Anna L. Kiss

Introduction Cavolae on the plasma membrane Internalization of caveolae Conclusion Endocytosis – the uptake of extracellular ligands, soluble molecules, protein and lipids from the extracellular surface – is a vital process, comprising multiple mechanisms, including phagocytosis, macropinocytosis, clathrin‐dependent and clathrin‐independent uptake such as caveolae‐mediated and non‐caveolar raft‐dependent endocytosis. The best‐studied endocytotic pathway for internalizing both bulk membrane and specific proteins is the clathrin‐mediated endocytosis. Although many papers were published about the caveolar endocytosis, it is still not known whether it represents an alternative pathway with distinct cellular compartments to avoid lysosomal degradation or ligands taken up by caveolae can also be targeted to late endosomes/lysosomes. In this paper, we summarize data available about caveolar endocytosis. We are especially focussing on the intracellular route of caveolae and providing data supporting that caveolar endocytosis can join to the classical endocytotic pathway.

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.

Caveolae‐mediated endocytosis is a highly regulated endocytic pathway that exists in parallel to other forms of clathrin‐dependent and ‐independent endocytosis. Internalized caveolae accumulate in intermediate organelles called caveosomes. Here we addressed the further fate of internalized caveolae by inducing caveolae‐mediated uptake of albumin by HepG2 cells. We followed the route of internalized caveolin‐1 by immunogold labelling of ultrathin frozen sections and by Western blot analyses of purified membrane fractions. Long‐term (1 and 3 hrs) albumin treatment resulted in the appearance of albumin‐containing caveolae in special multi‐caveolar complexes (consisting of multiple caveolae clustered together) connected to the plasma membrane and caveosome‐like structures in the cytoplasm. In addition, numerous CD63 (LIMP‐1) positive late endosomes/multi‐vesicular bodies were found positive for caveolin‐1, suggesting that upon albumin incubation, caveolin‐1 is endocytosed and enters the degradative pathway. Surprisingly, the number of caveolae at the plasma membrane increased after addition of albumin. This increase was blocked by cycloheximide treatment, indicating that albumin internalization also stimulates de novo protein synthesis, which is necessary for new caveolae formation. Together, our results show that during long‐term albumin uptake, caveolin‐1 travels to late endosomes and is replaced by newly synthesized caveolin‐1 at the plasma membrane.