InteractiveFly: GeneBrief

Rho GTPase activating protein at 19D: Biological Overview | References

Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6-aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. This study used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion (Fic, 2021).

The form and function of epithelial cells depends on their polarization into distinct apical, lateral, and basal domains by conserved polarity factors. This polarity is then maintained by mutual antagonism between apical polarity factors such as atypical PKC (aPKC) and lateral factors such as Lethal (2) giant larvae (Lgl) and Par-1. While many aspects of the polarity machinery are now well understood, it is still unclear how the apical domain is initiated and what role cell division control protein 42 (Cdc42) plays in this process. Cdc42 was identified for its role in establishing polarity in budding yeast, where it targets cell growth to the bud tip by polarizing the actin cytoskeleton and exocytosis toward a single site. It has subsequently been found to function in the establishment of cell polarity in multiple contexts. For example, Cdc42 recruits and activates the anterior PAR complex to polarize the anterior-posterior axis in the Caenorhabditis elegans zygote and the apical-basal axis during the asymmetric divisions of Drosophila neural stem cells. Cdc42 also plays an essential role in the apical-basal polarization of epithelial cells, where it is required for apical domain formation. Cdc42 is active when bound to GTP, which changes its conformation to allow it to bind downstream effector proteins that control the cytoskeleton and membrane trafficking. An important Cdc42 effector in epithelial cells is the Par-6-aPKC complex. Par-6 binds directly to the switch 1 region of Cdc42 GTP through its semi-CRIB domain (Cdc42 and Rac interactive binding). This induces a change in the conformation of Par-6 that allows it to bind to the C-terminus of another key apical polarity factor, the transmembrane protein Crumbs, which triggers the activation of aPKC's kinase activity. As a result, active aPKC is anchored to the apical membrane, where it phosphorylates and excludes lateral factors, such as Lgl, Par-1, and Bazooka (Baz). In addition to this direct role in apical-basal polarity, Cdc42 also regulates the organization and activity of the apical cytoskeleton through effectors such as neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin polymerization, and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK; Genghis khan [Gek] in Drosophila), which phosphorylates the myosin regulatory light chain to activate contractility (Fic, 2021).

This crucial role of active Cdc42 in specifying the apical domain raises the question of how Cdc42-GTP itself is localized apically. In principle, this could involve activation by Cdc42 guanine nucleotide exchange factors (Cdc42GEFs) that are themselves apical or lateral inactivation by Cdc42GAPs. The Cdc42GEFs Tuba, intersectin 2, and Dbl3 have been implicated in activating Cdc42 in mammalian epithelia. Only Dbl3 localizes apical to tight junctions, however, as Tuba is cytoplasmic and enriched at tricellular junctions and intersectin 2 localizes to centrosomes. Thus, GEF activity may not be exclusively apical, suggesting that it is more important to inhibit Cdc42 laterally. Although nothing is known about the role of GAPs in restricting Cdc42 activity to the apical domain of epithelial cells, this mechanism plays an instructive role in establishing radial polarity in the blastomeres of the early C. elegans embryo. In this system, the Cdc42GAP PAC-1 is recruited by the cadherin adhesion complex to sites of cell-cell contact, thereby restricting active Cdc42 and its effector the Par-6-aPKC complex to the contact-free surface (Fic, 2021).

This study has analyzed the roles of Cdc42GAPs in epithelial polarity using the follicle cells that surround developing Drosophila egg chambers as a model system. By generating mutants in a number of candidate Cdc42GAPs, this study identified the Pac-1 orthologue, RhoGAP19D, as the GAP that restricts active Cdc42 to the apical domain. In the absence of RhoGAP19D, lateral Cdc42 activity leads to an expansion of the apical domain and a high frequency of epithelial invasion into the germline tissue, a phenotype that mimics the early steps of carcinoma formation (Fic, 2021).

