarity. GEF-H1 (also known as ARHGEF2 and Lfc) was shown to be inactive at mature tight junctions. In this case, the tight junction protein Cingulin forms a complex with GEF-H1, preventing it from activating RhoA. Instead, GEF-H1 is thought to promote junction disassembly and cell proliferation, presumably through an association with the mitotic spindle. GEF-H1 was also implicated in the morphogenesis of the vertebrate neural tube, and in the regulation of RhoA activity during cytokinesis. Like GEF-H1, p190RhoGEF has been shown to associate with microtubules. GEF-H1 and AKAP-13 were also found to serve additional functions independent of their RhoGEF activity. Whether and how Cyst might consolidate the functions of its various mammalian orthologues remains to be explored (Silver, 2019 and references therein).

Distinct RhoGEFs Activate Apical and Junctional Contractility under Control of G Proteins during Epithelial Morphogenesis

Small RhoGTPases direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of actomyosin contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. This general problem was examined in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein-coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (Galpha and Gbetagamma) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized. However, the mechanisms responsible for polarizing Rho1 activity are unclear. This study reports that distinct guanine exchange factors (GEFs) activate Rho1 in these two cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1 but is recruited both medial-apically and at junctions by Galpha(12/13)-GTP, also called Concertina (Cta) in Drosophila. On the other hand, Dp114RhoGEF (Dp114), a newly characterized RhoGEF, is required for cell intercalation in the extending ectoderm, where it activates Rho1 specifically at junctions. Its localization is restricted to adherens junctions and is under Gbeta13F/Ggamma1 control. Furthermore, Gbeta13F/Ggamma1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. Finally, Dp114RhoGEF was absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results clarify the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia (Garcia De Las Bayonas, 2019).

Critical aspects of cell mechanics are governed by spatial-temporal control over Rho1 activity during Drosophila embryo morphogenesis. This work sheds new light on the mechanisms underlying polarized Rho1 activation during intercalation in the ectoderm. Rho1 activity was found to be driven by two complementary RhoGEFs under spatial control of distinct heterotrimeric G protein subunits. Notably, a regulatory module was uncovered specific for junctional Rho1 activation (Garcia De Las Bayonas, 2019).

Dp114RhoGEF was identified as a novel activator of junctional Rho1 in the extending ectoderm. Hence, two RhoGEFs, Dp114RhoGEF and RhoGEF2, coordinate independently the modular Rho signaling during tissue extension of the ectoderm. This has important implications, as it allows refinement of the nature of the interconnection between the two pools of Myo-II in this tissue. It has been shown previously that medial pulses of Myo-II flow toward and merge with the Myo-II pool at vertical junctions. However, to what extent these 'fusion' events contribute to junctional Myo-II was unclear. This study genetically uncoupled the regulation of both pools of Myo-II and showed that the loss of one pool does not compromise activation of Myo-II in the other. Indeed, junctional Myo-II levels and planar polarity are not affected in RhoGEF2 shRNA embryos or in RhoGEF2 germline clone where medial Myo-II is lost. This rules out the possibility of medial pulses being the main source of junctional Myo-II accumulation. Instead, it is concluded that actomyosin flow toward junctions contributes to junction shrinkage because it serves a distinct and direct mechanical function in junction remodeling rather than working by proxy by fueling junctional Myo-II (Garcia De Las Bayonas, 2019).

The division of labor in the molecular mechanisms of Rho1 activation in distinct cellular compartments lends itself to differential quantitative regulation. The activation kinetics of these different GEFs and nucleotide exchange catalytic efficiencies are likely to differentially impact Rho1 activity and therefore Myo-II activation at the junctional and medial-apical compartments. For example, RhoGEF2 mammalian orthologs, LARG and PDZ-RhoGEF, show a catalytic activity that is two orders of magnitude higher as compared with the Dp114RhoGEF orthologs subfamily. This may help to establish specific contractile regimes of actomyosin in given subcellular compartments. It is therefore important to tightly control RhoGEFs localization and activity to ensure a proper quantitative activation of the downstream GTPase (Garcia De Las Bayonas, 2019).

