Based on the X-ray structure of the human CYK-4 homolog (PDBID: 2OVJ), the Glu to Lys substitution would lead to a charge reversal, disrupting the network of salt-bridge and hydrogen-bond interactions that positions the arginine-finger (Arg 459), a conserved structural motif essential for GAP activity ( Fig. The two mutations result in a Glu to Lys change at residue 448 (CYK-4 GAP(E448K)) and a Thr to Ile change at residue 546 (CYK-4 GAP(T546I)) respectively ( Fig. Both were independently isolated using a forward genetics approach to identify conditional, embryonic-lethal alleles ( 20). elegans mutants containing amino acid substitutions within the CYK-4 GAP domain ( Fig. To study the role of the GAP domain in cytokinesis ( 19), we characterized two C. Supporting this hypothesis, haplo-insufficiency of Rac can suppress the rough-eye phenotype induced by RNAi of the Drosophila CYK-4 homolog (although cytokinesis was not examined) ( 14), suggesting that in some contexts CYK-4 may oppose Rac activation. However, it is also possible that RhoA is not the critical target of the CYK-4 GAP domain. However, since RhoA is the only family member essential for cytokinesis, it is assumed that Centralspindlin GAP activity acts on RhoA by either promoting RhoA cycling ( 18), or by inactivating RhoA after cytokinesis to promote contractile ring disassembly ( 1). In vitro, the GAP domains of CYK-4 and its homologs are active towards all three subclasses of Rho family GTPases: RhoA, Rac, and CDC-42 ( 11, 16, 17). The role of this domain in cytokinesis and the identity of the Rho family GTPase that it targets are unknown ( 11, 13– 15). The CYK-4 C-terminus contains a conserved GAP domain, predicted to inactivate Rho family GTPases ( Fig. D) Time-lapse montage of the furrow region in embryos expressing a GFP::plasma membrane marker. C) Plot of mean furrow diameter versus time. B) The E448K and T546I substitutions destabilize CYK-4 GAP domain structure. 1)-failure to form a central spindle accompanied by a defect in contractile ring constriction ( 9– 12).ĬYK-4 GAP domain mutants phenocopy Centralspindlin loss-of functionĪ) Residues changed in CYK-4 and ZEN-4 mutant proteins. Mutant forms of CYK-4 and ZEN-4 that disrupt Centralspindlin assembly lead to a cytokinesis phenotype similar to that resulting from depletion of Centralspindlin subunits by RNAi ( Fig. Formation of the hetero-tetrameric Centralspindlin complex requires an interaction between the central region of ZEN-4 and the N-terminal region of CYK-4 ( Fig. The current dominant model proposes that Centralspindlin contributes directly to the equatorial activation of RhoA by targeting ECT-2 to the central spindle ( 3, 4, 7). elegans), is critical for central spindle assembly and contractile ring constriction ( 1, 6). elegans) and two molecules of a protein containing a GAP domain that targets Rho family GTPases (CYK-4 in C. Centralspindlin, a conserved heterotetrameric complex consisting of two molecules of kinesin-6 (ZEN-4 in C. RhoA signaling is controlled by the central spindle- an array of anti-parallel microtubule bundles that forms between the separating chromosomes during anaphase ( 1). Consistent with a central role in cytokinesis, RhoA and its activating GEF, ECT-2, are essential for contractile ring assembly and constriction ( 3– 5). Anaphase spindle signaling is mediated by activation of the small GTPase RhoA ( 1, 2). Understanding how the anaphase spindle communicates with the cell cortex during cytokinesis is a major current challenge. To coordinate cell division with chromosome segregation, the anaphase spindle directs the assembly and constriction of a contractile ring composed of filamentous-actin and myosin II that physically divides the cell ( 1). Cytokinesis completes mitosis, partitioning a single cell into two.
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