Decision letter: The RhoGAP activity of CYK-4/MgcRacGAP functions non-canonically by promoting RhoA activation during cytokinesis

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Cytokinesis requires activation of the GTPase RhoA. ECT-2, the exchange factor responsible for RhoA activation, is regulated to ensure spatiotemporal control of contractile ring assembly. Centralspindlin, composed of the Rho family GTPase-activating protein (RhoGAP) MgcRacGAP/CYK-4 and the kinesin MKLP1/ZEN-4, is known to activate ECT-2, but the underlying mechanism is not understood. We report that ECT-2-mediated RhoA activation depends on the ability of CYK-4 to localize to the plasma membrane, bind RhoA, and promote GTP hydrolysis by RhoA. Defects resulting from loss of CYK-4 RhoGAP activity can be rescued by activating mutations in ECT-2 or depletion of RGA-3/4, which functions as a conventional RhoGAP for RhoA. Consistent with CYK-4 RhoGAP activity contributing to GEF activation, the catalytic domains of CYK-4 and ECT-2 directly interact. Thus, counterintuitively, CYK-4 RhoGAP activity promotes RhoA activation. We propose that the most active form of the cytokinetic RhoGEF involves complex formation between ECT-2, centralspindlin and RhoA. https://doi.org/10.7554/eLife.08898.001 eLife digest Cell division is a process in which a cell splits to form two daughter cells. In most cases, the cell first duplicates its genetic material and then the two copies are pulled to opposite ends of the cell. A ring of protein filaments—called the contractile ring—then assembles to form a band around the cell at the site of the division. This ring contracts and the force generated separates the cells in a step known as cytokinesis. A protein belonging to the Rho family, called RhoA, is essential for cytokinesis because it controls the formation of the contractile ring. Rho proteins are switched on by the activities of other proteins called guanine nucleotide exchange factors. Another group of proteins known as ‘GTPase activating proteins’ (or GAPs for short) generally act to promote the ability of Rho proteins to turn themselves off. In animals and other multicellular organisms, a GAP called CYK-4 largely concentrates on the spindle midzone, but some of the protein also moves to part of the cell membrane near the future site of cell division. It binds to a guanine nucleotide exchange factor called ECT-2 to switch RhoA on, which in turn promotes the formation of the contractile ring. However, it is not clear why a protein that activates RhoA is also able to trigger its inactivation. In this study, Zhang and Glotzer studied cell division in a roundworm called Caenorhabditis elegans. The experiments show that cells that lacked the GAP activity of CYK-4 were unable to complete cytokinesis because RhoA was not fully switched on. This requirement could be bypassed in cells with mutant forms of ECT-2 that were overactive. Therefore, an activity that was thought to inactivate RhoA actually promotes its activation. Further experiments show that the section (or ‘domain’) of CYK-4 that has GAP activity interacts directly with the guanine nucleotide exchange domain of ECT2. Zhang and Glotzer suggest that this interaction stimulates ECT2 and thereby promotes the activation of RhoA. Further experiments will reveal how CYK-4 stimulates ECT-2. In addition, it will be important to determine whether other proteins with GAP domains also work in this unconventional way. https://doi.org/10.7554/eLife.08898.002 Introduction RhoA activation during cytokinesis Cell division involves the myosin-mediated contraction of an actin-based contractile ring. In metazoans, contractile ring assembly involves activation of the GTPase RhoA. RhoA directly activates formin-mediated actin polymerization and indirectly promotes myosin activation. As the contractile ring must assemble at the correct position and at the correct time, between the segregating chromosomes in anaphase, RhoA activation is subject to multiple regulatory mechanisms (see Green et al., 2012 for review). The primary direct activator of RhoA during cytokinesis is the RhoGEF ECT-2. ECT-2 contains N-terminal BRCT domains and a C-terminal RhoGEF domain (Kim et al., 2005; Zou et al., 2014). Presumably as a consequence of being autoinhibited, ECT-2 function depends on activators. One of the important activators of ECT-2 during cytokinesis is the centralspindlin complex, which is a heterotetramer containing a dimeric kinesin, ZEN-4 (aka MKLP1, Pavarotti) and dimeric CYK-4 (aka MgcRacGAP, Tum/RacGAP50C) (Mishima et al., 2002). Centralspindlin organizes the spindle midzone and directly recruits numerous regulators of cytokinesis, including ECT-2, to this location (Burkard et al., 2009; Wolfe et al., 2009). ECT-2 and centralspindlin are conserved among—and restricted to—metazoans (Frédéric et al., 2013) (unpublished results). Though these proteins are conserved, their names are distinct in each organism. For simplicity, Caenorhabditis elegans names will be used throughout this manuscript with the exception that we will refer to RHO-1 with the more common name RhoA. Recruitment of ECT-2 to the spindle midzone involves regulated binding between ECT-2 and CYK-4. The BRCT domains of ECT-2 bind to CYK-4 phosphorylated by PLK-1 (Burkard et al., 2009; Wolfe et al., 2009). CYK-4 phosphorylation occurs in a cell cycle and microtubule-regulated manner. Furthermore, CDK-1 phosphorylation of ECT-2 inhibits the ECT-2-CYK-4 interaction during metaphase and inactivates a membrane binding motif within ECT-2 (Yüce et al., 2005; Su et al., 2011). The phosphorylation-dependent interaction between ECT-2 and centralspindlin is required for RhoA activation during cytokinesis in human cells (Burkard et al., 2007; Wolfe et al., 2009). Centralspindlin also localizes in trace, but biologically relevant, amounts on the cell membrane. Centralspindlin accumulation to the midzone and the membrane are independently regulated by its oligomerization, which is inhibited by a 14-3-3 protein and promoted by the chromosome passenger complex (Douglas et al., 2010; Basant et al., 2015). The role of the GAP activity of CYK-4 GAP has been controversial The CYK-4 subunit of centralspindlin contains an evolutionarily conserved Rho family GTPase-activating protein (RhoGAP) domain (Jantsch-Plunger et al., 2000). The function of this domain has been examined in a number of different contexts. In vitro, the GAP domain of CYK-4 efficiently activates the GTPase activity of the Rho-family GTPases, CED-10/Rac1 and CDC-42. CYK-4 also has GAP activity towards RhoA, but it is far less active towards RhoA as compared to Rac1 and CDC-42 (Touré et al., 1998; Jantsch-Plunger et al., 2000; Bastos et al., 2012). Despite extensive effort, there is no consensus for the biological role of this GAP activity (see White and Glotzer, 2012 for review). In some cell types, the GAP activity appears dispensable (Goldstein et al., 2005; Yamada et al., 2006), in others it appears to be important to negatively regulate Rac1 (D'Avino et al., 2004; Canman et al., 2008; Bastos et al., 2012), whereas in yet others it appears to promote RhoA activation (D'Avino et al., 2004; Zavortink et al., 2005; Loria et al., 2012). System-specific differences may underlie some of these diverse results. However, these studies differ in the mutations used to assess the function of the GAP domain, which is likely to affect the results. In addition, some of the controversy may be due to misinterpretation of indirect effects. The function of the RhoGAP domain has been examined in C. elegans embryos in some detail. These studies have focused largely on a temperature-sensitive, separation-of-function substitution mutation, E448K, that lies in the RhoGAP domain of CYK-4, cyk-4(or749ts) (Canman et al., 2008). The mutant protein can complex with the centralspindlin kinesin, ZEN-4, and bundle microtubules in the central spindle. However, cytokinesis does not proceed to completion in these embryos. Interestingly, depletion of CED-10/Rac1 or the actin nucleator subunit ARP-2 enables these embryos to complete cytokinesis (Canman et al., 2008). These genetic interactions have been interpreted to indicate that the GAP domain of CYK-4 is important to keep CED-10/Rac1 inactive and prevent the accumulation of branched actin in the equatorial region (Canman et al., 2008). However, subsequent analysis demonstrated that cyk-4(or749ts); ced-10(−) embryos are phenotypically abnormal (Loria et al., 2012). In particular, cyk-4; ced-10 mutant embryos have reduced accumulation of RhoA effectors as compared to ced-10 mutants alone (Loria et al., 2012). These results suggest that this mutation in CYK-4 affects more than the RhoGAP activity of CYK-4 or that CED-10/Rac1 is not the relevant target of the GAP domain, or both. Parallel mechanisms activate RhoA in the early C. elegans embryo Analysis of centralspindlin function in C. elegans embryos is impeded by the existence of a second, parallel pathway that promotes RhoA activation in the early embryo. Upon