RhoGEF2 is a major regulator of medial-apical Rho1 activity during Drosophila gastrulation. Originally characterized in the invaginating mesoderm, it was found that RhoGEF2 also activates Rho1 medial-apical activity in the elongating ectoderm. There, RhoGEF2 localizes both medial-apically and at junctions where it is also planar polarized. Although RhoGEF2 and active Rho1 are both planar polarized at junctions, in RhoGEF2 mutants, junctional Rho1-GTP is not affected and ectopic recruitment of RhoGEF2 following expression of Gα12/13Q303L does not cause ectopic junctional Rho1-GTP accumulation. Thus, RhoGEF2 localization at the membrane is not strictly indicative of its activation status. Interestingly, Gα12/13/Cta is necessary for RhoGEF2 to translocate from microtubules plus ends to the plasma membrane where it signals. To date, experimental evidence favor a model whereby the binding of active Gα12/13/Cta to the RhoGEF in the vicinity of the cell membrane triggers its conformational change and stabilizes it in an open conformation able to bind to lipids via its PH domain and signal at the plasma membrane. There is no evidence that Gα12/13/Cta-GTP actively destabilizes RhoGEF2-EB1 interaction, but this is a formal possibility to be tested. Importantly, Gα12/13/Cta alone does not account for the restricted activation of Rho1 medial-apically (Garcia De Las Bayonas, 2019).

It is hypothesized that additional factors must regulate the spatial distribution of RhoGEF2 activity. In principle, RhoGEF2 signaling activity could either be specifically induced medial-apically independent of RhoGEF2 recruitment or RhoGEF2 could be inhibited at junctions and laterally. Sequestration of inactive RhoGEFs at cell junctions has been reported previously in mammalian cell cultures, suggesting that such mechanism could be evolutionary conserved. Phosphorylation can control the activity of the RH-RhoGEFs subfamily. Therefore, phosphorylation could promote activation or inhibition of RhoGEF2 activity in specific subcellular compartments in the ectoderm. RhoGEF2 is reported to be phosphorylated in the gastrulating embryo (Garcia De Las Bayonas, 2019).

Complementary to RhoGEF2, Dp114RhoGEF activates junctional Rho1 in the ectoderm. Dp114RhoGEF strictly localizes at junctions, providing a direct explanation for its junctional-specific effect. Gβ13F/G&gamma1 is also enriched at adherens junctions, where it controls Dp114RhoGEF junctional recruitment together with additional upstream regulators. Therefore, it is suggested that Gβ13F/Gγ1-dependent tuning of junctional Rho1 activation could be achieved through its ability to concentrate the GEF at junctions. Gβ/Gγ-dependent regulation of RhoGEFs has been described in mammals. One study proposes that mammalian p114RhoGEF may bind and be activated by Gβ1/Gγ2. Interestingly, recent work demonstrates that Gα12 can also recruit p114RhoGEF at cell junctions under mechanical stress in mammalian cell cultures where it promotes RhoA signaling. However, the region of mammalian p114RhoGEF that binds to Gα12 is absent in invertebrate RhoGEFs. How Gβ13F/Gγ1 controls Dp114RhoGEF at junctions in the Drosophila embryo remains an open question. A recent study reports that Dp114RhoGEF localizes at adherens junctions in the Drosophila ectoderm through multiple mechanisms, including interactions with Baz/Par3 and the Crumbs complex. Therefore, investigating a possible connection between Gβ13F/Gγ1 signaling and Baz/Crumbs should help decipher the mechanisms of Dp114RhoGEF localization (Garcia De Las Bayonas, 2019).