In the absence of RhoGAP19D, both N-WASP and Gek are recruited to the lateral membrane, indicating that Cdc42 is ectopically activated there. This implies that RhoGAP19D is the major Cdc42GAP that represses Cdc42 laterally, because no other GAPs can compensate for its loss. This also suggests that the GEFs that activate Cdc42 are not restricted to the apical domain and can turn it on laterally once this repression is removed. This is consistent with the identification of multiple vertebrate GEFs with different localizations that contribute to apical Cdc42 activation. The current results therefore identify RhoGAP19D as a new lateral polarity factor. This leads to a revised network of polarity protein interactions in which RhoGAP19D functions as the third lateral factor that antagonizes the activity of apical factors, alongside Lgl, which inhibits aPKC, and Par-1, which excludes Baz/Par-3 (Fic, 2021).

The function of RhoGAP19D is very similar to that of its orthologue PAC-1, which inhibits Cdc42 at sites of cell contact in early C. elegans blastomeres to generate distinct apical and basolateral domains. Both RhoGAP19D and PAC-1 are recruited to the lateral domain by E-cadherin complexes, although the exact mechanism is slightly different. RhoGAP19D recruitment is strictly dependent on α-catenin, which links it through β-catenin to the E-cadherin cytoplasmic tail, whereas α-catenin (HMP-1) and p120-catenin (JAC-1) play partially redundant roles in recruiting PAC-1 to E-cadherin (HMR-1) in the worm. Nevertheless, in both cases, the recruitment of the Cdc42GAP translates the spatial cue provided by the localization of cadherin to sites of cell-cell contact into a polarity signal that distinguishes the lateral from the apical domain. Classic work on the establishment of polarity MDCK cells grown in suspension has revealed that the recruitment of cadherin (uvomorulin) to sites of cell-cell contact is the primary cue that drives the segregation of apical proteins from basolateral proteins. Furthermore, the expression of E-cadherin in unpolarized mesenchymal cells is sufficient to induce this segregation, although the mechanisms behind this process are only partially understood. The observation that RhoGAP19D directly links cadherin adhesion to the polarity system in epithelial cells extends the results of Klompstra (2015) in early blastomeres, strongly suggesting that PAC-1/RhoGAP19D plays an important role in the first steps in epithelial polarization (Fic, 2021).

Although PAC-1 and RhoGAP19D perform equivalent functions in early blastomeres and epithelial cells, there is one important difference between their mutant phenotypes. In pac-1 mutants, Par-6 and aPKC are mislocalized to the contacting surfaces of C. elegans blastomeres where Cdc42 is ectopically active. By contrast, Par-6 and aPKC are not mislocalized laterally in rhogap19d mutant Drosophila epithelial cells, even though lateral Cdc42-GTP does recruit two other Cdc42 effectors, N-WASP and Gek. Thus, lateral Cdc42 activity is sufficient to recruit Par-6/aPKC to the lateral domain in early blastomeres, but not in epithelial cells. Instead, it was observed that lateral Cdc42 activity in rhogap19d mutant follicle cells acts at a distance to expand the size of the apical domain. A likely explanation for this difference is the presence of Crumbs in epithelial cells. The interaction between Cdc42-GTP and Par-6 alters the conformation of Par-6 so that it can bind to Crumbs, which anchors the Par-6-aPKC complex to the apical membrane and activates aPKC's kinase activity. Although Par-6 presumably binds to Cdc42 laterally in rhogap19D mutants and undergoes the conformational change, it cannot be anchored laterally in the absence of Crumbs. This activated Par-6-aPKC complex can then diffuse until it is captured by Crumbs in the apical domain, thereby increasing apical aPKC activity, providing an explanation for why the apical domain expands in rhogap19d mutant cells. C. elegans has three Crumbs orthologues, but removal of all three simultaneously has no effect on viability or polarity. Thus, in contrast to Drosophila epithelial cells, C. elegans Crumbs proteins are not required for Par-6/aPKC localization and activation, suggesting that some other mechanism, such as Cdc42 binding, is sufficient to activate aPKC (Fic, 2021).