fertilization, embryos exhibit RhoA-dependent contractility that promotes embryo polarization and culminates in the formation of a transient furrow known as the pseudocleavage furrow. This wave of contractility requires a poorly conserved protein known as NOP-1 and is largely independent of centralspindlin (Tse et al., 2012). Cytokinetic contractility, on the other hand, involves both NOP-1 and centralspindlin. However, nop-1 is nonessential; loss of function mutants are viable and fertile (Rose et al., 1995). Cytokinesis proceeds to completion in the absence of NOP-1, although furrow initiation is slightly delayed and RhoA accumulates to lower levels at the cleavage furrow (Tse et al., 2012). Due to its role in RhoA activation, polarization in NOP-1-deficient embryos is also delayed. Mutational inactivation of NOP-1 permits direct analysis of centralspindlin-dependent furrow formation. Notably, when NOP-1 is inactivated, cyk-4(or749ts) embryos are completely defective in RhoA activation (Tse et al., 2012). Membrane-bound centralspindlin promotes RhoA activation The CYK-4 GAP domain is adjacent to a C1 domain that mediates membrane localization of centralspindlin (Lekomtsev et al., 2012). Given that the cyk-4(or749ts) substitution renders the protein thermosensitive, it is possible that these phenotypes are not the sole consequence of loss of GAP activity; this mutation could affect other functions of the GAP domain, the adjacent C1 domain may also be affected. To clarify these issues, we performed a targeted structure-function analysis of the C1 and GAP domains of CYK-4. We demonstrate that the cyk-4(or749ts) allele indeed affects its ability to associate with the membrane and show that this activity contributes to RhoA activation. We further show that the active site of the GAP domain contributes to the accumulation of downstream effectors of RhoA and RhoA-dependent contractility. Furthermore, we find that the catalytic domains of CYK-4 and ECT-2 directly interact in vitro. Finally, we show that hypomorphic defects in CYK-4-mediated RhoA dependent contractility can be suppressed by either loss of the RhoGAP activity provided by RGA-3/4 or by either of two activating mutations in ECT-2. These activating mutations in ect-2 rescue cyk-4(or749ts) and GAP-deficient CYK-4. Our results indicate that CYK-4 GAP activity is involved in ECT-2-mediated RhoA activation. Results The cyk-4(or749ts) allele, E448K, exhibits defects in membrane association We sought to conduct a structure-function analysis of CYK-4 to determine the individual contributions of the GAP and C1 domains of CYK-4. We established a rescue assay based on single copy integrants of GFP-tagged CYK-4 transgenes driven by the cyk-4 promoter inserted at a defined locus in the C. elegans genome using Mos1-mediated integration (Figure 1—figure supplement 1) (Frøkjaer-Jensen et al., 2012). The transgenes were expressed at consistent levels (Figure 1—figure supplement 2). The transgenes were rendered resistant to an RNAi construct that could effectively deplete endogenous CYK-4 by targeting the 3′ UTR and portions of the coding sequence (Figure 1—figure supplement 2). By combining the appropriate mutant transgene with RNAi to specifically deplete endogenous CYK-4, we obtained embryos that express CYK-4MUT. In this and all subsequent experiments, when a given variant is assayed, endogenous CYK-4 is depleted by RNAi; these will be referred to as cyk-4mut embryos. We first sought to validate the rescue assay, by assaying cyk-4E448K embryos and found that they closely phenocopy cyk-4(or749ts) embryos in which endogenous CYK-4 has the E448K substitution (Figure 1—figure supplement 3). This phenocopy indicates functional depletion of endogenous CYK-4. Additionally, a wild-type transgene was fully functional as it could complement a large deletion in CYK-4 to viability and fertility (Figure 6—figure supplement 1). Consistent with the C1 domain promoting membrane association, CYK-4∆C1 does not accumulate on ingressing cleavage furrows (Figure 1A,B). As cyk-4(or749ts) is temperature sensitive, we considered the possibility that the thermosensitivity also destabilizes the C1 domain that lies adjacent to the CYK-4 GAP domain (Figure 1—figure supplement 1). Indeed, at the restrictive temperature, CYK-4E448K exhibits a similar defect in localization as CYK-4∆C1 (Figure 1A,B). CYK-4 also associates with the membrane in the germline and indirect evidence suggests that this localization is compromised in embryos expressing