CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis

Abstract

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.

Figure 1

cyk-4(t1689ts) mutant embryos fail to complete cytokinesis. Wild-type embryos (A) and embryos from cyk-4 mutant hermaphrodites (B) were dissected from young adults, mounted on agarose pads, and observed by time-lapse Nomarski microscopy. The ingressing cleavage furrow is indicated (arrows). Bar, 10 μm.

Figure 2

cyk-4 mutant embryos produce ingressing cleavage furrows, but do not form a prominent central spindle. Wild-type (A and C) and cyk-4 mutant embryos (B and D) were fixed and stained for actin (green) and DNA (blue) (A and B), and tubulin (green) and DNA (blue) (C and D). Bar, 10 μm.

Figure 3

Positional cloning of the cyk-4 locus. (A) A schematic of LGIII showing the positions of various loci and the extent of the deficiencies. cyk-4 is uncovered by tDf10, tDf6, and ctDf2, but not ctDf3. (B) An enlargement of the physical map from the unc-64 locus until the end of LGIII. The ability of various cosmid pools to rescue cyk-4 is indicated. (C) A map of the predicted genes on K08E3. All the predicted genes except for K08E3.1 and K08E3.5 were inactivated by RNAi and only RNAi of K08E3.6 produced multinucleate embryos. (D) A schematic representation of the domain structure of CYK-4 and its human orthologue. The position of the point mutation identified in cyk-4(t1689ts) is indicated.

Figure 5

CYK-4 enhances GTP hydrolysis by Rho, Rac, and Cdc42. The GTPases were preloaded with α-[³²P]GTP, and then GST-CYK-4-GAP or GST was added at the indicated concentration. Samples were taken at 2-min intervals, the labeled nucleotide was resolved by thin layer chromatography, and the fraction of GTP and GDP was quantitated. This graph shows the fraction of GTP hydrolyzed at the 2-min time point as a function of CYK-4 concentration. These data are representative of at least three independent experiments.

Figure 4

cyk-4(RNAi) causes disorganization of the proximal gonad and affects formation of the central spindle. Young adults were injected with cyk-4 dsRNA, and the injected animals were analyzed 30 h after injection (B). The gonad of an uninjected worm is shown for comparison (A). Wild-type embryos (C) and embryos from cyk-4(RNAi)–injected animals (D) were fixed and stained for tubulin. Bars, 10 μm.

Figure 6

rhoA(RNAi) causes cytokinesis defects and cdc42 (RNAi) causes defects in spindle positioning. Young adults were injected with the indicated dsRNAs (Table ), and the embryos produced by the injected worms were analyzed by time-lapse Nomarski microscopy. Sequential images from a (A) wild-type, (B) rhoA(RNAi), and (C) a cdc42(RNAi) embryo are shown. Bar, 10 μm.

Figure 7

CYK-4 localizes to the central spindle and division remnants. (A–F) Wild-type embryos were fixed and stained for CYK-4 (green), tubulin (red), and DNA (blue). The localization of CYK-4 to the central spindle (arrow) in a single cell (D) and a two cell embryo (F) is shown. The localization of CYK-4 to the division remnant from the polar body (A), and between the AB and P1 blastomeres is indicated with arrows (E). (G) An embryo from a line expressing CYK-4:GFP is stained with anti-GFP antibodies. The same structures are seen as with CYK-4 antibodies (arrow). (H) The intrinsic fluorescence of the gonad of a worm expressing CYK-4:GFP. CYK-4 is seen at the incomplete membranes of the syncytial gonad (arrow) and in oocyte nuclei. Bars, 10 μm.

Figure 8

Time-lapse analysis of CYK-4:GFP. An embryo from a line expressing CYK-4:GFP was imaged using low light level microscopy. The central spindle localization of CYK-4 is observed before furrow ingression. Bar, 10 μm.

Figure 9

CYK-4 and ZEN-4/CeMKLP1 colocalize and are interdependent for their localization. An embryo expressing CYK-4:GFP stained for GFP (A), ZEN-4/CeMKLP1 (B), and the merged image (C). CYK-4 and ZEN-4/CeMKLP1 colocalize at division remnants (arrow) and central spindle (arrowheads) structures. ZEN-4 localization to the central spindle is CYK-4–dependent (arrow). Wild-type (D) and cyk-4(t1689ts) embryos (E) were fixed and stained for ZEN-4/CeMKLP1 (green), tubulin (red), and DNA (blue). AIR-2 localization to the central spindle is CYK-4–independent (arrow). Wild-type (F) and cyk-4(t1689ts) embryos (G) were fixed and stained for AIR-2 (green), tubulin (red), and DNA (blue). CYK-4 maintenance to division remnants (arrowheads) is ZEN-4/CeMKLP1–dependent. zen-4(or153ts) worms were maintained at 16°C and either fixed immediately (H) or shifted to 25°C for 18 min (I). Embryos were fixed and stained for CYK-4 (green), tubulin (red), and DNA (blue). Bars, 10 μm.

Figure 10

Model for the function of CYK-4 in central spindle formation and cytokinesis. See text for details.