title: rpS6 regulates blood-testis barrier dynamics through Arp3-mediated actin microfilament organization in rat sertoli cells. An in vitro study.
abstract: In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.
authors.affiliation: The Mary M. Wohlford Laboratory for Male Contraceptive Research (K.-W.M., H.C., C.Y.C.), Center for Biomedical Research, Population Council, New York, New York 10065; and School of Biological Sciences (W.M.L.), University of Hong Kong, Hong Kong, China.
authors.forename: Ka-Wai
authors.initials: KW
authors.lastname: Mok
authors.forename: Haiqi
authors.initials: H
authors.lastname: Chen
authors.forename: Will M
authors.initials: WM
authors.lastname: Lee
authors.forename: C Yan
authors.initials: CY
authors.lastname: Cheng
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D051378:Actin-Related Protein 3 / Q000378:metabolism*; D000818:Animals; D001814:Blood-Testis Barrier / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D066298:In Vitro Techniques; D008297:Male; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000378:metabolism*; D051058:Oncogene Protein v-akt / Q000235:genetics; D051381:Rats; D038601:Ribosomal Protein S6 / Q000235:genetics*; D012708:Sertoli Cells / Q000378:metabolism*; D013090:Spermatocytes / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D051378:Actin-Related Protein 3; D046912:Multiprotein Complexes; D038601:Ribosomal Protein S6; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D000076222:Mechanistic Target of Rapamycin Complex 1; D051058:Oncogene Protein v-akt; D058570:TOR Serine-Threonine Kinases