Sak 57, an intermediate filament keratin present in intercellular bridges of rat primary spermatocytes.
We have previously reported the purification of Sak 57 (for spermatogenic cell/sperm-associated keratin of molecular mass 57 kDa) from outer dense fibers of rat sperm tails. Internal protein sequence analysis of Sak 57 revealed 70-100% homology to the 1A and 2A regions of the alpha-helical rod domain of human, mouse, and rat keratins. A multiple antigen peptide was synthesized using the KQYEDIAQK sequence corresponding to the 2A region and a polyclonal antibody was produced in rabbit to detect Sak 57. During spermiogenesis, Sak 57 associates with the microtubular manchette before becoming a component of para-axonemal keratin structures of the developing tail. We now report that during late meiotic prophase, intercellular bridges linking late pachytene-diplotene spermatocytes display a distinct ribbon containing a Sak 57/beta-tubulin complex, separated by a nonimmunoreactive midzone. Indirect immunofluorescence demonstrates that the ribbon is the final stage of a three-step developmental sequence: (1) a spindlelike arrangement radiating from equidistant spherical centers in early pachytene spermatocytes, (2) an ectoplasmic shell-like framework in mid-to-late pachytene spermatocytes, and (3) a Sak 57/beta-tubulin-containing ribbon found in intercellular bridges linking adjacent late pachytene-diplotene spermatocytes. Shear forces causing a breakdown of one of the conjoined spermatocytes do not disrupt the cytoskeletal ribbon. Results of this work, together with previous observations during spermiogenesis, show that Sak 57 associates with cytoplasmic microtubules in a timely fashion. Upon completion of late meiotic prophase, the Sak 57/microtubule complex behaves as an intercellular ligament and contributes to both the strength of intercellular bridges and the cohesiveness of members of a spermatocyte lineage.
Tres
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Department of Cell Biology and Anatomical Sciences, City University of New York Medical School, New York 10031, USA.
Rivkin
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Kierszenbaum
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D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D007633:Keratins / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D008858:Microscopy, Phase-Contrast; D008954:Models, Biological; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013090:Spermatocytes / Q000737:chemistry* / Q000201:enzymology; D013737:Testis / Q000737:chemistry; D014404:Tubulin / Q000378:metabolism
D005456:Fluorescent Dyes; D007381:Intermediate Filament Proteins; D010446:Peptide Fragments; D014404:Tubulin; D007633:Keratins; C490219:PLK4 protein, human; D017346:Protein Serine-Threonine Kinases