Figure 6. Condensin components colocalize with centromere components and are required for the restricted distribution of centromere proteins toward the spindle poles during mitosis. (A-F) Wild-type prometaphase (A-C) and metaphase (D-F) chromosomes stained with DNA dye (blue), and antibodies against SMC-4 (green) and centromere protein HCP-3 (red). Merged images (C,F) show that SMC-4 and HCP-3 colocalize. The magnified focus of staining from the metaphase plate shows overlap of the two proteins (F, inset). (G-I) Metaphase chromosome stained with DNA dye (blue), and antibodies to SMC-4 (green) and the kinetochore centrosome-associated protein CeMCAK (red). In the merged image and magnified inset (I), CeMCAK partially overlaps (yellow) SMC-4 and extends more poleward (red) than SMC-4 on metaphase chromosomes. Unlike SMC-4, CeMCAK stains the centrosomes (H, arrowheads). (J) Wild-type and (K)smc-4
(RNAi) two-cell embryos stained with tubulin antibody (blue), DNA dye (red), and HCP-3 antibody (green; yellow represents overlap between HCP-3 and DNA). HCP-3 associates with mitotic chromosomes when SMC-4 is depleted, but is not present in a restricted pattern facing the spindle poles. (L) Wild-type and (M) hcp-3
(RNAi) one-cell embryos stained with tubulin antibody (blue), DNA dye (red), and SMC-4 antibody (green; yellow represents overlap between SMC-4 and DNA). When HCP-3 is depleted, SMC-4 associates with DNA but in a disorganized pattern.