Figure 4.

Dynamic interaction between SUN5 and DNAJB13 during spermiogenesis. A, FLAG–SUN5 partially recruited MYC–DNAJB13 to the nuclear envelope when overexpressed in HeLa cells. FLAG–SUN5 localized to the nuclear envelope (red, upper panel). MYC–DNAJB13 randomly distributed itself throughout the cell when overexpressed in HeLa cells (green, middle panel). When FLAG–SUN5 and MYC–DNAJB13 were co-transfected in HeLa cells, MYC–DNAJB13 was partially recruited to the nuclear envelope (bottom panel). DNA in the cells was stained with DAPI (blue). B, dynamic distribution of DNAJB13 in developing WT and Sun5-null spermatids was revealed by IF. In WT spermatids, DNAJB13 signals (green) were tightly attached to the implantation fossa during the maturation of the spermatid. The red arrow in the WT spermatids indicate the implantation fossa (upper two panels). In Sun5-null spermatids, although DNAJB13 (green) was enriched to the coupling apparatus, DNAJB13 signals were absent from the spermatid nucleus (lower two panels). The red segment in the Sun5-null spermatid indicates a gap between nucleus and DNAJB13. The spermatid nuclear DNA was stained with DAPI (blue). C, co-localization of SUN5 and DNAJB13 in WT and Sun5-null spermatids from testis. In WT spermatids, co-localization of SUN5 (green) and DNAJB13 (red) in the sperm head-to-tail coupling apparatus was observed (upper panel), but in Sun5-null late spermatids, DNAJB13 signal could be found only at the top of headless tails (lower panel). The spermatid nuclear DNA was stained with DAPI (blue). D, distribution of SUN5 and DNAJB13 in mature spermatozoa from epididymis. In WT mature spermatozoa, DNAJB13 (red) migrated to the flagella, and SUN5 (green) stayed in the HTCA (upper panel). In Sun5-null mature spermatozoa, DNAJB13 could be found only in the tip of the acephalic flagella (lower two panels). The sperm nuclear DNA was stained with DAPI (blue).