C. elegans strains and genetics

The C. elegans N2 Bristol strain was used as the wild-type background and worms were cultured under standard conditions as described in (Brenner, 1974). Temperature sensitive strains (ts) were grown at the permissive temperature of 15°C and transferred to the restrictive condition of 25°C at the L4 stage. 24 hr post-L4 worms were then analyzed for the mutant phenotypes. The wild type worms used for comparisons with these mutants were all subjected to the same temperature shifts and examined at the same times as the mutants. The following mutations and chromosome rearrangements were used: LG II: ect-2(e1778)/dyp-10(e128) II, ect-2(ax751) II, unc-4(e120) ect-2(zh8) II/mIn1 [dpy-10(e128) mIs14], unc-4(e120) II, ect-2(gk44) II; unc-119(ed3) III; xnIs162 [ect-2::GFP + unc-119(+)], ect-2(ax751) II; unc-119(ed3) III; xnIs162 [ect-2::GFP + unc-119(+)], ect-2(zh8) II/mIn1 [dpy-10(e128) mIs14]; mpk-1(ga111) III, ect-2(ax751) II; let-60(ga89) IV; ect-2(ax751) II; cosa-1(me13) III; LG III: mpk-1(ga117)/dpy-17(e164) unc-79(e1068) III, mpk-1(ku1) unc-32(e189) III, unc-32(e189) III, mpk-1(ga111) III, mels9(unc-119(+) pie-1promoter::gfp::syp-3); unc-119(ed3) III; cosa-1(me13) mpk-1(ga111) III; cosa-1(me13)III; syp-2(rj16(S25A) IV; LG IV: lip-1(zh15) IV, let-60(ga89) IV; and LG V: syp-2(ok307) V/nT1 [unc-?(n754) let-?(m435)] (IV;V), wgIs227 [syp-2::TY1::EGFP::3xFLAG(92C12) + unc-119, syp-2(rj16(S25A) V, and SYP-2(rj17(S25D).

ect-2 was identified in a targeted RNAi screen on 168 germline-enriched genes designed to identify novel components regulating chromosome remodeling and short/long arm identity on bivalents. Specifically, we utilized a GFP-tagged AIR-2 containing line (ojIs50 (pie-1p::GFP::AIR-2 + unc-119(+)]) to screen for candidates that when depleted resulted in the mislocalization of the Aurora B kinase, AIR-2, which localizes to the short arms of the bivalents during late prophase I of meiosis to ensure accurate chromosome segregation. Criteria for the selection of these genes are detailed in (Colaiácovo et al., 2002) and were applied to germline-enriched genes identified by microarray analysis in (Reinke et al., 2004).

ect-2(ax751rf) mutants phenocopied the ect-2 (RNAi) phenotype at the non-permissive temperature with AIR-2 failing to load onto the chromosomes (Figure 1A). However, unlike ect-2(RNAi), where 100% of animals (N=44/44) showed failure in AIR-2 loading on the chromosomes, only 52% of the ect-2(ax751rf) mutants (N=13/25) exhibited this defect, which could be due to the fact that ect-2(ax751) is not a null mutant.

Since the ect-2(zh8gf) and the mpk-1(ku1) mutants are in the unc-4 and unc-32 mutant backgrounds, respectively, we analyzed SC dynamics in unc-4 and unc-32 mutants. We did not find any defects in either SC assembly or disassembly in unc-4 and unc-32 mutants (Figure 2—figure supplement 2). Analysis of mpk-1(ga117) null mutants revealed defects in SC assembly, indicated by the formation of polycomplexes at 20°C (15%, n=20, where n is the number of gonads scored) and 25°C (66%, n=16).