Importantly, neither Gβ13F/Gγ1 nor Dp114RhoGEF are themselves planar polarized at junctions. Hence, their distribution alone cannot explain polarized Rho1 activity at junctions. Strikingly, an increase in Gβ13F/Gγ1 dimers was found to hyperpolarize Rho1 activity and Myo-II at vertical junctions. Gβ13F/Gγ1 overexpression also leads to an overall increase in Dp114RhoGEF levels at junctions, although Dp114RhoGEF is not planar polarized in this condition. This indicates that recruitment at the plasma membrane and activation of Dp114RhoGEF are independently regulated, similar to RhoGEF2. In contrast, Dp114RhoGEF overexpression increases Myo-II at both transverse and vertical junctions, although a slightly stronger accumulation is observed at vertical junctions. Therefore, although Dp114RhoGEF junctional levels are increased in both experiments, only Gβ13F/Gγ1 overexpression leads to an increased planar polarization of Rho1-GTP and Myo-II at vertical junctions. This points to a key role for Gβ13F/Gγ1 subunits in the planar-polarization process associated with but independent from the sole recruitment of Dp114RhoGEF at junctions. In principle, Gβ13F/Gγ1 could bias junctional Rho1 signaling either by promoting its activation at vertical junctions or by inhibiting it at transverse junctions (e.g., RhoGAP polarized activation). Gβ13F/Gγ1 could also control active Rho1 distribution independent of its activation. For instance, a scaffolding protein binding to Rho1-GTP at junctions could be polarized by Gβ13F/Gγ1 to bias Rho1-GTP distribution downstream of its activation. Anillin, a Rho1-GTP anchor known to stabilize Rho1 signaling at cell junctions is a potential candidate in the ectoderm. Last, Toll receptors control Myo-II planar polarity in the ectoderm. Whether Gβ13F/Gγ1 and Tolls are part of the same signaling pathway is an important point yet to address in the future (Garcia De Las Bayonas, 2019).

Finally, this study sheds light on new regulatory differences underlying tissue invagination and tissue extension. This study found that Dp114RhoGEF localizes at junctions in the ectoderm, where it activates Rho1 and Myo-II. In contrast, maternally and zygotically supplied Dp114RhoGEF::GFP is not detected at junctions in the mesoderm. Little if any cytoplasmic signal is seen in this condition, suggesting that Dp114RhoGEF::GFP could be degraded in these cells. Thus, repression of Dp114RhoGEF protein in the mesoderm could be an important mechanism for cell apical constriction and proper tissue invagination. Of interest, Rho1 signaling is absent at junctions in the mesoderm. Therefore, it is tempting to suggest that the absence of Dp114RhoGEF at junction in the mesoderm accounts for cells' inability to activate Rho1 in this compartment. Importantly, the GPCR Smog and Gβ13F/Gγ1 subunits, found to control junctional Rho1 in the ectoderm, are common to both tissues. Dp114RhoGEF differential expression and/or subcellular localization could be a key element to bias signaling toward junctional compartment in the ectoderm (Garcia De Las Bayonas, 2019).

Cell contractility necessitates activation of the Rho1-Rock-MyoII core pathway. During epithelial morphogenesis, tissue- and cell-specific regulation of Rho1 signaling requires the diversification of Rho1 regulators, in particular RhoGEFs, as shown in this study, and RhoGAPs. Some of them are tissue specific with given subcellular localizations and activation mechanisms. The identification of signaling modules, namely Gα12/13-RhoGEF2 and Gβ13F/Gγ1-Dp114RhoGEF, provides a simple mechanistic framework for explaining how tissue-specific modulators control Rho1 activity in a given subcellular compartment in a given cell type. Therefore, it is suggested that the variation of (1) ligands, GPCRs, and associated heterotrimeric G proteins and (2) types of RhoGEFs and RhoGAPs as well as their combination, activation, and localization by respective co-factors underlies the context-specific control of Rho1 signaling during tissue morphogenesis. How developmental patterning signals ultimately control Rho regulators is an exciting area for future investigations (Garcia De Las Bayonas, 2019).

A Mechanosensitive RhoA Pathway that Protects Epithelia against Acute Tensile Stress

Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. This study reports a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. This study identified Myosin VI/Jaguar as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric Galpha12 (Concertina) protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly (Acharya, 2018).

Epithelia are subject to tensile forces that can challenge their cell-cell integrity. . This is exemplified by the observation that monolayers fracture at junctions when monolayer contractility is acutely increased by calyculin. Similarly, overactivation of contractility during Drosophila gastrulation disrupts the actomyosin networks that couple cells together. The current experiments now identify a junctional mechanotransduction pathway that is responsible for sensing, and responding to, such tensile stresses. It is propose that Myosin VI is the key sensor of acute tensile stress applied to AJs. It is stabilized and accumulates at AJs when tensile forces are transmitted to E-cadherin. This promotes the formation of an E-cadherin-Gα12 complex that activates the p114 RhoGEF-RhoA pathway to increase the tensile strength of multicellular junctions via mDia1. Of note, RhoA signaling is active at the ZA, even under resting conditions, but this is mediated by other GEFs such as Ect2. Thus, the Myosin VI-Gα12-p114 RhoGEF pathway that this study has identified can be considered a selective response to superadded tensile stress (Acharya, 2018).

