LRGUK-1 is required for basal body and manchette function during spermatogenesis and male fertility.

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

Recent progress in histochemistry and cell biology.

Studies published in Histochemistry and Cell Biology in the year 2011 represent once more a manifest of established and newly sophisticated techniques being exploited to put tissue- and cell type-specific molecules into a functional context. The review is therefore the Histochemistry and Cell Biology's yearly intention to provide interested readers appropriate summaries of investigations touching the areas of tissue biology, developmental biology, the biology of the immune system, stem cell research, the biology of subcellular compartments, in order to put the message of such studies into natural scientific-/human- and also pathological-relevant correlations.

Histochemistry and cell biology: the annual review 2010.

This review summarizes recent advances in histochemistry and cell biology which complement and extend our knowledge regarding various aspects of protein functions, cell and tissue biology, employing appropriate in vivo model systems in conjunction with established and novel approaches. In this context several non-expected results and discoveries were obtained which paved the way of research into new directions. Once the reader embarks on reading this review, it quickly becomes quite obvious that the studies contribute not only to a better understanding of fundamental biological processes but also provide use-oriented aspects that can be derived therefrom.

Desmocollin 3-mediated binding is crucial for keratinocyte cohesion and is impaired in pemphigus.

Desmocollin (Dsc) 1-3 and desmoglein (Dsg) 1-4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.

Endothelial barrier stabilization by a cyclic tandem peptide targeting VE-cadherin transinteraction in vitro and in vivo.

Inflammatory stimuli result in vascular leakage with potentially life threatening consequences. As a key barrier component, loss of vascular endothelial (VE-) cadherin-mediated adhesion often precedes endothelial breakdown. This study aimed to stabilize VE-cadherin transinteraction and endothelial barrier function using peptides targeting the VE-cadherin adhesive interface. After modelling the transinteracting VE-cadherin structure, an inhibiting single peptide (SP) against a VE-cadherin binding pocket was selected, which specifically blocked VE-cadherin transinteraction as analyzed by single molecule atomic force microscopy (AFM). The tandem peptide (TP) consisting of two SP sequences in tandem was designed to strengthen VE-cadherin adhesion by simultaneously binding and cross-bridging two interacting cadherin molecules. Indeed, in AFM experiments TP specifically rendered VE-cadherin transinteraction resistant against an inhibitory monoclonal antibody. Moreover, TP reduced VE-cadherin lateral mobility and enhanced binding of VE-cadherin-coated microbeads to cultured endothelial cells, but acted independently of the actin cytoskeleton. TP also stabilized endothelial barrier properties against the Ca(2+) ionophore A23187 and the inhibitory antibody. Finally, TP abolished endothelial permeability increase induced by tumour necrosis factor-alpha in microperfused venules in vivo. Stabilization of VE-cadherin adhesion by cross-bridging peptides may therefore be a novel therapeutic approach for the treatment of vascular hyperpermeability.

Peptides Targeting the Desmoglein 3 Adhesive Interface Prevent Autoantibody-induced Acantholysis in Pemphigus.

Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the self-designed cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment.

Mitochondrial and nuclear localization of kanadaptin.

Kanadaptin has originally been isolated as a kidney Cl-/HCO3- anion exchanger 1 (kAE1)-binding protein. Initial studies suggested, that in the kidney of the rabbit kanadaptin is expressed exclusively in all epithelial cells of the collecting duct. Transcripts of kanadaptin were also found in tissues not expressing kAE1, indicating additional roles for kanadaptin. With respect to this, we could recently demonstrate translocation of kanadaptin into the nucleus of mammalian cells in a nuclear localization sequence- and importin-dependent manner (Hübner et al., Biochem. J. 361, 287-296, 2002). In this study, we provide evidence, that kanadaptin is widely expressed in many tissues and that expression of kanadaptin in the mouse occurs early in embryonic development. In rat kidney we found the most intense immunofluorescence for kanadaptin in cells of the proximal tubule, consistent with the detection by in situ hybridization of high amounts of kanadaptin messenger RNA in proximal tubule cells. Immunostaining revealed localization of kanadaptin in two subcellular locations, nuclei and mitochondria. Whereas nuclear localization was demonstrated in virtually all cells, mitochondrial staining was restricted to certain cell types. Nuclear staining was only seen in cryosections, whereas mitochondrial staining was observed in both cryosections and semithin sections of freeze-dried plastic-embedded tissue. In the kidney mitochondrial staining was particularly prominent in proximal tubular epithelium. Most surprisingly, in the collecting duct epithelium (including acid-secreting intercalated cells) only negligible immunostaining, if at all, could be observed. Immunoelectron microscopy showed immunolabelling of the entire cross-sectional profile of mitochondria (matrix/inner membrane). Mitochondrial localization of kanadaptin was further documented by immunoblotting of mitochondria-enriched cellular fractions. Utilizing an interspecies heterokaryon assay, we could further demonstrate that kanadaptin has nuclear export activity. Thus, kanadaptin can be regarded to be a highly mobile nucleocytoplasmic shuttling and multilocalizing protein, but its role in mammalian cells remains still obscure.