indirect immunofluorescence microscopy. Testis paraffin sections of adult mice were stained using an affinity-purified anti-Sun3 antiserum (green). DNA was labeled with 33258-Hoechst. Sun3 is detectable in round (A) as well as in elongated (B) spermatids but not in other spermatogenic cell types. Scale bar, 50 mm. Found at: doi:10.1371/journal.pone.0012072.s002 (1.22 MB TIF) Figure S2 Posterior polarization of Sun3 in round and elongated spermatids. Localization of Sun3 in round (A and B) and elongated (C and D) spermatids was investigated by indirect immunofluor- escence microscopy followed by 3D reconstruction. Testis paraffin sections of adult mice were stained using an affinity-purified anti- Sun3 antiserum. DNA was labeled with 33258-Hoechst (A’-D’). Arrows indicate the region of the implanation fossa. Images of sequenced single sections were taken by confocal laser scanning microscopy and 3D reconstruction of the scans was calculated using AmiraH Software. Scale bars, 5 mm. Found at: doi:10.1371/journal.pone.0012072.s003 (0.46 MB TIF) Figure S3 Sun1 expression during mammalian spermatogenesis. Localization of Sun1 within seminiferous tubules was analyzed by indirect immunofluorescence microscopy. Testis paraffin sections of adult mice were stained using an affinity-purified anti-Sun1 antiserum (green). DNA was labeled with 33258-Hoechst (blue). Sun1 is present in spermatocytes (Sc; punctured distribution References 1. Fridkin A, Penkner A, Jantsch V, Gruenbaum Y (2009) SUN-domain and KASH-domain proteins during development, meiosis and disease. Cell Mol Life Sci 66: 1518–1533. 2. Starr DA (2009) A nuclear-envelope bridge positions nuclei and moves chromosomes. J Cell Sci 122: 577–586. 3. Burke B, Roux KJ (2009) Nuclei take a position: managing nuclear location. Dev Cell 17: 587–597. 4. Crisp M, Liu Q, Roux K, Rattner JB, Shanahan C, et al. (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J Cell Biol 172: 41–53. 5. Stewart-Hutchinson PJ, Hale CM, Wirtz D, Hodzic D (2008) Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness. Exp Cell Res 314: 1892–1905. 6. Hagan I, Yanagida M (1995) The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability. J Cell Biol 129: 1033–1047. 7. Malone CJ, Fixsen WD, Horvitz HR, Han M (1999) UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development. Development 126: 3171–3181. 8. Hodzic DM, Yeater DB, Bengtsson L, Otto H, Stahl PD (2004) Sun2 is a novel mammalian inner nuclear membrane protein. J Biol Chem 279: 25805–25812. 9. Padmakumar VC, Libotte T, Lu W, Zaim H, Abraham S, et al. (2005) The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope. J Cell Sci 118: 3419–3430. 10. Hasan S, Gu¨ttinger S, Mu¨hlha¨usser P, Anderegg F, Bu¨ rgler S, et al. (2006) Nuclear envelope localization of human UNC84A does not require nuclear lamins. FEBS Lett 580: 1263–1268. 11. Tzur YB, Wilson KL, Gruenbaum Y (2006) SUN-domain protein's: "V''elcro that links the nucleoskeleton to the cytoskeleton. Nat Rev Mol Cell Biol 7: 782–788. 12. Liu Q, Pante N, Misteli T, Elsagga M, Crisp M, et al. (2007) Functional association of Sun1 with nuclear pore complexes. J Cell Biol 178: 785–798. 13. Starr DA, Fischer JA (2005) KASH ’n Karry: the KASH domain family of cargo-specific cytoskeletal adaptor proteins. Bioessays 27: 1136–1146. 14. Apel ED, Lewis RM, Grady RM, Sanes JR (2000) Syne-1, a dystrophin- and Klarsicht-related protein associated with synaptic nuclei at the neuromuscular junction. J Biol Chem 275: 31986–31995. 15. Zhang Q, Skepper JN, Yang F, Davies JD, Hegyi L, et al. (2001) Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues. J Cell Sci 114: 4485–4498. 16. Padmakumar VC, Abraham S, Braune S, Noegel AA, Tunggal B, et al. (2004) Enaptin, a giant actin-binding protein, is an element of the outer nuclear membrane and the actin cytoskeleton. Exp Cell Res 295: 330–339. 17. Zhen YY, Libotte T, Munck M, Noegel AA, Korenbaum E (2002) NUANCE, a giant protein connecting the nucleus and actin cytoskeleton. J Cell Sci 115: 3207–3222. 