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Inhibition of Rac by the GAP Activity of Centralspindlin Is Essential for Cytokinesis

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Science  05 Dec 2008:
Vol. 322, Issue 5907, pp. 1543-1546
DOI: 10.1126/science.1163086

Abstract

During cytokinesis, the guanosine triphosphatase (GTPase) RhoA orchestrates contractile ring assembly and constriction. RhoA signaling is controlled by the central spindle, a set of microtubule bundles that forms between the separating chromosomes. Centralspindlin, a protein complex consisting of the kinesin-6 ZEN-4 and the Rho family GTPase activating protein (GAP) CYK-4, is required for central spindle assembly and cytokinesis in Caenorhabditis elegans. However, the importance of the CYK-4 GAP activity and whether it regulates RhoA remain unclear. We found that two separation-of-function mutations in the GAP domain of CYK-4 lead to cytokinesis defects that mimic centralspindlin loss of function. These defects could be rescued by depletion of the GTPase Rac or its effectors, but not by depletion of RhoA. Thus, inactivation of Rac by centralspindlin functions in parallel with RhoA activation to drive contractile ring constriction during cytokinesis.

Cytokinesis completes mitosis, partitioning a single cell into two. To coordinate cell division with chromosome segregation, the mitotic apparatus directs the assembly and constriction of a contractile ring composed of filamentous actin and myosin II that physically divides the cell (1). Understanding how the mitotic apparatus communicates with the cell cortex during cytokinesis is a major challenge. One critical mediator of this signaling is the small guanosine triphosphatase (GTPase) RhoA (1, 2). RhoA signaling is thought to be controlled by an array of antiparallel microtubule bundles, called the central spindle, that forms between the separating chromosomes during anaphase (1). Consistent with a central role in cytokinesis, RhoA and its activating guanine nucleotide exchange factor (GEF) ECT-2 are essential for contractile ring assembly and constriction (35).

Centralspindlin, a conserved heterotetrameric complex consisting of two molecules of kinesin-6(ZEN-4 in C. elegans) and two molecules of a protein containing a GTPase activating protein (GAP) domain (CYK-4 in C. elegans), is critical for central spindle assembly and cytokinesis (1, 6). The current dominant model proposes that centralspindlin targets the ECT-2 GEF to the central spindle, thereby contributing to the equatorial activation of RhoA (3, 4, 7). Formation of the heterotetrameric centralspindlin complex requires an interaction between the N-terminal region of CYK-4 and the central region of ZEN-4 (Fig. 1A) (6, 8). Mutant forms of CYK-4 and ZEN-4 that disrupt centralspindlin assembly lead to a phenotype similar to that resulting from depletion of centralspindlin subunits by RNA interference (RNAi) (Fig. 1, C and D): failure to form a central spindle, accompanied by a defect in contractile ring constriction (912).

Fig. 1.

CYK-4 GAP domain mutants phenocopy centralspindlin loss of function. (A) Residues changed in CYK-4 and ZEN-4 mutant proteins. (B) The E448K and T546I substitutions destabilize CYK-4 GAP domain structure. (C) Plot of mean furrow diameter versus time. Error bars denote SEM. (D) Time-lapse montage of the furrow region in embryos expressing a GFP::plasma membrane marker. Series begin at anaphase onset. Scale bar, 20 μm. Amino acid abbreviations: D, Asp; E, Glu; H, His; K, Lys; I, Ile; N, Asn; S, Ser; T, Thr; Y, Tyr.

The conserved GAP domain in the CYK-4 C terminus is predicted to inactivate Rho family GTPases (Fig. 1A) (11). However, the role of the CYK-4 GAP domain in cytokinesis and the identity of the Rho family GTPase that it targets are unknown (11, 1315). In vitro, the GAP domains of CYK-4 and its homologs are active toward all three subclasses of Rho family GTPases: RhoA, Rac, and CDC-42 (11, 16, 17). It has been assumed that the CYK-4 GAP activity acts on RhoA because it is the only Rho family member essential for cytokinesis (11). CYK-4 has been proposed to promote RhoA cycling (18) or to inactivate RhoA after cytokinesis during contractile ring disassembly (1). It is also possible that RhoA is not the critical target of the CYK-4 GAP domain. In support of this hypothesis, haplo-insufficiency of Rac can suppress the rough-eye phenotype induced by RNAi of the Drosophila CYK-4 homolog (14). Although cytokinesis was not examined, this result indicates that in some contexts CYK-4 may oppose Rac activation.

