Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner

Members of the Rho/Rac/Cdc42 superfamily of GTPases and their upstream activators, guanine nucleotide exchange factors (GEFs) , have emerged as key regulators of actin and microtubule dynamics. In their GTP bound form, these proteins interact with downstream effector molecules that alter actin and microtubule behavior. During Drosophila embryogenesis, a Galpha subunit (Concertina) and a Rho-type guanine nucleotide exchange factor (DRhoGEF2) have been implicated in the dramatic epithelial-cell shape changes that occur during gastrulation and morphogenesis . Using Drosophila S2 cells as a model system, we show that DRhoGEF2 induces contractile cell shape changes by stimulating myosin II via the Rho1 pathway. Unexpectedly, we found that DRhoGEF2 travels to the cell cortex on the tips of growing microtubules by interaction with the microtubule plus-end tracking protein EB1. The upstream activator Concertina, in its GTP but not GDP bound form, dissociates DRhoGEF2 from microtubule tips and also causes cellular contraction. We propose that DRhoGEF2 uses microtubule dynamics to search for cortical subdomains of receptor-mediated Galpha activation, which in turn causes localized actomyosin contraction associated with morphogenetic movements during development.     

DRhoGEF2 and diaphanous regulate contractile force during segmental groove morphogenesis in the Drosophila embryo

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis.     

DRhoGEF2 regulates cellular tension and cell pulsations in the Amnioserosa during Drosophila dorsal closure

Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction.     

Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis

The mechanism that positions the cytokinetic contractile ring is unknown, but derives from the spindle midzone. We show that an interaction between the Rho GTP exchange factor, Pebble, and the Rho family GTPase-activating protein, RacGAP50C, connects the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. We propose that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis.     

The pebble GTP exchange factor and the control of cytokinesis

Several G proteins of the Rho family have been shown to be required for cytokinesis. The activity of these proteins is regulated by GTP exchange factors (GEFs), which stimulate GDP/GTP exchange, and by GTPase activating proteins (GAPs), which suppress activity by stimulating the intrinsic GTPase activity. The role of Rho family members during cytokinesis is likely to be determined by their spatial and temporal interactions with these factors. Here we focus on the role of the pebble (pbl) gene of Drosophila melanogaster, a RhoGEF that is required for cytokinesis. We summarise the evidence that the primary target of PBL is Rho1 and describe genetic approaches to elucidating the function of PBL and identifying other components of the PBL-activated Rho signalling pathway.     

Rnd proteins: Multifunctional regulators of the cytoskeleton and cell cycle progression

Rnd3/RhoE has two distinct functions, regulating the actin cytoskeleton and cell proliferation. This might explain why its expression is often altered in cancer and by multiple stimuli during development and disease. Rnd3 together with its relatives Rnd1 and Rnd2 are atypical members of the Rho GTPase family in that they do not hydrolyse GTP. Rnd3 and Rnd1 both antagonise RhoA/ROCK‐mediated actomyosin contractility, thereby regulating cell migration, smooth muscle contractility and neurite extension. In addition, Rnd3 has been shown to have a separate role in inhibiting cell cycle progression by reducing translation of cell cycle regulators, including cyclin D1 and Myc. We propose that Rnd3 could act as a tumour suppressor to limit proliferation, but when mutations bypass this activity of Rnd3, it can promote cancer invasion through its effects in the actin cytoskeleton.     

The Drosophila melanogaster Rab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function

To identify novel regulators of nonmuscle myosin II (NMII) we performed an image-based RNA interference screen using stable Drosophila melanogaster S2 cells expressing the enhanced green fluorescent protein (EGFP)-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. We identified the Rab-specific GTPase-activating protein (GAP) RN-tre as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1 and a decrease in phosphomyosin-positive cells by immunostaining, while expression of constitutively active Rho or Rho-kinase (Rok) rescues the punctate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as overexpression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, our results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility.     

