Ocadaic acid retains caveolae in multicaveolar clusters.

Caveola-mediated endocytosis exists parallel to other forms of endocytosis. Being ligand-triggered, caveolar endocytosis provides a more selective and highly regulated way for uptake of specified substances. Internalized caveolae accumulate in intermediate organelles called caveosomes. It is still debated whether caveosomes are independent organelles or the downstream caveosomes interact with the classical endocytotic compartments. In our work caveola internalization was stimulated with a serine/threonine phosphatase (PP1 and PP2A) inhibitor (ocadaic acid-OA). To find out whether caveolar clusters are really independent organelles or they are still connected to the cell surface we used an electron dense surface marker, ruthenium red (Ru red). Since we were especially interested in the fate of caveolar clusters, the cells were treated with OA for longer time. Stimulating caveola-mediated endocytosis, OA treatment resulted in a significant increase in the number of caveolar cluster. Most of these clusters were found Ru red positive indicating that they were still conneted to the cell surface. Our double labeling experiments on ultrathin frozen sections clearly showed that in OA-treated cells caveolae are not transported to late endosomes instead they are accumulted in large multicaveolar clusters. We think that PP2A can be one of the key components to regulate the fusion of various endocytotic compartments and /or the trafficking along the microtubules.