Cooperative assembly of CYK-4/MgcRacGAP and ZEN-4/MKLP1 to form the centralspindlin complex

Abstract

Cytokinesis in metazoan cells requires a set of antiparallel microtubules that become bundled upon anaphase onset to form a structure known as the central spindle. Bundling of these microtubules requires a protein complex, centralspindlin, that consists of the CYK-4/MgcRacGAP Rho-family GTPase-activating protein and the ZEN-4/MKLP1 kinesin-6 motor protein. Centralspindlin, but not its individual subunits, is sufficient to bundle microtubules in vitro. Here, we present a biochemical and genetic dissection of centralspindlin. We show that each of the two subunits of centralspindlin dimerize via a parallel coiled coil. The two homodimers assemble into a high-affinity heterotetrameric complex by virtue of two low-affinity interactions. Conditional mutations in the regions that mediate complex assembly can be readily suppressed by numerous second site mutations in the interacting regions. This unexpected plasticity explains the lack of primary sequence conservation of the regions critical for this essential protein-protein interaction.

Figure 1.

ZEN-4 D520N impairs microtubule bundling in vitro and is responsible for the in vivo phenotype of zen-4(or153ts). (A) zen-4(or153ts) D520N fails to rescue zen-4(w35) at 25°C. Simple transgenic arrays marked by rol-6(su1006) were prepared and the efficiency of zen-4 D520N and D735N versus wt ZEN-4 to rescue zen-4(w35) was assayed. The rescue efficiency was scored by counting the blistered [bli-6(sc16)] animals, both at the restrictive (25°C; gray) and the permissive temperature (16°C; black) as described previously (Mishima et al., 2004). At 25°C ZEN-4 D520N could not rescue the null, whereas both wt and ZEN-4 D735N efficiently rescued the null. (B) The D520N point mutation in zen-4(or153ts) inhibits MT bundling. MT bundling assays were performed with constituents of centralspindlin alone, and with reconstituted centralspindlin complex, comparing wt and mutant complexes. The quantification of results was performed by the analysis of 10 random fields, followed by integrating pixel intensities above a fixed threshold. (C) Schematic representation of ZEN-4 (light gray) and CYK-4 (dark gray) proteins. The CYK-4/ZEN-4 interaction domains are indicated with a dotted line above the illustrations. The plots represent the predictions of the coiled coils of ZEN-4 and CYK-4, by COILS (Lupas et al., 1991) and PAIRCOILS (Berger et al., 1995).

Figure 2.

The primary defect of zen-4(or153ts) is due to the failure of ZEN-4 D520N to form a stable complex with CYK-4. (A) The ability of ZEN-4 D520N to form a dimer was tested by size exclusion chromatography. ZEN-4(1-603)D520N eluted in the same fraction as wt ZEN-4(1-603), thus ZEN-4 D520N is a dimer. (B) CYK-4(1-120) and ΔCYK-4(34-120) fragments were expressed as ³⁵S-labeled proteins by in vitro translation. Also shown (far right lanes) are parallel reactions of ZEN-4 translation reactions with [³⁵S]methionine, as controls for the efficiency of translation. Unlabeled ZEN-4(434-775) wt and D520N fragments, and the negative control ΔZEN-4(475-775), were added and incubated for 1 h. ZEN-4 was subsequently immunoprecipitated with an anti-ZEN-4 antibody. Whereas wt ZEN-4 can bind wt CYK-4, ZEN-4 D520N cannot. Therefore, ZEN-4 D520N is defective in CYK-4 binding, yet is a dimer (A). (C) The affinity and the rates of interaction between ZEN-4 and CYK-4 were measured through SPR. Sensorgrams of triplicate runs representing 100 nM ZEN-4 injected are shown. The affinity of ZEN-4 D520N for CYK-4 is reduced by 60-fold from wt ZEN-4/CYK-4 affinity at 25°C. However, at 16°C the mutant's affinity for CYK-4 improves.

Figure 3.

Isolation of zen-4(or153ts) suppressors. (A) Schematic view of the zen-4(or153ts) suppressor screen. Homozygous zen-4(or153ts) animals were mutagenized at 16°C. After recovery, the F1 embryos were isolated by bleaching and allowed to self-fertilize at permissive temperature (16°C). When F2 animals reached adulthood, the population was shifted to 25°C and fertile animals were selected. Star depicts a suppressor mutation. (B) Approximately 500,000 F1 genomes were screened and 14 suppressor mutations (point mutations indicated in green) were isolated, all of which were viable at 25°C. Some mutations were isolated multiple times. The D520N mutation of zen-4(or153ts) is indicated in red. (C) CYK-4 fragments purified as GST fusions, and retained on the glutathione Sepharose beads, were used in a standard pull-down assay. Recombinant wild type or D520N ZEN-4(1-585) were added to the beads in a 100-μl reaction, and incubated for 2 h at 25°C. The beads were subsequently washed and the bound fraction was analyzed by SDS-PAGE and Coomassie Blue staining. CYK-4 fragments containing the suppressor substitutions (G12S and G12D) bound to both ZEN-4 D520N and wild-type ZEN-4, whereas wild-type CYK-4 could only bind to wild-type ZEN-4. The negative control, GST, did not bind either form of ZEN-4.

Figure 4.