To study the role of the CYK-4 GAP domain in cytokinesis (19), we characterized two conditional C. elegans alleles (20) that lead to residue substitutions within the GAP domain—a Glu → Lys change at residue 448 (CYK-4GAP(E448K)) and a Thr → Ile change at residue 546 (CYK-4GAP(T546I)), respectively (Fig. 1, A and B). On the basis of the x-ray structure of the human CYK-4 homolog (PDB ID: 2OVJ), the charge reversal resulting from the Glu → Lys substitution would disrupt a network of salt-bridge and hydrogen-bond interactions that positions the arginine finger (Arg459), a conserved residue essential for GAP activity (Fig. 1B) (17, 21, 22). Although the effects of the Thr → Ile substitution are less clear, the larger isoleucine side chain may clash with surrounding residues and also interfere with arginine finger positioning. Both alleles are strictly recessive (fig. S1), temperature-sensitive, and fast-inactivating, showing a fully penetrant cytokinesis defect within 1 min of shifting to the restrictive temperature. Thus, these are loss-of-function alleles affecting the CYK-4 GAP domain.

We used quantitative live-imaging assays to characterize cytokinesis in the CYK-4 GAP mutant embryos. Although no obvious defects were evident during the initial stages of contractile ring assembly (fig. S2, B and C), the GAP mutants exhibited a severe defect in contractile ring constriction. The constriction rate was slower than in control embryos by a factor of ∼3, and cleavage furrows only ingressed to ∼50% of the initial cell diameter before regressing. This ingression defect mimicked that in the centralspindlin-assembly mutants and in embryos depleted of CYK-4 by RNAi (12) (Fig. 1, C and D, fig. S3, and movie S1). Thus, the CYK-4 GAP domain is fundamental to the role of centralspindlin in promoting contractile ring constriction.

Because centralspindlin is also required for assembly of the central spindle (911), we next examined central spindle structure in the GAP mutant embryos. In contrast to the absence of a central spindle after depletion of the kinesin-6 ZEN-4, there were no clear defects in central spindle organization or ZEN-4 targeting in the GAP mutants (Fig. 2A). The recruitment of a green fluorescent protein (GFP) fusion with AuroraBAIR-2 kinase (the enzymatic component of the chromosomal passenger complex) to the central spindle was also not affected in the GAP mutants (Fig. 2B and movie S2). Disrupting central spindle structure in C. elegans embryos leads to abrupt centrosome separation after anaphase onset (23). This phenotype was not observed in the CYK-4 GAP mutants, thereby confirming a mechanically intact central spindle (Fig. 2C). Thus, CYK-4GAP(E448K) and CYK-4GAP(T546I) are separation-of-function mutants that uncouple the role of CYK-4 in contractile ring constriction from its role in central spindle assembly.

Fig. 2.

Central spindle assembly is not disrupted in the GAP mutants. (A) Immunofluorescence staining for ZEN-4, tubulin, and DNA. (B) Images of GFP::AuroraBAIR-2 40 s after anaphase onset. (C) Plot of mean centrosome separation versus time. Error bars denote SEM. Scale bars, 10 μm.

CYK-4 GAP activity is expected to down-regulate a Rho family GTPase; therefore, RNAi-mediated depletion of the target GTPase should rescue the cytokinesis defect in CYK-4 GAP mutant embryos. There are five Rho family members in C. elegans: RhoARHO-1, RacCED-10, RacRAC-2, RhoGMIG-2, and CDC-42 (24). Because RhoARHO-1 depletion results in a cytokinesis defect on its own (11, 16, 17), we tested whether partial depletion of RhoARHO-1 or its activating ECT-2 GEF could suppress the GAP mutant phenotype. Instead of suppressing, partial depletion of either RhoARHO-1 or ECT-2 enhanced the CYK-4GAP(E448K) cytokinesis defect, which suggests that RhoARHO-1 is not the main target of CYK-4 GAP activity (Fig. 3, A and B, and fig. S4A).

Fig. 3.

Rac depletion suppresses the cytokinesis defect in CYK-4GAP(E448K) embryos, as shown by time-lapse montages of the furrow region in embryos expressing a GFP::plasma membrane marker. Series begin at anaphase onset. (A) CYK-4GAP(E448K) embryos exhibit partial furrow ingression followed by regression. (B) Partial depletion of RhoARHO-1 enhances the CYK-4GAP(E448K) cytokinesis defect. (C) RNAi of other Rho family members does not disrupt cytokinesis. (D) RNAi of RacCED-10 or RacRAC-2 rescues cytokinesis success in CYK-4GAP(E448K) embryos. Scale bar, 20 μm.

