Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes Eva Go¨ b, Johannes Schmitt¤, Ricardo Benavente, Manfred Alsheimer* Department of Cell and Developmental Biology, Biocenter, University of Wu¨ rzburg, Wu¨ rzburg, Germany Abstract Background: LINC complexes are nuclear envelope bridging protein structures formed by interaction of SUN and KASH proteins. They physically connect the nucleus with the peripheral cytoskeleton and are critically involved in a variety of dynamic processes, such as nuclear anchorage, movement and positioning and meiotic chromosome dynamics. Moreover, they are shown to be essential for maintaining nuclear shape. Findings: Based on detailed expression analysis and biochemical approaches, we show here that during mouse sperm development, a terminal cell differentiation process characterized by profound morphogenic restructuring, two novel distinctive LINC complexes are established. They consist either of spermiogenesis-specific Sun3 and Nesprin1 or Sun1g, a novel non-nuclear Sun1 isoform, and Nesprin3. We could find that these two LINC complexes specifically polarize to opposite spermatid poles likely linking to sperm-specific cytoskeletal structures. Although, as shown in co-transfection/ immunoprecipitation experiments, SUN proteins appear to arbitrarily interact with various KASH partners, our study demonstrates that they actually are able to confine their binding to form distinct LINC complexes. Conclusions: Formation of the mammalian sperm head involves assembly and different polarization of two novel spermiogenesis-specific LINC complexes. Together, our findings suggest that theses LINC complexes connect the differentiating spermatid nucleus to surrounding cytoskeletal structures to enable its well-directed shaping and elongation, which in turn is a critical parameter for male fertility. Citation: Go¨ b E, Schmitt J, Benavente R, Alsheimer M (2010) Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes. PLoS ONE 5(8): e12072. doi:10.1371/journal.pone.0012072 Editor: Mary Bryk, Texas A&M University, United States of America Received May 7, 2010; Accepted July 14, 2010; Published August 10, 2010 Copyright: 2010 Go¨ b et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Deutsche Forschungsgemeinschaft Grant Al 1090/1-1 (http://www.dfg.de), the Graduate School GK 1048 of the University of W u¨rzburg (http://www.gk-1048.uni-wuerzburg.de) and the Elitenetzwerk Bayern (https://www.elitenetzwerk.bayern.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alsheimer@biozentrum.uni-wuerzburg.de ¤ Current address: Laboratory of Molecular Hepatology, Clinic for Gastroenterology and Hepatology, University Hospital Zurich (USZ), Zurich, Switzerland Introduction Anchorage and active positioning of the nucleus plays a fundamental role during diverse developmental processes such as fertilization, cell migration, establishment of polarity and is critical for differentiation of various cell types [1–3]. It essentially requires a direct interaction between the nucleus, in particular the nuclear envelope (NE), and the cytoskeleton. In this context, the so-called LINC complexes (linker of nucleoskeleton and cytoskeleton) gained more and more importance as they actually define the molecular basis to physically connect the nucleus to the peripheral cytoskeleton. LINC complexes are formed across the NE by the interaction of members of two transmembrane (TM) protein families: SUN and KASH domain proteins [4,5]. SUN domain proteins are an evolutionary conserved family of inner nuclear membrane (INM) proteins that share a common C- terminal motif, the SUN (Sad1p/Unc84 homology) domain [6,7]. The mammalian genome codes for at least five SUN proteins. The two major SUN proteins, Sun1 and Sun2, are widely expressed in different cell types [8,9]. Sun3, Sun4 and Sun5 appear to have a more restricted, most likely testis-specific expression, but as yet remained rather uncharacterized [10–12]. SUN proteins are integral inner nuclear membrane proteins with an N-terminal nucleoplasmic region separated by a TM domain from the C- terminal part that extends into the perinuclear space (PNS) [8,9]. Within the PNS, SUN proteins directly interact via their terminal SUN domain with the C-terminal KASH (Klarsicht/Anc1/Syne1 homology) domain of respective KASH protein partners [2,13]. Mammals contain at least four KASH proteins, which are called nesprins [14–19]. Nesprins are outer nuclear membrane (ONM) proteins containing a long cytoplasmic N-terminus that has the ability to bind to the cytoskeleton [2]. Two of them, Nesprin1 and 2, are large actin-binding proteins [15,20], whereas Nesprin3, a smaller molecule, binds to plectin that in turn links to the intermediate filament system and/or actin [18,21]. Contrary, Nesprin4 is restricted to few cell types and binds to kinesin, a microtubule associated protein [19]. Tethering of nesprins to the ONM depends on the localization of Sun1 and Sun2 in the INM [4,20,21]. Since both Sun1 and Sun2 also interact with A-type lamins and other components of the INM (i.e. emerin) the SUN- KASH-interaction within the PNS forms a functional cross linkage of the nucleoskeleton and the cytoskeleton [4,22]. Besides their primary function in connecting nucleoplasmic to cytoplasmic structures, LINC complexes are supposed to be PLoS ONE | www.plosone.org 1 August 2010 | Volume 5 | Issue 8 | e12072