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J. Biol. Chem., Vol. 280, Issue 9, 8452-8463, March 4, 2005

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Modulation of the Actin Cytoskeleton via Gelsolin Regulates Vacuolar H⁺-ATPase Recycling^*

Valérie Beaulieu, Nicolas Da Silva, Nuria Pastor-Soler, Christopher R. Brown, Peter J. S. Smith, Dennis Brown¶, and Sylvie Breton¶||

From the Program in Membrane Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, the ^¶Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, and the BioCurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543

The role of the actin cytoskeleton in regulating membrane protein trafficking is complex and depends on the cell type and protein being examined. Using the epididymis as a model system in which luminal acidification is crucial for sperm maturation and storage, we now report that modulation of the actin cytoskeleton by the calcium-activated actin-capping and -severing protein gelsolin plays a key role in regulating vacuolar H⁺-ATPase (V-ATPase) recycling. Epididymal clear cells contain abundant V-ATPase in their apical pole, and an increase in their cell-surface V-ATPase expression correlates with an increase in luminal proton secretion. We have shown that apical membrane accumulation of V-ATPase is triggered by an elevation in cAMP following activation of bicarbonate-regulated soluble adenylyl cyclase in response to alkaline luminal pH (Pastor-Soler, N., Beaulieu, V., Litvin, T. N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L. R., and Breton, S. (2003) J. Biol. Chem. 278, 49523-49529). Here, we show that clear cells express high levels of gelsolin, indicating a potential role in the functional activity of these cells. When jasplakinolide was used to overcome the severing action of gelsolin by polymerizing actin, complete inhibition of the alkaline pH- and cAMP-induced apical membrane accumulation of V-ATPase was observed. Conversely, when gelsolin-mediated actin filament elongation was inhibited using a 10-residue peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate-binding region (phosphoinositide-binding domain 2) of gelsolin, significant V-ATPase apical membrane mobilization was induced, even at acidic luminal pH. In contrast, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) and the phospholipase C inhibitor U-73122 inhibited the alkaline pH-induced V-ATPase apical accumulation. Thus, maintenance of the actin cytoskeleton in a depolymerized state by gelsolin facilitates calcium-dependent apical accumulation of V-ATPase in response to luminal pH alkalinization. Gelsolin is present in other cell types that express the V-ATPase in their plasma membrane and recycling vesicles, including kidney intercalated cells and osteoclasts. Therefore, modulation of the actin cortex by this severing and capping protein may represent a common mechanism by which these cells regulate their rate of proton secretion.

Received for publication, November 10, 2004

^* This work was supported by National Institutes of Health Grants HD40793 (to S. B.), DK38452 (to D. B. and S. B.), DK42956 (to D. B.), P41-RR001395 (to P. J. S. S.), and KO8-HD45524 (to N.P.-S.) and National Research Service Award HD08684 (to N. P.-S.). The work performed in the Microscopy Core Facility of the Massachusetts General Hospital Program in Membrane Biology was supported by Center for the Study of Inflammatory Bowel Disease Grant DK43351 and Boston Area Diabetes and Endocrinology Research Center Award DK57521. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence and reprint requests should be addressed: Program in Membrane Biology, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-5785; Fax: 617-726-5669; E-mail: sbreton{at}partners.org.

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