CYK-4 and ZEN-4 form parallel coiled coil dimers, yet dimerization is not strictly required for their interaction. (A) Purified fragments of ZEN-4(1-603) and (1-539) were subjected to size exclusion chromatography (refer to Figure 2A, because these samples were analyzed in the same experiment). ZEN-4(1-539) eluted in a much earlier fraction than ZEN-4(1-603), thus it behaves as a monomer. (B) Summary of size exclusion chromatography with a panel of ZEN-4 deletion derivatives. The peak of the elution fractions of ZEN-4 derivatives are plotted versus log (MW) and compared with standards. (C) CBD ZEN-4 fragments (1-539), (1-555), and (1-603) and GST CYK-4(1-120) were coexpressed from a bicistronic vector. GST-CYK-4 was purified on glutathione Sepharose beads and eluted with reduced glutathione, followed by incubation of the eluates with chitin beads and elution with TEV protease. Both monomeric ZEN-4(1-539) and dimeric ZEN-4(1-555 or 1-603) copurified with CYK-4. (D) Recombinant ZEN-4 fragments containing a cysteine residue at position 547 were incubated under oxidizing conditions. The reaction was quenched and the ability of ZEN-4 to form disulfide bridges was analyzed. ZEN-4(603) as well as ZEN-4(1-555) efficiently formed dimers, whereas ZEN-4(1-539) did not. Thus, ZEN-4 forms a parallel coiled coil dimer. (E) CYK-4 preparations (36-119 with an N-terminal cysteine shown here), were incubated under oxidizing conditions. The reaction was quenched and the ability of CYK-4 to form disulfide bridges was analyzed. CYK-4 also forms a parallel coiled coil dimer.

Figure 5.

Coiled coils in CYK-4 and ZEN-4 play a stabilizing role in centralspindlin complex formation. (A) Binding of monomeric ZEN-4(1-539) to dimeric CYK-4(1-120) was measured using ITC. Measurements were performed at 25°C using the working buffer as a reference. The protein concentration was 10 μM, and the ligand concentration (ZEN-4) was 100 μM. Using a simple one-site model, the affinity of monomeric ZEN-4 for dimeric CYK-4 was calculated at 800 nM. (B) CYK-4 fragments purified as MBP fusions and retained on the amylose beads were used in a standard pull-down assay. Recombinant ZEN-4(1-585) GFP was added to the beads in a 100-μl reaction, and it was incubated at room temperature for 2 h. The beads were subsequently washed, and the bound fraction was analyzed by SDS-PAGE and Coomassie Blue staining. CYK-4 containing a coiled coil, able to form dimers, could bind ZEN-4, whereas CYK-4 lacking a coiled coil could not, nor could the negative control, MBP. S, supernatant; P, pellet. (C) CYK-4 fragments purified as GST fusions and retained on the glutathione Sepharose beads were used in a standard pull-down assay. Recombinant ZEN-4(1-585), ZEN-4(1-539) GCN4, and ZEN-4(1-539) were added to the beads in a 100-μl reaction, and they were incubated at room temperature for 2 h. The beads were subsequently washed, and the bound fraction was analyzed by SDS-PAGE and Coomassie Blue staining. ZEN-4 containing a coiled coil, either native or engineered, could bind CYK-4, whereas ZEN-4 lacking a coiled coil could not. The negative control, GST, did not bind any of the ZEN-4 fragments. (D) Comparison of binding affinities between monomeric and dimeric ZEN-4/CYK-4 constructs. Symbols indicate the method by which the binding constant was determined: ♣, ITC; ♦, pull-down assay; ♥, pull-down assay with fluorescence intensity measured; ♠, SPR; and 1, approximate K_D.

Figure 6.

Centralspindlin: functional insights from structural aspects of the complex. (A) Sequence comparison of the CYK-4/ZEN-4 interaction domains among vertebrates and invertebrates. The original temperature-sensitive mutations are indicated by red dots; suppressors of zen-4(or153ts) are indicated by green dots; and suppressors of cyk-4(t1689ts) are indicated by black dots (Mishima et al., 2002). The accession numbers for the aligned sequences are as follows: ZEN-4/MKLP1[C_elegans (NP_741411.1); C_briggsae (emb CAE61601.1); H_sapiens (AAH51826.1); Danio_rerio (AAF00594.1); Strong_purp (AAG18582.1); Dros_melano (CAA12181.1); Mus_musc (NP_077207); Canis_famil (XP_535528.2); Chinese_hamster (CAA58558.1); Xen_trop (NP_001011104.1); Gallus_gallus (NP_001025725.1); Anopheles_gamb (XP_308170.2); Apis_mellifera (XP_624886.1); Xen_laevis (AAH84928.1); Tetraodon_nigriv (CAF99883.1); Rattus_norv (XP_236313.3); Bos_taurus (XP_593824.2)]; CYK-4/MgcRacGAP [C_Briggsae (CAE71360.1); D_melanog (CAB96203.1); C_elegans (NP_499845.1); M_mus (NP_036155.1); H_sapiens (NP_037409.2); Rattus_nov (XP_235650.2); Canis_famil (XP_543675.2); Bos_taurus (XP_592496.2); Xen_laevis (AAH70771.1); Xen_trop (NP_001001236.1); Tetraodon_nigrov (CAG03964.1); Gallus_gallus (XP_424490.1); Stron_purp (XP_783360.1); Danio_rerio (AAH95799.1); Pan_trog (XP_509531.1); Apis_mellifera (XP_393627.2); Anopholes_gamb (XP_319660.2)]. (B) Schematic representation of ZEN-4 (a) and the centralspindlin complex (b). Model of possible mode of action of the complex, and the structural effects that CYK-4 may have on ZEN-4 (c).