If the failure of active Cdc42 to recruit aPKC laterally in rhogap19d mutant cells is due to the absence of Crumbs in this region, there must be a mechanism to exclude Crumbs from the lateral domain. One proposed mechanism depends on Yurt (Moe and EPB41L5 in vertebrates), which is restricted to the lateral domain by aPKC and binds to Crumbs to antagonize its activity. However, no lateral recruitment of aPKC was observed in rhogap19d;yurt double-mutant cells. Thus, there must be some parallel mechanism that excludes Crumbs, Par-6, and/or aPKC from the lateral domain (Fic, 2021).

Although loss of RhoGAP19D only leads to a partial disruption of polarity, it causes the follicular epithelium to invade the adjacent germline tissue with 40% penetrance. This invasive behavior is not driven by an epithelial-to-mesenchymal transition, because the cells retain their apical adherens junctions and epithelial organization. Instead, the deformation of the epithelium seems to be driven by the combination of an increase in lateral contractility and an expansion of the apical domain, because reducing the dosage of Gek, which activates myosin II to drive the contractility, significantly reduces the frequency of this phenotype, as does halving the dosage of any of the apical polarity factors. The expansion of the apical domain makes the domain too long for the cells to adopt the lowest-energy conformation, giving them a tendency to become wedge shaped, which could drive the evagination. It is also possible that buckling of the epithelium contributes to invasion. Recent work has shown that epithelial monolayers under compressive stress and constrained by a rigid external scaffold have a tendency to buckle inward. The follicular cell layer is surrounded by an ECM that constrains the shape of the egg chamber and that should therefore resist expansion. In addition, the pulses of lateral contractility are likely to generate compressive stress because transiently reducing cell height while maintaining a constant volume will increase the cells' cross-sectional area, thereby exerting a pushing force on the neighboring cells. This compression coupled to the tendency to become wedge shaped due to apical expansion could therefore trigger the rare buckling events that initiate invasion. In support of this view, lateral contractility has been shown to drive the folding of the imaginal wing disc between the prospective hinge region and the pouch. This phenotype provides an example of how a partial disruption of polarity can induce cell shape changes that lead to major alterations in tissue morphogenesis (Fic, 2021).

The rhogap19d phenotype resembles the defects earliest observed in the development of ductal carcinoma in situ. In flat epithelial atypia (FEA), the ductal cells are still organized into an epithelial layer, but they display apical protrusions that are strongly labeled by the apical polarity factor Par-6. This suggests that the apical domain has expanded and bulges out of the cell, just as was observed in the rhogap19d mutant follicular cells. In the next stage, atypical ductal hyperplasia (ADH), the ductal cells start to invade the lumen of the duct while retaining aspects of normal apical-basal polarity. This again resembles the invasive phenotype of rhogap19d mutants, although overproliferation of the ductal cells probably also contributes to invasion in this case. Thus, these abnormalities, which can sometimes progress to ductal carcinoma in situ and breast cancer, mirror the effects of lateral Cdc42 activation. The RhoGAP19D human orthologues, ARHGAP21 and ARHGAP23, have been shown to bind directly to α-catenin and localize to cell-cell junctions. Furthermore, low expression of ARHGAP21 or ARHGAP23 correlates with reduced survival rates in several cancers of epithelial origin. It would therefore be interesting to determine whether these orthologues perform the same functions in epithelial polarity as RhoGAP19D and if their loss contributes to tumor development (Fic, 2021).