Unlike depletion of RhoARHO-1, RNAi of the other Rho family members does not affect cytokinesis in control embryos (Fig. 3C). RNAi of CDC-42 or RhoGMIG-2 also did not ameliorate the CYK-4GAP(E448K) cytokinesis phenotype (Fig. 3D). However, RNAi of RacCED-10 or RacRAC-2 led to substantial rescue, allowing 70% and 24%, respectively, of CYK-4GAP(E448K) embryos to successfully complete the first cytokinesis (Fig. 3, A and D). Simultaneous RNAi of RacCED-10 and RacRAC-2 did not increase the efficiency of rescue over RNAi of RacCED-10 alone (fig. S4D). These results indicate that CYK-4 GAP activity promotes furrow ingression by down-regulating Rac. Consistent with a role for CYK-4 in Rac inactivation, the overexpression of a GAP-dead CYK-4 mutant in dividing mammalian cells was previously found to increase the level of active Rac at the cell equator (25).

If CYK-4 GAP activity is critical for the role of centralspindlin in contractile ring constriction, RacCED-10 depletion should also rescue the furrow ingression defect resulting from inhibiting centralspindlin assembly. Indeed, 85% of furrows in ZEN-4CSA(D520N) embryos ingressed fully when RacCED-10 was depleted (fig. S4B). However, RacCED-10 depletion was less efficient in rescuing cytokinesis completion in the ZEN-4CSA(D520N) mutant relative to the CYK-4 GAP mutant (fig. S4B). This result suggests an additional role for either centralspindlin or the central spindle in the completion of cytokinesis that is independent of CYK-4 GAP activity. RacCED-10 depletion had no effect on furrow ingression in AuroraBAIR-2(P265L) embryos, which have a spectrum of defects in central spindle assembly and furrow ingression similar to that resulting from centralspindlin disruption (12) (fig. S4C); this result confirmed the specificity of the rescue.

Why is inhibition of Rac signaling important for contractile ring constriction? Rac promotes activation of the Arp2/3 complex via its effectors WASpWSP-1 and WAVEWVE-1, resulting in the formation of a branched meshwork of short cross-linked actin filaments (26). Possibly, these Rac effectors interfere with actin filaments nucleated by the cytokinesis formin, CYK-1, which functions with myosin II to drive cytokinesis. To test this idea, we determined whether depletion of WASpWSP-1/WAVEWVE-1 or the Arp2/3 complex could also rescue the CYK-4GAP(E448K) cytokinesis defect (27, 28) (Fig. 4, A and B). Although their individual depletions did not markedly suppress the CYK-4GAP(E448K) cytokinesis defect, co-depletion of WASpWSP-1 and WAVEWVE-1 led to substantial rescue: 69% of furrows ingressed fully and 38% of embryos successfully completed cytokinesis (Fig. 4A). RNAi of Arp2ARX-2 also suppressed the CYK-4GAP(E448K) cytokinesis defect, with 74% of furrows ingressing fully and 52% of embryos completing division (Fig. 4B). Thus, cytokinesis depends on CYK-4 GAP to inactivate Rac, thereby reducing activation of the Arp2/3 complex via WASp and WAVE. Active Arp2/3 complex in the furrow region may disrupt contractile ring constriction by branching formin-nucleated actin filaments. Alternatively, active Arp2/3 complex could nucleate the formation of an independent branched actin network that competes for essential contractile ring components or presents a structural barrier to furrow ingression.

Fig. 4.

CYK-4 GAP inactivates Rac and its effectors, WASp/WAVE and the Arp2/3 complex, to promote cytokinesis. Cytokinesis in CYK-4GAP(E448K) embryos is rescued by (A) co-depletion of WASpWSP-1 and WAVEWVE-1 or (B) depletion of Arp2ARX-2. Scale bar, 20 μm.

We have uncovered a negative regulation cascade that is essential for successful cytokinesis. Although negative regulation has been proposed to be important during cytokinesis, previous models have emphasized inhibition of cortical contractility by astral microtubules that contact the polar regions of the cell (29, 30). A requirement for negative regulation of an inhibitory pathway at the cell equator has not been widely considered. Our findings lead to a model in which inactivation of Rac by CYK-4 GAP functions in parallel with activation of RhoA to drive contractile ring constriction during cytokinesis (fig. S7).

Supporting Online Material

www.sciencemag.org/cgi/content/full/322/5907/1543/DC1

Materials and Methods

Figs. S1 to S7

Movies S1 and S2

References

References and Notes

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