Rat kinesin light chain 3 associates with spermatid mitochondria

AbstractWe recently discovered that in rat spermatids, kinesin light chain KLC3 can associate with outer dense fibers, major sperm tail components, and accumulates in the sperm midpiece. Here, we show that mitochondria isolated from rat-elongating spermatids have bound KLC3. Immunoelectron microscopy indicates that the association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the plasma membrane to the developing midpiece. KLC3 is able to bind in vitro to mitochondria from spermatids as well as somatic cells employing a conserved kinesin light chain motif, the tetratrico-peptide repeats. Expression of KLC3 in fibroblasts results in formation of large KLC3 clusters close to the nucleus, which also contain mitochondria: no other organelles were present in these clusters. Mitochondria are not present in KLC3 clusters after deletion of KLC3's tetratrico-peptide repeats. Our results indicate that the rat spermatid kinesin light chain KLC3 can associate with mitochondria

Developmental Expression of the 84-kDa ODF Sperm Protein: Localization to both the Cortex and Medulla of Outer Dense Fibers and to the Connecting Piece

AbstractOuter dense fibers (ODF) are specialized cytoskeletal elements of the mammalian sperm tail which are composed of several prominent proteins. We previously reported the isolation of a cDNA (111-450) encoding a putative 84-kDa ODF protein. Here we demonstrate by independent cDNA isolations and by translational/immunoprecipitation of testicular mRNAs using anti-ODF 84 antibodies that 111-450 cDNA encodes the 84-kDa protein. We then analyzed the testicular expression of the ODF 84 mRNA and protein. Riboprobes generated from the clones recognized four testicular-specific transcripts of 1.6, 2.2, 2.4, and 2.8 kb in both rat and bull of which the immunoprecipitable product of the 2.4-kb mRNA comigrates with ODF 84 protein. Developmental Northerns indicated that the 2.2- and 2.4-kb mRNAs are first transcribed during meiotic prophase while the other two species are first expressed in round spermatids. The levels of all the transcripts steadily increased up to elongated spermatids. Immunocytochemistry revealed that the anti-84 reactive ODF proteins were synthesized and assembled in the cytoplasm of elongated spermatids (steps 9–18) with peak activity occurring in step 16 of spermiogenesis. Immunogold labeling was selective to the assembling ODF and connecting piece of the tail and to granulated bodies of the cytoplasmic lobe. Both the striated collar and capitulum of the connecting piece were immunolabeled as well as the basal plate of the implantation fossa. A combination of pre- and postembedding immunogold labeling provided evidence that the 84-kDa ODF protein is localized to both the cortex and medulla of the ODF in contrast to the sole medullary localization of the major 27-kDa ODF protein. Thus the 84-kDa ODF protein, encoded by the 2.4 transcript, is translationally regulated, packaged after synthesis into granulated bodies, assembled in a proximal to distal direction along the axoneme and may interact by means of leucine zippers specifically with the 27-kDa ODF protein during assembly. Its localization to both the cortex and medulla of the ODF, as opposed to exclusive medullary localization of the 27-kDa ODF protein, and the presence of two leucine zippers, only one of which interacts with the 27-kDa ODF, suggests that it could act as a link between proteins of the two regions of the ODF

Gene trap mutation of murine Outer dense fiber protein-2 gene can result in sperm tail abnormalities in mice with high percentage chimaerism

<p>Abstract</p> <p>Background</p> <p>Outer dense fiber protein 2, Odf2, is a major component of the outer dense fibers, ODF, in the flagellum of spermatozoa. ODF are associated with microtubule doublets that form the axoneme. We recently demonstrated that tyrosine phosphorylation of Odf2 is important for sperm motility. In the course of a study of Odf2 using Odf2 mouse knockout lines we observed that males of a high percentage chimaerism, made using XL169 embryonic stem cells, were infertile, whereas mice of low-medium percentage chimaerism were fertile.</p> <p>Results</p> <p>XL169 ES cells have a β-geo gene trap cassette inserted in the Odf2 gene. To determine possible underlying mechanisms resulting in infertility we analyzed epididymal sperm and observed that >50% displayed bent tails. We next performed ultrastructural analyses on testis of high percentage XL169 chimaeric mice. This analysis showed that high percentage XL169 chimaeric mice produce elongating spermatids that miss one or more entire outer dense fibers in their midpiece and principal piece. In addition, we observed elongating spermatids that show thinning of outer dense fibers. No other obvious abnormalities or defects are present in elongating spermatids. Spermatozoa from the caput and cauda epididymis of XL169 mice of high percentage chimaerism show additional tail defects, including absence of one or more axonemal microtubule doublets and bent tails. Sperm with bent tails display abnormal motility.</p> <p>Conclusions</p> <p>Our results document the possible impact of loss of one Odf2 allele on sperm tail structure and function, resulting in a novel sperm tail phenotype.</p