Mind bomb 2 promotes cell migration and epithelial structure by regulating adhesion complexes and the actin cytoskeleton

Cell migration is essential in animal development and co-opted during metastasis and inflammatory diseases. Some cells migrate collectively, which requires them to balance epithelial characteristics such as stable cell-cell adhesions with features of motility like rapid turnover of adhesions and dynamic cytoskeletal structures. How this is regulated is not entirely clear but important to understand. While investigating Drosophila oogenesis, it was found that the putative E3 ubiquitin ligase, Mind bomb 2 (Mib2), is required to promote epithelial stability and the collective cell migration of border cells. Through biochemical analysis, components of Mib2 complexes were identified, includeing E-cadherin and α- and β-catenins, as well as actin regulators. Three Mib2 interacting proteins, RhoGAP19D, Supervillin, and Myosin heavy chain-like, affect border cell migration. mib2 mutant main body follicle cells have drastically reduced E-cadherin-based adhesion complexes and diminished actin filaments. It is concluded that Mib2 acts to stabilize E-cadherin-based adhesion complexes and promote a robust actin cytoskeletal network, which is important for maintenance of epithelial integrity. The interaction with cadherin adhesion complexes and other cytoskeletal regulators contribute to its role in collective cell migration. Since Mib2 is well conserved, it may have similar functional significance in other organisms (Trivedi, 2022).

The elmo-mbc complex and rhogap19d couple Rho family GTPases during mesenchymal-to-epithelial-like transitions

Many metazoan developmental processes require cells to transition between migratory mesenchymal- and adherent epithelial-like states. These transitions require Rho GTPase-mediated actin rearrangements downstream of integrin and cadherin pathways. A regulatory toolbox of GEF and GAP proteins precisely coordinates Rho protein activities, yet defining the involvement of specific regulators within a cellular context remains a challenge due to overlapping and coupled activities. This study demonstrated that Drosophila dorsal closure is a powerful model for Rho GTPase regulation during transitions from leading edges to cadherin contacts. During these transitions a Rac GEF elmo-mbc complex regulates both lamellipodia and Rho1-dependent, actomyosin-mediated tension at initial cadherin contacts. Moreover, the Rho GAP Rhogap19d controls Rac and Rho GTPases during the same processes and genetically regulates the elmo-mbc complex. This study presents a fresh framework to understand the inter-relationship between GEF and GAP proteins that tether Rac and Rho cycles during developmental processes (Toret, 2018).

Rho family GTPases play crucial roles during Drosophila dorsal closure, but the GEFs and GAPs that regulate epidermal MET (mesenchymal-to-epithelial transition)-related processes are not well defined. A dorsal closure defect was originally described for myoblast city (mbc) mutants, an established Rac GEF that is in a complex with ELMO (Ced-12 - FlyBase). Based on the cell migration function of the ELMO-DOCK complex, its mammalian ortholog, the ELMO-MBC complex is thought to drive dorsal epidermis migration. However, an ELMO-DOCK complex also regulates Rho GTPases transiently downstream of cadherin contact initiation in mammals (Erasmus, 2015; Toret, 2014). With roles downstream of both integrins and cadherins, the ELMO-DOCK complex is well positioned to regulate Rho GTPases during MET-like processes. Therefore, this study investigated the specific roles of the ELMO-MBC complex and a novel GAP protein in Drosophila dorsal closure (Toret, 2018).

This study defines precisely dorsal closure Rac and Rho activities in time and space that occur at the epidermal leading edge and initial cadherin contacts. Moreover, it identifies roles for the atypical Rac GEF, the ELMO-MBC complex and the Rho GAP RhoGAP19D in the coordination of these Rac and Rho cycles during in vivo MET-like transitions (Toret, 2018).

ELMO-MBC complex mutants were defective in the number of epidermal leading edge lamellipodia, but only showed a late closure defect. This suggests that lamellipodia are dispensable for the majority of the dorsal closure process and agrees with recent models where amnioserosa provides the bulk force for dorsal closure rather than epidermal migration. Additionally, Rho1, myosin II and tension regulation at new epidermal cadherin contacts were perturbed and cadherin contacts were flattened in ELMO-MBC complex mutants. Similarly, an ELMO-DOCK complex drives Rac activation, Rho inactivation and actin rearrangements upon E-cadherin engagement in mammalian cells, but the actin reorganization role and consequences could not be addressed (Toret, 2014). Lamellipodial ELMO-DOCK-mediated Rac activation drives membrane extension, and an analogous role at new cadherin contacts would drive contact heightening. A membrane extension role accounts for the cadherin flattening observed at new DE-cadherin contacts in elmo mbc mutants, and may explain why initial E-cadherin contacts collapse in Elmo2-depleted MDCK cells (Toret, 2014). The conserved functions reveal that the ELMO-MBC activities identified in this study likely generally apply to MET-like processes (Toret, 2018).

