Figure 3. Sun3 polarizes to the posterior pole in differentiating spermatid nuclei colocalizing with manchette microtubules. (A–F) Distribution of Sun3 in round (A, C, E) and elongating (B, D, F) spermatids was investigated by indirect confocal immunofluorescence microscopy. Testis paraffin sections were co-stained using affinity- purified anti-Sun3 antiserum (A–F) and mAb 13d4 against LAP2 (A’, B’), pAb against Lamin B3 (C’, D’) or pAb against acrosomal protein Cage1 (E’, F’). Overlays are shown in A’’–F’’. DNA was labeled with 33258- Hoechst (A’’’–F’’’). Arrows indicate the fossa region. (G, H) Relation between the distribution of Sun3 and the localization of manchette microtubules in round (G) and elongating (H) spermatids was analyzed by indirect confocal immunofluorescence microscopy. Double-label immunofluorescence was performed on testis paraffin sections with affinity-purified anti-Sun3 antiserum (G, H) and mAbs against a- (H’) or b-tubulin (G’). Overlays are seen in G’’ and H’’. DNA was labeled with 33258-Hoechst (G’’’, H’’’). Arrows indicate the implantation fossa. Manchette microtubules extending to flagellar regions are denoted by arrowheads. Scale bars: 5 mm. doi:10.1371/journal.pone.0012072.g003 LINC in Sperm Head Formation performed immunofluorescence microscopy on testis paraffin sections using antibodies against yet known mammalian KASH proteins: Nesprins 1, 2, 3 and 4. Whereas Nesprins 2 and 4 were not detectable in any postmeiotic stage (not shown), Nesprins 1 and 3, both are actually present in spermatids as evidenced by immunofluorescence analysis (Figure 4A,B; see also below). Strikingly, Nesprin1 specifically localized to posterior regions of the NE during the nuclear elongation process and, moreover, entirely colocalized with Sun3 (Figure 4B). Notably, like Sun3, Nesprin1 was not detectable in the fossa region but was present only along lateral regions of developing spermatids. These observations strongly point to a direct interaction between Sun3 and Nesprin1 in differentiating spermatids. There- fore, we next prepared co-transfection/immunoprecipitation experiments using truncated recombinant SUN and KASH protein constructs co-expressed in the mammalian HeLa cell culture system referring to the approaches described by Stewart- Hutchinson et al. [5]. We co-expressed Myc-tagged Sun3 together with the KASH domain of Nesprin1 or Nesprin3 coupled to an N- terminal EGFP. As control we used N-terminally Myc-tagged Sun1 lacking the nucleoplasmic domain together with an EGFP- Nesprin3 construct (Figure 4C). To analyze the interaction of these proteins in HeLa cells, immunoprecipitations were conducted with anti-myc antibodies and EGFP-nesprin proteins were detected using anti-EGFP antibodies. The ability of Sun3 to interact with KASH domain partners is displayed in Figure 4D showing that Myc-Sun3 co-immunoprecipitates with EGFP-Nesprin1 as well as EGFP-Nesprin3. This demonstrates an interaction of Sun3 and Nesprin1 in transfected mammalian cells, and moreover, sub- stantiates our assumption based on the immunofluorescence experiments. As expected, under the same experimental conditions we were able to co-immunoprecipitated Myc-Sun1 with EGFP- Nesprin3 (Figure 4D) which is consistent with previous findings concerning a Sun1-Nesprin3 interaction [5]. Our results clearly demonstrate that Sun3 can participate in LINC complex formation through its interaction with Nesprin1. Moreover, Sun3 and Nesprin1, both jointly polarize to the posterior pole within the spermatid NE, a behaviour which is maintained throughout the sperm elongation process. Hence, our findings strongly indicate that Sun3 and Nesprin1 assemble a spermiogenesis-specific LINC complex which polarizes to the posterior pole, thereby facing sites where cytoplasmic manchette microtubules contact the NE. Sun1 is expressed during sperm head formation, but shows an exceptional behaviour Because of their function in nuclear and chromosome dynamics and their suggested role in maintaining nuclear morphology [2,30–32,53], mammalian SUN proteins Sun1 and Sun2 are good candidates to have a major role in nuclear shaping during sperm head formation, too. Consistent with previous reports, we were unable to detect Sun2 in postmeiotic stages (not shown); Sun1, however, appears to be present during entire mammalian gametogenesis (Figure S3; see also [25]). In order to unravel the spatial distribution of Sun1 in spermatids, we performed double-label immunofluorescence analysis using affinity purified antibodies to Sun1 together with antibodies to Lap2, Lamin B3 and Cage1. In round spermatids Sun1 mainly localizes to the posterior pole of the nucleus, a localization which was already visible in early stages, and, in principle, resembles that of other NE proteins including Sun3. However, contrary to lamin B3 and Lap2 but conform to Sun3, Sun1 is excluded from the implantation fossa (Figure 5, Movie S1). Moreover, in addition to the posterior polarization, we found an PLoS ONE | www.plosone.org 5 August 2010 | Volume 5 | Issue 8 | e12072