KLC3 is involved in sperm tail midpiece formation and sperm function

AbstractKinesin light chain 3 (KLC3) is the only known kinesin light chain expressed in post-meiotic male germ cells. We have reported that in rat spermatids KLC3 associates with outer dense fibers and mitochondrial sheath. KLC3 is able to bind to mitochondria in vitro and in vivo employing the conserved tetratrico-peptide repeat kinesin light chain motif. The temporal expression and association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the spermatid cell periphery to the developing midpiece suggesting a role in midpiece formation. In fibroblasts, expression of KLC3 results in formation of large KLC3 aggregates close to the nucleus that contain mitochondria. However, the molecular basis of the aggregation of mitochondria by KLC3 and its role in sperm tail midpiece formation are not clear.Here we show that KLC3 expression from an inducible system causes mitochondrial aggregation within 6h in a microtubule dependent manner. We identified the mitochondrial outer membrane porin protein VDAC2 as a KLC3 binding partner. To analyze a role for KLC3 in spermatids we developed a transgenic mouse model in which a KLC3ΔHR mutant protein is specifically expressed in spermatids: this KLC3 mutant protein binds mitochondria and causes aggregate formation, but cannot bind outer dense fibers. Male transgenic mice display significantly reduced reproductive efficiency siring small sized litters. We observed defects in the mitochondrial sheath structure in a number of transgenic spermatids. Transgenic males have a significantly reduced sperm count and produce spermatozoa that exhibit abnormal motility parameters. Our results indicate that KLC3 plays a role during spermiogenesis in the development of the midpiece and in the normal function of spermatozoa

Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association.

Na,K-ATPase is a membrane protein that catalyzes ATP to maintain transmembrane sodium and potassium gradients. In addition, Na,K-ATPase also acts as a signal-transducing receptor for cardiotonic steroids such as ouabain and activates a number of signalling pathways. Several studies report that ouabain affects cell migration. Here we used ouabain at concentrations far below those required to block Na,K-ATPase pump activity and show that it significantly reduced RPE cell migration through two mechanisms. It causes dephosphorylation of a 130 kD protein, which we identify as p130cas. Src is involved, because Src inhibitors, but not inhibitors of other kinases tested, caused a similar reduction in p130cas phosphorylation and ouabain increased the association of Na,K-ATPase and Src. Knockdown of p130cas by siRNA reduced cell migration. Unexpectedly, ouabain induced separation of nucleus and centrosome, also leading to a block in cell migration. Inhibitor and siRNA experiments show that this effect is mediated by ERK1,2. This is the first report showing that ouabain can regulate cell migration by affecting nucleus-centrosome association

Targeted Disruption of the Testicular SPAG5/Deepest Protein Does Not Affect Spermatogenesis or Fertility

In an effort to define the molecular basis for morphogenesis of major sperm tail structures, including outer dense fibers, we recently cloned the Spag5 gene by virtue of its strong and specific leucine-zipper-mediated interaction with Odf1, the 27-kDa major outer dense fiber protein. Spag5 is expressed during meiosis and in round spermatids and is similar, if not identical, to Deepest, a putative spindle pole protein. Here we report the disruption of the Spag5 gene by homologous recombination. Spag5-null mice lack Spag5 mRNA and protein. However, male mice are viable and fertile. Analysis of the process of spermatogenesis and sperm produced in Spag5-null mice did not reveal a major phenotype as a consequence of the knockout event. This result suggests that if Spag5 plays a role in spermatogenesis it is likely compensated for by unknown proteins

Primary cilia in fibroblast-like type B synoviocytes lie within a cilium pit, a site of endocytosis

The synovium is a thin connective tissue that lines the joint space of free moving articulations. In this report, the expression, structure, and composition of non-motile (primary) cilia in fibroblast-like synoviocytes (FLS) that populate the synovium have been studied. Primary cilia are non-motile, microtubule-based organelles that have been found in a variety of vertebrate cell types. We document that primary cilia are expressed in normal human synovium FLS, cultured human FLS, and FLS cells present in human synovial fluid, and that the cellular region occupied by the primary cilium shows a similar and highly defined architecture within these FLS. This architecture includes the presence of a unique structure that surrounds the lower portion of the cilium shaft. This structure, given the term cilium-pit, includes a space, the pit reservoir. Actin filament bundles surround the cilium-pit, and when these bundles are removed experimentally the volume of the cilium-pit and its continuity with the extracellular environment changes. Finally, this study documents that the cilium-pit is a site of endocytosis and is also the site for the localization of receptors (TNF receptors TNFR1 and TNFR2) associated with synoviocyte function. Taken together, the results of the present study suggest that the FLS cilium-pit functions to regulate the exposure of the primary cilium, both spatially and temporally to extracellular molecules and to couple primary cilium based signaling pathways with those linked to endocytosis

Ouabain affects actin polymerization.

<p>To analyze the effect of ouabain on actin polymerization we analyzed actin stress fibers as follows. Primary fibroblasts (HS68) were untreated, or treated with 50 nM ouabain for 24 hrs, fixed and stained with phalloidin. Panels 1 and 2, examples of untreated cells; panel 3 and 4, examples of cells treated with ouabain, showing a loss of stress fibers. Arrows point to stress fibers.</p