spermiogenesis-specific Cage1 (Figure 6B) and, more important, that of Sun3 (Figure 2C,D) demonstrating that Sun1g expression is restricted to spermiogenesis. To obtain information about Sun1g protein, we isolated mature spermatozoa obtained from epididymes of adult mice, separated sperm heads from tails and prepared protein suspensions from the sperm head fraction. Suspensions from isolated sperm heads were compared with whole testicular protein suspensions in immuno- blotting experiments using specific antibodies against Sun1 (Figure 6C). Remarkably, the antibodies recognized only one single protein band in the sperm head fraction with a molecular mass of 72 kDa which is consistent with the calculated mass of Sun1g. This protein band was also detectable in whole testis suspension, albeit and as expected, higher molecular mass bands corresponding to full-length Sun1 or other Sun1 isoforms were present, too. Remarkably, these bands could not be detected in mature spermatozoa indicating that Sun1g is the only Sun1 isoform expressed at the end of sperm differentiation (Figure 6C). Our results obtained by RT-PCR and immunoblotting strongly suggest that the newly identified Sun1 isoform, Sun1g, is testis- specific and selectively expressed during spermiogenesis. Remark- ably, missing exon seven to ten, Sun1g lacks sequences proposed to be required for nuclear retention (see also [12,22]). Sun1g expression correlates with the appearance of an anterior non-nuclear LINC complex LINC complexes are present in many, if not all cell types; however, all yet identified LINC complexes specifically localize to the NE. As shown above, at the anterior pole of spermatids Sun1 localizes in a non-nuclear manner (Figure 5), a distribution which strongly correlates with the expression of Sun1g. To investigate if non-nuclear Sun1 forms a distinct LINC complex, we analyzed presence and behavior of respective KASH binding partners. As mentioned above, we detected both, Nesprin1 and 3 at the NE of spermatids, with Nesprin1 entirely colocalizing with Sun3 (Figure 4B). In contrast, Nesprin3 showed a quite different localization and its behavior remarkably resembled that of Sun1 (Figure 4A). Like Sun1, Nesprin3 was initially found at the posterior pole of round and early elongating spermatids and also at the anterior side of the sperm head (Figure 4A a–c). In late elongated spermatids, however, both proteins were exclusively found at the anterior pole (Figure 4A d–f). Notably, Nesprin3 and Sun3 although being able to interact with each other (Figure 4D) did not colocalize at all, but rather appeared to be mutually exclusive (Figure 4B d–f). Since the C-terminal SUN domain of Sun1 is capable to directly interact with the KASH domain of Nesprin3 (Figure 4D; [12]) it can be assumed that during sperm head formation at the anterior pole Sun1 (most likely Sun1g; see above) and Nesprin3 together form a functional, however non- nuclear LINC complex. In summary, during sperm head formation SUN and KASH domain proteins appear to form distinct LINC complexes that, strikingly, polarize to opposite poles with Sun1g/Nesprin3 localizing to the anterior and Sun3/Nesprin1 to the posterior side. Discussion In eukaryotes, cellular and nuclear shaping is crucial for multiple events including cell migration, division and differentia- tion of diverse cell types. Also spermiogenesis, the differentiation of motile, fertilization competent spermatozoa, is a matter of fundamental morphogenic restructuring. Initially round nuclei are deliberately deformed to achieve their compact and elongated structure. The molecular mechanisms responsible for this LINC in Sperm Head Formation morphological remodeling are largely unknown yet far. However, the cytoskeleton appears to be of fundamental importance since spermatids establish specialized cytoskeletal structures that are suggested to play a role in sperm-specific nuclear shaping; either in providing forces to actively elongate nuclei or in positioning nuclei to be shaped by exogenous forces [34,38,52]. While spermatids differentiate these cytoskeletal elements become visible at opposite regions of spermatid nuclei. At the anterior side develops the acroplaxome a thin cytoplasmic sheet mainly built-up by F-actin between elongating nucleus and acrosome whereas the posterior half of the nucleus is covered by the spermatid-specific manchette a calyx-like structure formed of bundles of microtubules [33–35] (Figure 7). Remarkably, both structures tightly appose to differentiating nuclei almost anchoring directly to the NE. Taking these facts to account, consequently, the question arises how these cytoskeletal elements and forces could be connected to the elongating nucleus. Here we show for the first time that LINC complexes are also established in male postmeiotic germ cells. We also provide compelling evidence that these complexes are good candidates for anchoring spermatid head components to sur- rounding cytoskeletal structures, thus enabling deliberate sperm head shaping. Two novel LINC complexes, Sun1g/Nesprin3 and Sun3/ Nesprin1, during sperm head formation In the present study we demonstrated that differentiating spermatids express two novel LINC complexes: Sun1g/Nesprin3 and Sun3/Nesprin1. Our immunocytochemical and biochemical experiments clearly evidence that mammalian Sun1g and Sun3 actually are testis-specific proteins with an expression profile exactly correlating with postmeiotic sperm differentiation. Furthermore, we characterize Sun3 as a Nesprin1 interacting protein that can participate in LINC complex formation thus enlarging the potential of mammalian cells to establish multiple LINC complex variants. Notably, our immunofluorescence data indicate that in spermatids these two novel LINC complexes are not homogenously distributed in the NE as in somatic cells. However, they accumulate specifically in distinct regions of the elongating sperm head: LINC complexes consisting of Sun3/Nesprin1 polarize to the posterior pole of spermatid nuclei whereas LINC complexes formed by Sun1g/Nesprin3 show an atypical non-nuclear localization at the anterior pole (Figure 7). Directed polarization of NE components per se reflects a general feature of mammalian spermiogenesis: profound modula- tion of the entire NE which is suggested to influence nuclear elongation [42,43,45]. However, our experiments uncovered remarkable divergences between single NE components concern- ing their individual polarization. Particularly Sun3 differed strikingly from all other NE proteins analyzed thus far. NE proteins (including Sun1), like nuclear pore complexes, progres- sively redistribute to the posterior pole before they apparently disappear as spermiogenesis progresses ([42–44,54] and this study). However, Sun3 although resembling in principle the posterior polarization of the other NE components, differs due to its one-to- one colocalization with manchette microtubules over the time and its absence from the fossa region. Therefore, we suggest that during different stages of sperm differentiation subsets of NE proteins occupy distinct and distinguishable NE territories thus enabling fundamentally new roles for NE proteins, especially for LINC complex components. Our results strongly suggest that LINC complexes established in the male postmeiotic germline are distinctly formed by either Sun1g and Nesprin3 or Sun3 and Nesprin1. Interestingly, Sun1g and Nesprin1 both are present at the same time in the same cell but, although having the competence to bind each other ([5]; PLoS ONE | www.plosone.org 8 August 2010 | Volume 5 | Issue 8 | e12072