At first sight, it seemed paradoxical that stimulation of RhoA would be used to preserve epithelial integrity. RhoA promotes actomyosin assembly at AJs under resting conditions and also in calyculin-stimulated cells. Both F-actin and NMII (Zipper) increased at bicellular junctions upon treatment with calyculin, and this was abrogated by p114 RhoGEF KD. This p114 RhoGEF-stimulated increase in actomyosin might be expected to promote junctional rupture by increasing the line tension in bicellular junctions and enhancing the forces acting to disrupt epithelial integrity, especially those focused on multicellular junctions. One possibility was that enhanced actomyosin also increased the stiffness of junctions to resist tensile stress. However, simulations in a mechanical model predicted that increasing stiffness alone would accelerate monolayer fracture rather than retarding it (Acharya, 2018).

Instead, it is considered that the protective effect of the p114 RhoGEF pathway is better explained by an increase in the tensile strength of AJs. In simulations of the vertex model, increasing tensile strength protected monolayer integrity against calyculin-induced stresses, even if junctional stiffness was also increased. Experimentally, it is suggested that this protective effect is especially important at the multicellular vertices. Physical considerations identify vertices as the junctional sites where cellular forces will be greatest, and, indeed, vertices were the principal sites where cell separation first began in these experiments. The accelerated onset of fracture that was seen in p114 RhoGEF KD cells thus implied that tension-activated p114 RhoGEF-RhoA signaling might reinforce vertices against stress (Acharya, 2018).

RhoA signals to both NMII and F-actin. However, calyculin appeared to maximally stimulate NMII, and this was not reduced by p114 RhoGEF KD. In contrast, p114 RhoGEF signaling was necessary to stimulate actin assembly at vertices in response to calyculin, an effect that was mediated by the RhoA-sensitive formin, mDia1. In turn, mDia1 was required to reinforce E-cadherin at vertices and for monolayers to resist tensile stress. Thus, p114 RhoGEF-RhoA-mediated actin assembly appeared to be key to preserving epithelial integrity in these experiments, although NMII regulation may also be relevant when tension is increased by other means. Without excluding possible roles for other membrane proteins found at vertices, it is therefore proposed that tension-activated RhoA signaling increases the tensile strength of monolayers by stimulating mDia1-dependent actin assembly to reinforce E-cadherin adhesion at vertices (Acharya, 2018).

It was noteworthy that RhoA signaling was selectively increased at cell-cell junctions but not at other adhesive sites, especially cell-substrate interactions. This highlights a key role for mechanisms that can confer spatial specificity on the mechanotransduction response. Two elements appear to be responsible for junctional selectivity in this instance. First, Gα12 can interact directly with E-cadherin, and this is necessary for junctional RhoA to be stimulated by tensile stress. The current working model is that Gα12, preactivated by S1P2 (Sphingosine 1-phosphate), is recruited to E-cadherin upon application of mechanical stress, where it then recruits and activates p114 RhoGEF to drive RhoA signaling (Acharya, 2018).

Second, this study identified Myosin VI as the force sensor that promotes the E-cadherin-Gα12 association. This requires both the ability of Myosin VI to associate with E-cadherin and also its pronounced capacity to anchor to actin filaments in response to load. The findings suggest that Myosin VI interacts transiently with E-cadherin under steady-state conditions. However, it is stabilized by load-sensitive anchorage when acute tensile stresses are transmitted through E-cadherin. In contrast, the functional impact of Myosin VI was abrogated by the L310G mutant, which retains processive motor function but has defective nucleotide gating linked to load-sensitivity. How increased F-actin anchorage promotes association of Myosin VI with E-cadherin remains to be determined. One possibility is that the increased dwell time of Myosin VI facilitates post-translational modifications that stabilize its binding to E-cadherin. This stabilized Myosin VI-cadherin complex may then promote the recruitment of Gα12 through conformational changes or accessory proteins. Irrespective, Myosin VI appears to exert its signaling effects via E-cadherin-Gα12, since tension-activated RhoA was abolished if Gα12 was unable to bind cadherin (Acharya, 2018).

