Abstract
The fibroblast-like synoviocyte is a CD13-positive cell-type containing numerous caveolae, both single and interconnected clusters. In unstimulated cells, all single caveolae at the cell surface and the majority of those localized deeper into the cytoplasm were freely accessible from the medium, as judged from electron microscopy of synoviocytes exposed to the membrane impermeable marker Ruthenium Red. Caveolar internalization could be induced by a CD13 antibody or by cholera toxin B subunit (CTB). Thus, in experiments using sequential labeling with Alexa 488- and 594-conjugated CTB, about 50% of CTB-positive caveolae were internalized by 5 min of chase, and these remained inaccessible from the cell surface for periods up to 24 h. No colocalization with an endosomal marker, EEA1, or Lysotracker was observed, indicating that internalized caveolae clusters represent a static compartment. Vimentin was identified as the most abundant protein in detergent resistant membranes (DRM's), and by immunogold electron microscopy caveolae were seen in intimate contact with intermediate-size filaments. These observations indicate that vimentin-based filaments are responsible for the spatio-temporal fixation of caveolae clusters. RECK, a glycosylphosphatidylinositol-anchored protein acting as a negative regulator of cell surface metalloproteinases, was also localized to the caveolae clusters. We propose that these clusters function as static reservoirs of specialized lipid raft domains where proteins involved in cell-cell interactions, such as CD13, can be sequestered by binding to RECK in a regulatory manner.