An anillin-Ect2 complex stabilizes central spindle microtubules at the cortex during cytokinesis

Cytokinesis occurs due to the RhoA-dependent ingression of an actomyosin ring. During anaphase, the Rho GEF (guanine nucleotide exchange factor) Ect2 is recruited to the central spindle via its interaction with MgcRacGAP/Cyk-4, and activates RhoA in the central plane of the cell. Ect2 also localizes to the cortex, where it has access to RhoA. The N-terminus of Ect2 binds to Cyk-4, and the C-terminus contains conserved DH (Dbl homologous) and PH (Pleckstrin Homology) domains with GEF activity. The PH domain is required for Ect2's cortical localization, but its molecular function is not known. In cultured human cells, we found that the PH domain interacts with anillin, a contractile ring protein that scaffolds actin and myosin and interacts with RhoA. The anillin-Ect2 interaction may require Ect2's association with lipids, since a novel mutation in the PH domain, which disrupts phospholipid association, weakens their interaction. An anillin-RacGAP50C (homologue of Cyk-4) complex was previously described in Drosophila, which may crosslink the central spindle to the cortex to stabilize the position of the contractile ring. Our data supports an analogous function for the anillin-Ect2 complex in human cells and one hypothesis is that this complex has functionally replaced the Drosophila anillin-RacGAP50C complex. Complexes between central spindle proteins and cortical proteins could regulate the position of the contractile ring by stabilizing microtubule-cortical interactions at the division plane to ensure the generation of active RhoA in a discrete zone.     

The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila

The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, we discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF2^2(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it’s functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila.     

Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila

Epithelial morphogenesis requires cell movements and cell shape changes coordinated by modulation of the actin cytoskeleton. We identify a role for Echinoid (Ed), an immunoglobulin domain-containing cell-adhesion molecule, in the generation of a contractile actomyosin cable required for epithelial morphogenesis in both the Drosophila ovarian follicular epithelium and embryo. Analysis of ed mutant follicle cell clones indicates that the juxtaposition of wild-type and ed mutant cells is sufficient to trigger actomyosin cable formation. Moreover, in wild-type ovaries and embryos, specific epithelial domains lack detectable Ed, thus creating endogenous interfaces between cells with and without Ed; these interfaces display the same contractile characteristics as the ectopic Ed expression borders generated by ed mutant clones. In the ovary, such an interface lies between the two cell types of the dorsal appendage primordia. In the embryo, Ed is absent from the amnioserosa during dorsal closure, generating an Ed expression border with the lateral epidermis that coincides with the actomyosin cable present at this interface. In both cases, ed mutant epithelia exhibit loss of this contractile structure and subsequent defects in morphogenesis. We propose that local modulation of the cytoskeleton at Ed expression borders may represent a general mechanism for promoting epithelial morphogenesis.     

The Saccharomyces cerevisiae actin cytoskeletal component Bsp1p has an auxiliary role in actomyosin ring function and in the maintenance of bud-neck structure

Iqg1p is a component of the actomyosin contractile ring that is required for actin recruitment and septum deposition. Cells lacking Iqg1p function have an altered bud-neck structure and fail to form a functional actomyosin contractile ring resulting in a block to cytokinesis and septation. Here it is demonstrated that increased expression of the actin cytoskeleton associated protein Bsp1p bypasses the requirement for contractile ring function. This also correlates with reduced bud-neck width and remedial septum formation. Increased expression of this protein in a temperature-sensitive iqg1-1 background causes remedial septum formation at the bud neck that is reliant upon chitin synthase III activity and restores cell separation. The observed suppression correlates with a restoration of normal bud-neck structure. While Bsp1p is a component of the contractile ring, its recruitment to the bud neck is not required for the observed suppression. Loss of Bsp1p causes a brief delay in the redistribution of the actin cytoskeleton normally observed at the end of actin ring contraction. Compromise of Iqg1p function, in the absence of Bsp1p function, leads to a profound change in the distribution of actin and the pattern of cell growth accompanied by a failure to complete cytokinesis and cell separation.     

Wound-induced contractile ring: a model for cytokinesis.

The actomyosin-based contractile ring is required for several biological processes, such as wound healing and cytokinesis of animal cells. Despite progress in defining the roles of this structure in both wound closure and cell division, we still do not fully understand how an actomyosin ring is spatially and temporally assembled, nor do we understand the molecular mechanism of its contraction. Recent results have demonstrated that microtubule-dependent local assembly of F-actin and myosin-II is present in wound closure and is similar to that in cytokinesis in animal cells. Furthermore, signalling factors such as small Rho GTPases have been shown to be involved in the regulation of actin dynamics during both processes. In this review we address recent findings in an attempt to better understand the dynamics of actomyosin contractile rings during wound healing as compared with the final step of animal cell division.     