CYK-4E448K (Zhou et al., 2013). We generated strains in which both the endogenous cyk-4 and the GFP-tagged transgene contained the E448K mutation. At the permissive temperature, CYK-4E448K membrane recruitment was readily detected in the germline, however this localization was lost as animals were shifted to the restrictive temperature (Figure 1—figure supplement 4). Similarly, the C1 domain is required for membrane localization in the gonad (Figure 1—figure supplement 4). Thus, CYK-4E448K impairs membrane localization, and it is likely to impair the function of both its GAP and C1 domains. Figure 1 with 4 supplements see all Download asset Open asset CYK-4-dependent membrane binding promotes furrow ingression. (A) CYK-4 accumulates on the plasma membrane. Membrane accumulation is observed on ingressing cleavage furrows (boxed regions). CYK-4 membrane accumulation requires the C1 domain and is compromised by the E448K substitution in the cyk-4(or749ts) allele. (B) Membrane accumulation of CYK-4 variants. The accumulation was quantified as a ratio of the accumulation of CYK-4::GFP/mCherry::PH at the furrow tip as depicted in the schematic; the mean intensity ±s.e.m. are plotted. (N = 8–12 embryos *p < 0.05, one way ANOVA followed by Tukey multiple comparison). (C) Deletion of the C1 domain and the E448K substitution impair cleavage furrow ingression. Kymographs generated from time-lapse recordings of the equatorial region of embryos of the indicated genotypes expressing the membrane marker mCherry::PH. Kymographs begin at anaphase onset. (D) Deletion of the C1 domain and the E448K substitution abrogate centralspindlin-dependent furrow ingression. The progression of cytokinesis was assessed in embryos expressing CYK-4 variants in combination with a loss of function mutation in nop-1. (E) Mutation of CED-10/Rac1 slightly increases furrow ingression in CYK-4∆C1 embryos and allows complete, albeit delayed, furrow ingression in CYK-4E448K embryos. The progression of cytokinesis was assessed in embryos expressing CYK-4 variants in combination with a loss of function mutation in ced-10. (F–I) Quantification of furrow ingression rates in embryos of the indicated genotypes. Representative examples are shown in the accompanying kymographs. (N = 8–12 embryos; error bars, 95% confidence intervals). (J) The ability of ced-10/Rac1 to rescue cytokinesis in CYK-4E448K embryos requires the NOP-1 pathway for furrow ingression. Images shown reflect the maximal extent of furrow ingression in embryos of the indicated genotypes expressing the membrane marker mCherry::PH. Unless otherwise specified, all scale bars in all figures are 10 µm. https://doi.org/10.7554/eLife.08898.003 We compared the progression of cytokinesis in embryos expressing CYK-4WT, CYK-4∆C1, and CYK-4E448K. CYK-4∆C1 embryos exhibit a cytokinetic defect that is largely similar to that of cyk-4E448K embryos. In particular, furrow ingression is slow and incomplete (Figure 1C,F). However, subtle differences were detected; the onset of significant furrow ingression is delayed relative to controls in cyk-4E448K embryos but not in cyk-4∆C1 embryos. This suggests that the CYK-4E448K phenotype could reflect a compound defect, rather than a sole defect in the ability to associate with the membrane. Cleavage furrow ingression in C. elegans depends on the combined action of centralspindlin and a non-essential protein, NOP-1 (Tse et al., 2012). To determine whether the C1 domain is essential for centralspindlin-dependent furrow ingression, we expressed CYK-4WT, CYK-4∆C1, and CYK-4E448K in embryos that lack the NOP-1-dependent pathway for furrow ingression. As expected, CYK-4WT supports full furrow ingression in this sensitized background. In stark contrast, neither CYK-4∆C1 nor CYK-4E448K support detectable furrow ingression in the absence of NOP-1 activity (Figure 1D,G). Previous studies demonstrated that loss of function mutations in ced-10/rac-1 or depletion of the protein partially suppress the cytokinesis defect in cyk-4(or749ts) embryos (Figure 1E,H). However, as described above, it is important to examine the extent of suppression in the absence of the parallel, NOP-1-dependent, pathway. We therefore examined whether ced-10/rac-1 loss of function mutations could suppress the cytokinesis defect in cyk-4∆C1. Although cleavage furrows in cyk-4∆C1; ced-10(n1993) embryos ingress somewhat more deeply than cyk-4∆C1 embryos, they do not complete cytokinesis (Figure 1E,H). The simplest interpretation of these results is that although