This study identifies a new crucial physical state of late dorsal closure after epidermal DE-cadherin contacts form. In this region, a loss of tension at new DE-cadherin contacts is coordinated with an ELMO-MBC-dependent decrease in Rho activity and myosin localization. The novel cadherin contact that experiences little to no tension and has major implications for force-dependent cadherin interactions, such as vinculin. The formation of this tension-free zone has a major impact on embryo development and its absence results in a lateral expansion of new contacts. As new contacts form in elmo mbc mutants, the epidermis elongates and results in a leading edge that is progressively squeezed and creates the gaps observed in elmo mbc mutants (Toret, 2018).

Depletion of RhoGAP19D resulted in embryos that complete epidermal closure faster than wild type. RhoGAP19D-depleted embryos displayed complex epidermal cell phenotypes (a fragmented actomyosin cable, bimodal leading-edge tensions, transient Rac and lamellipodia states, and cadherin height defects). The fragmented actomyosin cable can explain the binary tensions. The RhoGAP19D-depleted actomyosin cable and tension behaviors resembled Rac over Rho biosensor data, which suggests a myosin-Rac link. The increased lamellipodia protrusions are consistent with the new epidermis migration. In wild-type embryos, the speed of the epidermal leading edge and the reduction of the amnioserosa were equal. This suggests that these two processes are normally coupled, whereas in RhoGAP19D mutants they were decoupled and the epidermal cells migrate faster over the unaffected amnioserosa. Lateral filopodial dynamics were decreased in elmo-mbc mutants and increased in RhoGAP19D mutants, which could be indirectly due to the associated dynamic lamellipodia changes or due to unexplored links with Cdc42. A transient regulation of Rac activity at new contacts, could also account for the over-heightened cadherin contacts upon RhoGAP19D depletion. Notably, mammalian Arhgap21 depletion results in faster cell migration. Additionally, mammalian Arhgap21 has an undefined cadherin role and localizes at E-cadherin contacts with kinetics that resemble Elmo2 and Dock1. In dorsal closure, RhoGAP19D has a MBC-dependent enrichment in the MET-like region. Together, these results suggest that, like the ELMO-DOCK complex, Arhgap21 also has conserved roles in metazoan MET-related processes (Toret, 2018).

Mammalian Arhgap21 was first reported to activate predominately Cdc42 in vitro, but later tissue culture studies favored RhoA and RhoC activation roles over Cdc42. The in vivo phenotype (faster closure) and Rac biosensor data favor a transient Rac GAP role for RhoGAP19D. In contrast, RhoGAP19D depletion also stabilized Rho sensor foci, and supports a Rho GAP function. Moreover, in the epidermis, loss of function of both RhoGAP19D and the ELMO-MBC complex mirrors the severest constitutively active Rho1 expression and is suppressed by dominant-negative Rho1. This argues that both proteins function in dual pathways that inactivate Rho1. The complex Rac and Rho regulation identified in this study is accounted for by transferring the inhibitory relationship between Rho and Rac to the Rho GAP and Rac GEF. RhoGAP19D loss results in an inability to stimulate Rho1 GTPase activity directly (persistent Rho activation), and also a failure to inhibit ELMO-MBC-mediated Rac activation. Improper transient Rac activity would directly or indirectly inhibit Rho processes like actomyosin-generated tension. The ELMO-MBC complex loss would prevent Rac activity, and thus not inhibit Rho until the Rho GAP, or other secondary mechanisms compensates. This explains the tapering off of Rho, myosin, and tension levels at new cadherin contacts in elmo-mbc mutants. Loss of both ELMO-MBC and RhoGAP19D would prevent Rac activation, but also all Rho inactivation. The mechanisms that underlie RhoGAP19D-mediated regulation of the ELMO-MBC complex may be direct or indirect, but the ELMO-MBC complex recruiting its negative regulator as the contact matures is a possibility (Toret, 2018).

