REGULATED INTERNALIZATION OF CAVEOLAE

Caveolae are specialized invaginations of the plasma membrane which ha ve been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to det ermine the fraction of internal versus plasma membrane caveolae. The G PI-anchored protein, alkaline phosphatase, was clustered in caveolae a fter antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phos phatase activity which was dependent on antibody-induced clustering. T he decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a p rotein kinase C activator. Internalization of clustered alkaline phosp hatase in the presence or absence of okadaic acid was blocked by cytoc halasin D and by the kinase inhibitor staurosporine. Electron microsco py confirmed that okadaic acid induced removal of caveolae from the ce ll surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell clo se to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clus ters was shown to be dependent on an intact microtubule network. Altho ugh the exact mechanism of internalization remains unknown, the result s show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and re quire an intact actin network.