CYK-4E448K diminishes membrane association of CYK-4, it may retain some function at the restrictive temperature such that it facilitates the abscission step in cyk-4(or749ts); ced-10(n1993) embryos, as CYK-4-mediated membrane association is essential for completion of cytokinesis in cultured human cells (Lekomtsev et al., 2012). Importantly, when NOP-1 activity is compromised, inactivation of CED-10/Rac1 does not suppress the defect in furrow ingression caused by CYK-4E448K (Figure 1J,I), suggesting that CYK-4 does not act directly on CED-10. Mutants in the active site of the CYK-4 GAP domain are cytokinesis-defective To begin to determine the role of the catalytic activity of CYK-4 during cytokinesis, we first studied the consequence of mutating the highly conserved catalytic arginine that stabilizes the transition state during GTP hydrolysis. Substitution of the catalytic arginine with alanine strongly attenuates GAP activity against Rac in a variety of CYK-4 orthologs and is widely used to inactivate Rho family GAPs (Rittinger et al., 1997; Yamada et al., 2006; Miller and Bement, 2009; Bastos et al., 2012; Zanin et al., 2013). As expected, CYK-4R459A GAP domain retains the ability to bind to RhoA•GTP, demonstrating that the protein is well folded in vitro (Figure 2—figure supplement 1). While CYK-4 GAP exhibits GAP activity towards both RhoA and CED-10/Rac1, CYK-4R459A GAP lacks detectable GAP activity towards either GTPase (Figure 2—figure supplement 2). In order to determine if catalytic activity is required for viability, we used a strain heterozygous for a deletion mutant of CYK-4, cyk-4(ok1034). One quarter of the embryos from these heterozygous hermaphrodites contain maternally provided CYK-4 and lack zygotic CYK-4. These zygotic null embryos fail to hatch and arrest with a variety of terminal phenotypes (Figure 2A, left). Many, but not all, embryos contain muscle tissue and have undergone partial morphogenesis. The embryos also contain enlarged cells, likely due to defects in cytokinesis (Sugimoto et al., 2001). We introduced the GAP-defective transgene into this strain. Remarkably, cyk-4(ok1034); cyk-4R459A animals hatch and develop to adulthood. However, these animals are sterile (Figure 2A, middle). Thus, the GAP activity of CYK-4 is not essential for post embryonic development but it has an important role in gonad development, likely due to a requirement for post-embryonic cell proliferation in the germline. As a consequence, it is not possible to use classical genetic tools to obtain embryos in which CYK-4R459A is the sole form of CYK-4. Figure 2 with 2 supplements see all Download asset Open asset CYK-4 GAP activity is required for cytokinesis and viability. (A) cyk-4(ok1034) null embryos arrest at variable stages during embryogenesis (left). The GAP-deficient CYK-4R459A transgene rescues the embryonic lethality, but animals arrest as sterile adults (middle). GAP-deficient CYK-4R459A is recessive, no phenotypes is in the of scale µm. (B) GAP-deficient CYK-4R459A embryos fail to complete cytokinesis. Kymographs are generated as in Figure with the exception that the from the CYK-4 transgenes is also shown on CYK-4R459A accumulates more strongly at furrow as compared to CYK-4WT (C) The of furrow ingression in CYK-4WT and CYK-4R459A embryos. Results are quantified as described in Figure (D) Membrane accumulation of CYK-4WT and Results are quantified as described in Figure < by To the role of the CYK-4 GAP activity during we used the assay (Figure 1—figure supplement 1). expressing are largely during the stages of the first cell spindle chromosome central spindle assembly during anaphase, and CYK-4R459A highly on the spindle midzone (Figure Cleavage furrow initiation occurs and the furrow at near wild-type rates to near However, cytokinesis does not complete and the furrow this phenotype was fully (Figure these embryos also fail to complete cytokinesis not These results suggest that a step in cytokinesis is most to loss of CYK-4 GAP This phenotype is distinct from that of cyk-4(or749ts) furrows in cyk-4R459A embryos ingress more and more deeply than cyk-4E448K embryos (Figure We assessed the ability of CYK-4R459A to associate with membrane during furrow ingression (Figure CYK-4R459A accumulates on the membrane as compared to this localization suggests that CYK-4R459A is well folded in Therefore, the cytokinetic defect in this strain is to be an indirect consequence of a of CYK-4 to localize to the membrane. CYK-4 GAP domain mutations that prevent RhoA