(A) Schematic representation of the SYP-4 protein with the predicted Polo-like kinase phosphorylation site (S269) indicated. CC indicates the coiled-coil domains and aa indicates amino acid. (B) Low-magnification images of whole-mounted gonads depicting SYP-1 (green) and phosphorylated SYP-4 (pSYP-4; red) localization in wild type. pSYP-4 signal is observed starting at early-pachytene although SC assembly, as observed here by SYP-1 immunostaining, starts earlier at the leptotene/zygotene stage. PMT (premeiotic tip) and LZ (leptotene/zygotene). (C) High-magnification images of wild type germline nuclei at the indicated stages stained with DAPI (blue), anti-SYP-1 (green) and anti-pSYP-4 (red). 16 gonad arms were analyzed for wild type (B–C). Phosphorylated SYP-4 signal is absent at leptotene/zygotene, observed colocalizing with SYP-1 in mid-pachytene and acquiring a similar restricted localization as SYP-1 during the disassembly of the SC starting at late pachytene. (D) High-magnification images of mid-pachytene nuclei from wild type, plk-2(ok1936), plk-2(ok1936); plk-1(RNAi), and syp-4(S269A) mutants stained with DAPI (blue), anti-SYP-1 (green) and anti-pSYP-4 (red). Phosphorylated SYP-4 signal is absent in the syp-4(S269A) mutant indicating specificity of the phospho-specific antibody. Phosphorylated SYP-4 signal is reduced in mid-pachytene nuclei in plk-2 mutants and absent at that stage in plk-2; plk-1(RNAi) germlines. Note that an uneven SYP-1 signal intensity is observed along chromosomes in plk-2; plk-1(RNAi) germlines, but pSYP-4 signal is not detected even on chromosomes with strong SYP-1 signal. 22, 17, 16, and 18 gonad arms were analyzed for wild type, plk-2(ok1936), plk-2(ok1936); plk-1(RNAi), and syp-4(S269A) mutants, respectively. Scale bar, 20 μm for (B) and 3 μm for (C) and (D).