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.

Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

“…BK013, Denver, CO) as earlier described3942. In brief, Sertoli cell lysates obtained from cells in treatment vs. control groups were prepared in Tris lysis buffer [20 mM Tris, pH 7.5 at 22 °C, containing 20 mM NaCl and 0.5% Triton X-100, freshly supplemented with protease and phosphatase inhibitors] as described42. Cell debris was removed by centrifugation at 20,817  g for 1 h at 4 °C.…”

Transport of germ cells across the seminiferous epithelium during spermatogenesis—the involvement of both actin- and microtubule-based cytoskeletons

“…[18][19][20] Some are recently shown to be actively involved in F-actin organization at the basal ES at the Sertoli cell-cell interface, such as Akt1/2, 21,22 which was earlier shown to be a putative component of the apical and basal ES 23 in the rat testis. In short, the MT-based tracks are analogous to 2-way highways that modulate in-coming and outgoing traffic to maintain cellular homeostasis.…”

Regulation of the blood‐testis barrier by a local axis in the testis: role of laminin α2 in the basement membrane

“…Actin bundling assay using lysates of Sertoli cells was performed as previously described (25) by using Actin Binding Protein Spin‐Down Assay Biochem Kits from Cytoskeleton (Denver, CO, USA). In brief, 10 mlof celllysates from 6‐well dishes vs .…”

“…In brief, 10 mlof celllysates from 6‐well dishes vs . 10 ml Tris lysis buffer (serving as a negative control) was added to F‐actin microfilaments that were generated by using the kit at ∼21 μM, as detailed previously (25), to assess the ability of cell lysates to induce actin bundling. This mixture was centrifuged at 14, 000 g at 24°C for 5 min to sediment bundled F‐actin in the pellet, whereas linear and unbundled actin microfilaments remained in the supernatant.…”