In conclusion, these findings identify a mechanotransduction pathway that is selectively elicited to preserve epithelial integrity in response to tensile stress. The selectivity of this pathway implies that junctions may possess multiple mechanisms to sense mechanical signals that operate under different circumstances. Of note, α-catenin is necessary for the elemental force-sensitive association of cadherins with F-actin and also supports Ect 2-dependent RhoA signaling in steady-state AJs. Therefore, α-catenin may confer mechanosensitivity under baseline conditions, whereas the Myosin VI-dependent pathway that this study has identified is activated in response to superadded mechanical stress. Furthermore, the experiments tested the effects of acute application of tensile stress. Other mechanisms contribute when mechanical stresses are applied more slowly or are sustained longer, such as cellular rearrangements and oriented cell division. That epithelia possess such a diversity of compensatory mechanisms attests to the fundamental challenge of mechanical stress in epithelial biolog (Acharya, 2018).

Functions of Cysts orthologs in other species

ARHGEF18/p114RhoGEF Coordinates PKA/CREB Signaling and Actomyosin Remodeling to Promote Trophoblast Cell-Cell Fusion During Placenta Morphogenesis

Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. This study shows that p114RhoGEF/ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating protein kinase A (PKA) signaling, and is required for PKA-induced actomyosin remodeling, cAMP-responsive element binding protein (CREB)-driven gene expression of proteins required for trophoblast differentiation, and, hence, trophoblast cell-cell fusion. These data thus indicate that p114RhoGEF links actomyosin dynamics and cell-cell junctions to PKA/CREB signaling, gene expression and cell-cell fusion (Beal, 2021).

A Gα12-specific Binding Domain in AKAP-Lbc and p114RhoGEF

AKAP-Lbc is a Rho-activating guanine nucleotide exchange factor (RhoGEF) important in heart development and pro-fibrotic signaling in cardiomyocytes. Heterotrimeric G proteins of the G12/13 subfamily, comprising Gα12 and Gα13, are well characterized as stimulating a specialized group of RhoGEFs through interaction with their RGS-homology (RH) domain. Despite lacking an RH domain, AKAP-Lbc is bound by Gα12 through an unknown mechanism to activate Rho signaling. This study identified a Gα12-binding region near the C-terminus of AKAP-Lbc, closely homologous to a region of p114RhoGEF that was discovered to interact with Gα12. This binding mechanism is distinct from the well-studied interface between RH-RhoGEFs and G12/13 α subunits, as demonstrated by Gα12 mutants selectively impaired in binding either this AKAP-Lbc/p114RhoGEF region or RH-RhoGEFs. AKAP-Lbc and p114RhoGEF showed high specificity for binding Gα12 in comparison to Gα13, and experiments using chimeric G12/13 α subunits mapped determinants of this selectivity to the N-terminal region of Gα12. In cultured cells expressing constitutively GDP-bound Gα12 or Gα13, the Gα12 construct was more potent in exerting a dominant-negative effect on serum-mediated signaling to p114RhoGEF, demonstrating coupling of these signaling proteins in a cellular pathway. In addition, charge-reversal of conserved residues in AKAP-Lbc and p114RhoGEF disrupted Gα12 binding for both proteins, suggesting they harbor a common structural mechanism for interaction with this α subunit. These results provide the first evidence of p114RhoGEF as a Gα12 signaling effector, and define a novel region conserved between AKAP-Lbc and p114RhoGEF that allows Gα12 signaling input to these non-RH RhoGEFs (Martin, 2016).

Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF

Myosin II-driven mechanical forces control epithelial cell shape and morphogenesis. In particular, the circumferential actomyosin belt, which is located along apical cell-cell junctions, regulates many cellular processes. Despite its importance, the molecular mechanisms regulating the belt are not fully understood. This study characterize Lulu2, a FERM (4.1 protein, ezrin, radixin, moesin) domain-containing molecule homologous to Drosophila melanogaster Yurt, as an important regulator. In epithelial cells, Lulu2 is localized along apical cell-cell boundaries, and Lulu2 depletion by ribonucleic acid interference results in disorganization of the circumferential actomyosin belt. In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions. This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C. This study further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction. These findings therefore reveal a novel molecular system regulating the circumferential actomyosin belt in epithelial cells (Nakajima, 2011).

