title: Modulation of the actin cytoskeleton via gelsolin regulates vacuolar H+-ATPase recycling.
abstract: 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.
authors.affiliation: Program in Membrane Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
authors.forename: Valérie
authors.initials: V
authors.lastname: Beaulieu
authors.forename: Nicolas
authors.initials: N
authors.lastname: Da Silva
authors.forename: Nuria
authors.initials: N
authors.lastname: Pastor-Soler
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Brown
authors.forename: Peter J S
authors.initials: PJ
authors.lastname: Smith
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
authors.forename: Sylvie
authors.initials: S
authors.lastname: Breton
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002645:Chickens; D000242:Cyclic AMP / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D047630:Depsipeptides / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004822:Epididymis / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D018260:Gelsolin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D017830:Octoxynol / Q000494:pharmacology; D010010:Osteoclasts / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010477:Perfusion; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D011522:Protons; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013094:Spermatozoa / Q000378:metabolism; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
chemical_list: D000199:Actins; D002614:Chelating Agents; D047630:Depsipeptides; D004791:Enzyme Inhibitors; D004963:Estrenes; D018260:Gelsolin; D010455:Peptides; D011108:Polymers; D011522:Protons; D011760:Pyrrolidinones; C057531:jasplakinolide; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D017830:Octoxynol; D000242:Cyclic AMP; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D002118:Calcium