Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton

Live cell, time-lapse microscopy was used to study trafficking of caveolin-1-GFP in stably expressing CHO cells. Multiple cytological and biochemical tests verified that caveolin-1-GFP was a reliable marker for endogenous caveolin-1. At steady state, most caveolin-1-GFP was either at the cell surface associated with invaginated caveolae or near the centrosome in caveosomes. Live cell fluorescence imaging indicated that while much of the caveolin-1-GFP in caveolae at the cell surface was relatively sessile, numerous, highly motile caveolin-1-GFP-positive vesicles were present within the cell interior. These vesicles moved at speeds ranging from 0.3-2 μm/second and movement was abolished when microtubules were depolymerized with nocodazole. In the absence of microtubules, cell surface invaginated caveolae increased more than twofold and they became organized into linear arrays. Complete depolymerization of the actin cytoskeleton with latrunculin A, by contrast, triggered rapid and massive movements of caveolin-positive structures towards the centrosomal region of the cell. The caveolar membrane system of CHO cells therefore appears to be comprised of three caveolin-1-containing compartments. These include caveolae that are confined to the cell surface by cortical actin filaments, the peri-centrosomal caveosomes and caveolar vesicles, which we call 'cavicles', that move constitutively and bi-directionally along microtubules between the cell surface and caveosomes. The behavior of cavicles suggests that they function as transport intermediates between caveolae and caveosomes.