Stimulation of cortical myosin phosphorylation by p114RhoGEF drives cell migration and tumor cell invasion

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. This study demonstrates that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. These data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells (Terry, 2012).

Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis

Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. This study shows that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight-junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell-cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a complex containing myosin II, Rock II and the junctional adaptor cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis (Terry, 2011).


Search PubMed for articles about Drosophila Cysts

Acharya, B. R., Nestor-Bergmann, A., Liang, X., Gupta, S., Duszyc, K., Gauquelin, E., Gomez, G. A., Budnar, S., Marcq, P., Jensen, O. E., Bryant, Z. and Yap, A. S. (2018). A Mechanosensitive RhoA Pathway that Protects Epithelia against Acute Tensile Stress. Dev Cell 47(4): 439-452. PubMed ID: 30318244

Beal, R., Alonso-Carriazo Fernandez, A., Grammatopoulos, D. K., Matter, K. and Balda, M. S. (2021). ARHGEF18/p114RhoGEF Coordinates PKA/CREB Signaling and Actomyosin Remodeling to Promote Trophoblast Cell-Cell Fusion During Placenta Morphogenesis. Front Cell Dev Biol 9: 658006. PubMed ID: 33842485

di Pietro, F., Osswald, M., De Las Heras, J. M., Cristo, I., Lopez-Gay, J., Wang, Z., Pelletier, S., Gaugue, I., Leroy, A., Martin, C., Morais-de-Sa, E. and Bellaiche, Y. (2023). Systematic analysis of RhoGEF/GAP localizations uncovers regulators of mechanosensing and junction formation during epithelial cell division. Curr Biol 33(5): 858-874.e857. PubMed ID: 36917931

Garcia De Las Bayonas, A., Philippe, J. M., Lellouch, A. C. and Lecuit, T. (2019). Distinct RhoGEFs Activate Apical and Junctional Contractility under Control of G Proteins during Epithelial Morphogenesis. Curr Biol 29(20): 3370-3385. PubMed ID: 31522942

Martin, J. W., Cavagnini, K. S., Brawley, D. N., Berkley, C. Y., Smolski, W. C., Garcia, R. D., Towne, A. L., Sims, J. R. and Meigs, T. E. (2016). A Gα12-specific Binding Domain in AKAP-Lbc and p114RhoGEF. J Mol Signal 11: 3. PubMed ID: 31051012

Nakajima, H. and Tanoue, T. (2011). Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF. J Cell Biol 195(2): 245-261. PubMed ID: 22006950

Pinheiro, D., Hannezo, E., Herszterg, S., Bosveld, F., Gaugue, I., Balakireva, M., Wang, Z., Cristo, I., Rigaud, S. U., Markova, O. and Bellaiche, Y. (2017). Transmission of cytokinesis forces via E-cadherin dilution and actomyosin flows. Nature 545(7652): 103-107. PubMed ID: 28296858

Silver, J. T., Wirtz-Peitz, F., Simoes, S., Pellikka, M., Yan, D., Binari, R., Nishimura, T., Li, Y., Harris, T. J. C., Perrimon, N. and Tepass, U. (2019). Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly. J Cell Biol 218(10): 3397-3414. PubMed ID: 31409654

Simoes, S., Lerchbaumer, G., Pellikka, M., Giannatou, P., Lam, T., Kim, D., Yu, J., Ter Stal, D., Al Kakouni, K., Fernandez-Gonzalez, R. and Tepass, U. (2022). Crumbs complex-directed apical membrane dynamics in epithelial cell ingression. J Cell Biol 221(7). PubMed ID: 35588693

Terry, S. J., Elbediwy, A., Zihni, C., Harris, A. R., Bailly, M., Charras, G. T., Balda, M. S. and Matter, K. (2012). Stimulation of cortical myosin phosphorylation by p114RhoGEF drives cell migration and tumor cell invasion. PLoS One 7(11): e50188. PubMed ID: 23185572

Terry, S. J., Zihni, C., Elbediwy, A., Vitiello, E., Leefa Chong San, I. V., Balda, M. S. and Matter, K. (2011). Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis. Nat Cell Biol 13(2): 159-166. PubMed ID: 21258369

Biological Overview

date revised: 12 August 2023

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.