18. Wilhelmsen K, Litjens SHM, Kuikman I, Tshimbalanga N, Janssen H, et al. (2005) Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin. J Cell Biol 171: 799–810. LINC in Sperm Head Formation corresponding to meiotic telomeres), in round (rSp; cap like distribution at the posterior pole) and in elongated spermatids (eSp; cap like distribution at the anterior pole). Scale bar, 15 mm. Found at: doi:10.1371/journal.pone.0012072.s004 (0.78 MB TIF) Movie S1 Localization of Sun1 and acrosomal protein Cage1 in a 3D reconstructed round spermatid. Testis paraffin section of an adult mouse was co-stained for Sun1 (green) and acrosomal protein Cage1 (red). DNA was labeled with 33258-Hoechst (blue). Images of sequenced single sections were taken by confocal laser scanning microscopy and reconstruction of the scans was calculated using AmiraH Software. Found at: doi:10.1371/journal.pone.0012072.s005 (1.58 MB MPG) Acknowledgments The Nesprin3 antibody was kindly provided by Dr. Arnoud Sonnenberg (The Netherlands Cancer Inst., Amsterdam, The Netherlands). Author Contributions Conceived and designed the experiments: RB MA. Performed the experiments: EG JS MA. Analyzed the data: EG JS RB MA. Wrote the paper: EG MA. 19. Roux KJ, Crisp ML, Liu Q, Kim D, Kozlov S, et al. (2009) Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization. Proc Natl Acad Sci U S A 106: 2194–2199. 20. Haque F, Lloyd DJ, Smallwood DT, Dent CL, Shanahan CM, et al. (2006) SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton. Mol Cell Biol 26: 3738–3751. 21. Ketema M, Wilhelmsen K, Kuikman I, Janssen H, Hodzic D, et al. (2007) Requirements for the localization of nesprin-3 at the nuclear envelope and its interaction with plectin. J Cell Sci 120: 3384–3394. 22. Haque F, Mazzeo D, Patel JT, Smallwood D, Ellis JA, et al. (2010) Mammalian SUN protein interaction networks at the inner nuclear membrane and their role in laminopathy disease processes. J Biol Chem 285: 3487–3498. 23. Schmitt J, Benavente R, Hodzic D, Ho¨o¨g C, Stewart CL, et al. (2007) Transmembrane protein Sun2 is involved in tethering mammalian meiotic telomeres to the nuclear envelope. Proc Natl Acad Sci U S A 104: 7426– 7431. 24. Ding X, Xu R, Yu J, Xu T, Zhuang Y, et al. (2007) SUN1 is required for telomere attachment to nuclear envelope and gametogenesis in mice. Dev Cell 12: 863–872. 25. Chi YH, Cheng LI, Myers T, Ward JM, Williams E, et al. (2009) Requirement for Sun1 in the expression of meiotic reproductive genes and piRNA. Development 136: 965–973. 26. Grady RM, Starr DA, Ackerman GL, Sanes JR, Han M (2005) Syne proteins anchor muscle nuclei at the neuromuscular junction. Proc Natl Acad Sci U S A 102: 4359–4364. 27. Puckelwartz MJ, Kessler E, Zhang Y, Hodzic D, Randles KN, et al. (2009) Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice. Hum Mol Genet 18: 607–620. 28. Lei K, Zhang X, Ding X, Guo X, Chen M, et al. (2009) SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice. Proc Natl Acad Sci U S A 106: 10207–10212. 29. Me´jat A, Misteli T (2010) LINC complexes in health and disease. Nucleus 1: 40–52. 30. Olins AL, Hoang TV, Zwerger M, Herrmann H, Zentgraf H, et al. (2009) The LINC-less granulocyte nucleus. Eur J Cell Biol 88: 203–214. 31. Xiong H, Rivero F, Euteneuer U, Mondal S, Mana-Capelli S, et al. (2008) Dictyostelium Sun-1 connects the centrosome to chromatin and ensures genome stability. Traffic 9: 708–724. 32. Khatau SB, Hale CM, Stewart-Hutchinson PJ, Patel MS, Stewart CL, et al. (2009) A perinuclear actin cap regulates nuclear shape. Proc Natl Acad Sci U S A 106: 19017–19022. 33. Kierszenbaum AL, Rivkin E, Tres LL (2003) Acroplaxome, an F-actin-keratin- containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head. Mol Biol Cell 14: 4628–4640. 34. Russell LD, Russell JA, MacGregor GR, Meistrich ML (1991) Linkage of manchette microtubules to the nuclear envelope and observations of the role of the manchette in nuclear shaping during spermiogenesis in rodents. Am J Anat 192: 97–120. PLoS ONE | www.plosone.org 12 August 2010 | Volume 5 | Issue 8 | e12072