{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T22:18:06Z","timestamp":1773958686195,"version":"3.50.1"},"reference-count":70,"publisher":"American Physiological Society","issue":"1","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["American Journal of Physiology-Cell Physiology"],"published-print":{"date-parts":[[2011,7]]},"abstract":"<jats:p> Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H<jats:sup>+<\/jats:sup>-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of \u201cresting\u201d clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10\u201330 \u03bcM) and HA1077 (30 \u03bcM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 \u03bcg\/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 \u03bcM) or HA1077 (30 \u03bcM) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation. <\/jats:p>","DOI":"10.1152\/ajpcell.00198.2010","type":"journal-article","created":{"date-parts":[[2011,3,17]],"date-time":"2011-03-17T01:12:05Z","timestamp":1300324325000},"page":"C31-C43","source":"Crossref","is-referenced-by-count":30,"title":["Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells"],"prefix":"10.1152","volume":"301","author":[{"given":"Winnie Waichi","family":"Shum","sequence":"first","affiliation":[{"name":"Center for Systems Biology, Program in Membrane Biology\/Nephrology Division, Massachusetts General Hospital and"},{"name":"Harvard Medical School, Boston, Massachusetts"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicolas","family":"Da Silva","sequence":"additional","affiliation":[{"name":"Center for Systems Biology, Program 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