The role of Rho family proteins in controlling the migration of crawling cells

Migration of crawling cells (amoebae and some kinds of the tissue cells) is a process related to the dynamic reorganization of actomyosin cytoskeleton. That reorganization engages actin polymerization and de-polymerization, branching of actin network and interaction of myosin II with actin filaments. All those cytoskeleton changes lead to the cell progression, contraction and shifting of the uropod and the cell adhesion. Numerous external stimuli, which activate various surface receptors and signal transduction pathways, can promote migration. Rho family proteins play an important role in the regulation of actin cytoskeleton organization. The most known members of this family are Rho, Rac and Cdc42 proteins, present in all mammalian tissue cells. These proteins control three different stages of cell migration: progression of the frontal edge, adhesion which stabilizes the frontal area, and de-adhesion and shifting of the uropod. Cdc42 and Rac control cell polarization, lamellipodium formation and expansion, organization of focal complexes. Rho protein regulates contractile activity of actomyosin cytoskeleton outside the frontal area, and thus contraction and de-adhesion of the uropod.     

Dissecting the role of Rho-mediated signaling in contractile ring formation

In anaphase, microtubules provide a specification signal for positioning of the contractile ring. However, the nature of the signal remains unknown. The small GTPase Rho is a potent regulator of cytokinesis, but the involvement of Rho in contractile ring formation is disputed. Here, we show that Rho serves as a microtubule-dependent signal that specifies the position of the contractile ring. We found that Rho translocates to the equatorial region before furrow ingression. The Rho-specific inhibitor C3 exoenzyme and small interfering RNA to the Rho GDP/GTP exchange factor ECT2 prevent this translocation and disrupt contractile ring formation, indicating that active Rho is required for contractile ring formation. ECT2 forms a complex with the GTPase-activating protein MgcRacGAP and the kinesinlike protein MKLP1 at the central spindle, and the localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. In addition, we show that the bundled microtubules direct Rho-mediated signaling molecules to the furrowing site and regulate furrow formation. Our study provides strong evidence for the requirement of Rho-mediated signaling in contractile ring formation.     

Controlling the switches: Rho GTPase regulation during animal cell mitosis

Animal cell division is a fundamental process that requires complex changes in cytoskeletal organization and function. Aberrant cell division often has disastrous consequences for the cell and can lead to cell senescence, neoplastic transformation or death. As important regulators of the actin cytoskeleton, Rho GTPases play major roles in regulating many aspects of mitosis and cytokinesis. These include centrosome duplication and separation, generation of cortical rigidity, microtubule–kinetochore stabilization, cleavage furrow formation, contractile ring formation and constriction, and abscission. The ability of Rho proteins to function as regulators of cell division depends on their ability to cycle between their active, GTP-bound and inactive, GDP-bound states. However, Rho proteins are inherently inefficient at fulfilling this cycle and require the actions of regulatory proteins that enhance GTP binding (RhoGEFs), stimulate GTPase activity (RhoGAPs), and sequester inactive Rho proteins in the cytosol (RhoGDIs). The roles of these regulatory proteins in controlling cell division are an area of active investigation. In this review we will delineate the current state of knowledge of how specific RhoGEFs, RhoGAPs and RhoGDIs control mitosis and cytokinesis, and highlight the mechanisms by which their functions are controlled.     

Movement of a cytokinesis factor cdc12p to the site of cell division.

A key question in cytokinesis is how the plane of cell division is positioned within the cell. Although a number of cytokinesis factors involved in formation of the actomyosin contractile ring have been identified, little is known about how these factors are localized and assembled at the cell-division site. Cells of the fission yeast Schizosaccharomyces pombe divide using a medial actomyosin ring that assembles in early mitosis [1]. The S. pombe cdc12 gene encodes a formin, a member of a family of proteins that have functions in cytokinesis and cell polarity and that may bind Rho/Cdc42 GTPases, profilin and other actin-associated proteins [1] [2] [3] [4]. The cdc12 protein (cdc12p) is required specifically for medial-ring assembly during cytokinesis and is a component of this ring [2] [5]. In this study, cdc12p was found, during interphase, in a discrete, motile cytoplasmic spot that moved to the future site of cell division at the onset of mitosis. Three lines of evidence indicated that this cdc12p spot moved on both actin and microtubule networks: movement required either actin or microtubules; the spot was associated with actin and microtubule structures; and individual spots were seen to move along both microtubule and non-microtubule tracks. These findings demonstrate that a cytokinesis factor may travel on both microtubule and actin networks to the future site of cell division.