binding are highly defective in RhoA activation To these that CYK-4 must interact with Rho family during cytokinesis, and the possibility that the phenotype of CYK-4R459A is due to binding of CYK-4 to active RhoA, we mutations in CYK-4 that its binding to RhoA and other (Rittinger et al., 1997; et al., conserved, in the RhoA (Figure supplement 1) were to and in the rescue expressing expressing CYK-4R459A and CYK-4∆C1, exhibit fully embryonic (Figure supplement 1). exhibits reduced binding to RhoA in vitro (Figure 2—figure supplement and it does not exhibit membrane in (Figure Interestingly, embryos exhibit a furrow ingression defect than cyk-4R459A embryos, as furrow ingression is and less complete (Figure Importantly, NOP-1 depletion from embryos largely furrow ingression (Figure Thus, Rho GTPase binding by CYK-4 is essential for cytokinetic ingression. Figure with 1 supplement see all Download asset Open asset RhoA binding by CYK-4 is required for cytokinesis. (A) but not on the plasma membrane of the ingressing cleavage furrows (boxed regions). (B) Membrane accumulation of variants was quantified as described in Figure (N = 8–12 embryos; < by one way ANOVA followed by Tukey multiple comparison). (C) The Rho family GTPase binding defective variant of CYK-4, cytokinesis analysis of the progression of cytokinesis in embryos in the or absence of NOP-1 Kymographs were as described in to Figure (D) The of furrow ingression in embryos. Results are quantified as described in Figure (E) of CDC-42 and CED-10/Rac1, either alone or in does not cytokinesis defects in the sensitized nop-1 background. Kymographs were as described in to Figure that depletion of CDC-42 results in cleavage furrow ingression. The of furrow ingression in embryos in nop-1 CDC-42 CED-10/Rac1 Results are quantified as described in Figure We sought to determine the Rho family GTPase to which CYK-4 must bind to its function in furrow formation is dependent on CYK-4 binding to either CED-10/Rac1 or CDC-42 to a regulatory complex, then inactivation of these be to a phenotype at as as a mutation that the GTPase binding site of CYK-4. However, mutation of CED-10/Rac1, or depletion of does not affect the of cleavage furrow ingression (Jantsch-Plunger et al., 2000; Loria et al., 2012), when combined with mutations in NOP-1 (Figure Indeed, cytokinesis occurs efficiently and proceeds to completion in embryos in which NOP-1, CED-10/Rac1, and CDC-42 are (Figure We that RhoA is the relevant GTPase that CYK-4 binds to promote cleavage furrow formation. Due to its direct role in furrow ingression, it is not possible to RhoA in the manner. CYK-4 GAP domain mutations are defective in centralspindlin-dependent We sought to determine how the GAP activity of CYK-4 promotes cytokinesis. Previous studies at for the phenotype in cyk-4R459A embryos. the GAP domain could function as GAP that on RhoA, CYK-4 GAP-deficient embryos fail to complete cytokinesis because of a requirement for RhoA inactivation at cytokinesis. CED-10/Rac1 could be an important target of CYK-4 GAP CYK-4 GAP embryos to accumulate Rac1 activity that with cytokinesis. although it is CYK-4 GAP activity could promote RhoA activation, and therefore the CYK-4 GAP embryos may fail cytokinesis due to incomplete RhoA activation. We sought to between these The first and opposite for the of experiments in which RhoA levels are (Figure the to complete cytokinesis in cyk-4R459A embryos is due to of RhoA, as be from the for the function of a RhoA then a in active RhoA levels the if the GAP active site promotes RhoA activation, then the of RhoA activity be to the phenotype of To between these we reduced RhoA levels by As expected, all control embryos complete cytokinesis (Figure cyk-4R459A mutant embryos exhibit furrow furrows in these embryos less than of (Figure This supports in which CYK-4 GAP activity is involved in RhoA activation. Figure 4 with supplements see all Download asset Open asset The GAP activity of CYK-4 is required for centralspindlin-dependent independent of (A) of the known regulators of RhoA. (B) GAP defective CYK-4 cytokinesis defects that are by loss of NOP-1 this defect is not fully suppressed by mutation of analysis of the progression of cytokinesis in CYK-4R459A embryos in the or absence of CED-10/Rac1 NOP-1 The of furrow ingression in and ced-10(n1993) embryos are shown for Kymographs were as described in to Figure (C) The of furrow ingression in CYK-4R459A embryos. Results are quantified as described in Figure The of furrow ingression in no