LINC in Sperm Head Formation Figure 4. Appearance of two novel spermiogenesis-specific LINC complexes correlates with sperm head formation. (A, B) Distribution of Nesprins1 and 3 in spermatids. (A) Testis paraffin sections were co-stained using pAb against Nesprin3 together with affinity-purified anti-Sun1 antiserum. (B) Co-staining of testis paraffin sections using affinity-purified anti-Sun3 antiserum and pAb against Nesprin1 (b) or pAb against Nesprin3 (e). DNA was labeled with 33258- Hoechst. Overlays are seen in A, c, f and in B, c, f. Arrowheads denote the non-nuclear acrosome-associated Sun1/Nesprin3 localization. Ar- rows indicate the fossa region. Scale bars: 5 mm. (C) Schematic illustration of recombinant constructs used for co-transfection/immu- noprecipitation experiments. (D) HeLa cells were transiently co- transfected with Myc-Sun1 and EGFP-Nesprin3 (top), Myc-Sun3 and EGFP-Nesprin1 (middle) or Myc-Sun3 and EGFP-Nesprin3 (bottom). Cells lysed in RIPA buffer were co-immunoprecipitated with anti-myc mAbs and immunoblotted with either anti-EGFP or anti-myc mAbs. For control, unspecific mouse IgG antibodies were used. doi:10.1371/journal.pone.0012072.g004 exceptional and unexpected localization of Sun1 at the anterior pole of the sperm head (Figure 5). Even more striking was the finding that at the anterior pole Sun1 localized outside the nucleus which became quite evident by double staining against Sun1 and the acrosomal marker Cage1. The Sun1 signal appeared anterior, but right on top of the acrosome indicating that it is part of the anterior acrosomal membrane system (Figure 5, Movie S1; see also below). Notably, acrosome-associated localization strictly differs from all other yet described NE proteins expressed during mammalian spermiogenesis, i.e. Lap2, LBR, lamins B3 and B1 and Sun3 (Figure 3, Figure 5). Remarkably, as spermiogenesis progresses, we found a striking change in Sun1 distribution in that it gradually disappeared from the posterior pole, whereas the signal at the anterior pole of the sperm head became progressively stronger (Figure 4A, Figure 5B, Figure S3). In elongated spermatids, however, Sun1 was only detectable at anterior regions covering most parts of the acrosome Figure 5. Sun1 displays a dual localization in early spermatid nuclei. Distribution of Sun1 in postmeiotic cells was investigated by indirect immunofluorescence microscopy. Testis paraffin sections were stained using affinity-purified anti-Sun1 antiserum (A-C). Same cells were co-stained for mAb 13d4 against LAP2 (A’), pAb against lamin B3 (B’) or pAb against acrosomal protein Cage1 (C’). Overlays are seen in A’’-C’’. DNA was labeled with 33258-Hoechst (A’’’-C’’’). Arrowheads indicate non-nuclear acrosome-associated localization of Sun1 (A-C). Arrow denotes the implanation fossa. Scale bars: 5 mm. doi:10.1371/journal.pone.0012072.g005 PLoS ONE | www.plosone.org 6 August 2010 | Volume 5 | Issue 8 | e12072