Substrate recognition by SMK-1, but not its interaction with HCP-4 CENP-C , is required for timely congression and error-free segregation of chromosomes. (A) Schematic of SMK-1 and sequence alignment in the highly conserved EVH1 domain. The tyrosine and tryptophan residues required for FxxP motif binding are highlighted. (B) Schematic of HCP-4CENP-C showing the location of FxxP motifs and overview of HCP-4 and SMK-1 mutations evaluated in pull-down assays. Mutating FxxP motifs to AxxA prevents their interaction with the EVH1 domain, as indicated in the cartoon. (C) Coomassie Blue-stained protein gels showing purified recombinant proteins (Input) and proteins eluted from glutathione agarose resin after GST pull-down. Molecular weight of size markers (M) is indicated in kilodaltons (kD). (D) Selected images from time-lapse movies of one-cell embryos co-expressing GFP::histone H2B (HIS-58) and GFP::γ-tubulin (TBG-1). Time is relative to NEBD. Numbers refer to transgene-encoded wild-type and mutant HCP-4, as described in B. Scale bar, 5 µm. (E) Chromosome span (mean of n embryos ± SEM) versus time relative to NEBD. Measurements were performed in time-lapse movies such as those shown in D. (F) Selected images from time-lapse movies of one-cell embryos co-expressing endogenously tagged wild-type or mutant SMK-1::GFP together with transgenic mCherry::histone H2B (HIS-58). The number of anaphases with chromatin (chr) bridges relative to the total number of anaphases examined is indicated. Scale bar, 5 µm. (G) Intensity of the chromosomal SMK-1::GFP signal just prior to NEBD in the one-cell embryo (mean ± 95% CI), normalized to the mean of wild-type SMK-1::GFP, measured in embryos such as those shown in F. Statistical significance was determined by the Mann-Whitney test. **P < 0.01. (H) Chromosome span (mean of n embryos ± SEM) versus time relative to NEBD. Measurements were performed in time-lapse movies such as those shown in F. Source data are available for this figure: SourceData F4.
