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.
Highlights
EXPERIMENTAL PROCEDURESTissue Fixation and Immunofluorescence—Adult male SpragueDawley rats (Charles River Laboratories, Wilmington, MA) were anesthetized using an intraperitoneal sodium pentobarbital injection of 10 mg/100 g of body weight
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 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
Summary
Tissue Fixation and Immunofluorescence—Adult male SpragueDawley rats (Charles River Laboratories, Wilmington, MA) were anesthetized using an intraperitoneal sodium pentobarbital injection of 10 mg/100 g of body weight. After incubation with chicken anti-V-ATPase antibody and donkey anti-chicken IgG coupled to fluorescein isothiocyanate, the sections were incubated with anti-HRP polyclonal antibody (Jackson ImmunoResearch Laboratories, Inc.) at a 1:5000 dilution, followed by Cy3-conjugated goat anti-rabbit IgG. Triton X-100-soluble and -insoluble fractions were prepared based on a previous publication [70] The epithelia from both control and jasplakinolide-treated vas deferens were freeze-thawed three times in 100 l of PBS containing protease inhibitors (Complete mini, Roche Diagnostics) and centrifuged at 16,000 ϫ g for 17 min. Western blotting was performed using monoclonal antibody raised against chicken gizzard actin (1:2000 dilution), followed by HRP-conjugated goat antimouse IgG (1:10,000 dilution; Pierce). Effect of Jasplakinolide on V-ATPase Expression—The vas deferens and distal cauda epididymidis were perfused in vivo as described above with PBS containing either 10 M jasplakinolide or the vehicle alone (methanol). The same membranes were blotted with ␣-tubulin monoclonal antibody (1:20,000 dilution; Chemicon International, Inc.)
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