This study identifies a new GEF-GAP protein partnership as a major regulator of the inverse relationship between the Rac and Rho cycles during integrin-to-cadherin transitions. Curiously, mammalian Arhgap21 is an EMT protein, but how the MET-related functions are linked to cadherin contact disruption and leading edge establishment remains unclear. Other cadherin-associated GEF and GAP proteins may act during non-MET-related processes such as mature junction or other cadherin contact expansions (McCormack et al., 2013). The parallels between Drosophila and mammalian systems, despite the mechanistic differences between dorsal closure, wound healing and cell pairs that form cadherin contacts, demonstrate that dorsal closure is a powerful model for MET-like processes (Toret, 2018).

Search PubMed for articles about Drosophila

Erasmus, J. C., Welsh, N. J. and Braga, V. M. (2015). Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion. Cell Signal 27(9): 1905-1913. PubMed ID: 25957131

Ferreira, S. M., Costa-Junior, J. M., Kurauti, M. A., Leite, N. C., Ortis, F., Rezende, L. F., Barbosa, H. C., Boschero, A. C. and Santos, G. J. (2020). ARHGAP21 Acts as an Inhibitor of the Glucose-Stimulated Insulin Secretion Process. Front Endocrinol (Lausanne) 11: 599165. PubMed ID: 33324349

Fic, W., Bastock, R., Raimondi, F., Los, E., Inoue, Y., Gallop, J. L., Russell, R. B. and St Johnston, D. (2021). RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion. J Cell Biol 220(4). PubMed ID: 33646271

Klompstra, D., Anderson, D. C., Yeh, J. Y., Zilberman, Y. and Nance, J. (2015). An instructive role for C. elegans E-cadherin in translating cell contact cues into cortical polarity. Nat Cell Biol 17(6): 726-735. PubMed ID: 25938815

Pissarra, M. F., Torello, C. O., Gomes, R. G. B., Shiraishi, R. N., Santos, I., Vieira Ferro, K. P., Lopes, M. R., Bergamo Favaro, P. M., Olalla Saad, S. T. and Lazarini, M. (2021). Arhgap21 Deficiency Results in Increase of Osteoblastic Lineage Cells in the Murine Bone Marrow Microenvironment. Front Cell Dev Biol 9: 718560. PubMed ID: 34917608

Soares, G. M., Zangerolamo, L., Costa-Junior, J. M., Vettorazzi, J. F., Carneiro, E. M., Saad, S. T., Boschero, A. C. and Barbosa-Sampaio, H. C. (2019). Whole-Body ARHGAP21-Deficiency Improves Energetic Homeostasis in Lean and Obese Mice. Front Endocrinol (Lausanne) 10: 338. PubMed ID: 31191459

Toret, C. P., Collins, C. and Nelson, W. J. (2014). An Elmo-Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion. J Cell Biol 207(5): 577-587. PubMed ID: 25452388

Toret, C. P., Shivakumar, P. C., Lenne, P. F. and Le Bivic, A. (2018). The elmo-mbc complex and rhogap19d couple Rho family GTPases during mesenchymal-to-epithelial-like transitions. Development [Epub ahead of print]. PubMed ID: 29437779

Trivedi, S., Bhattacharya, M. and Starz-Gaiano, M. (2022). Mind bomb 2 promotes cell migration and epithelial structure by regulating adhesion complexes and the actin cytoskeleton. Dev Biol 491: 94-104. PubMed ID: 36067835

date revised: 10 April 2023

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