Caveolin-1 recruitment to the trailing edge of motile cells results in focal adhesion disassembly and nascent interaction with actin stress fibers.

摘要

The protein caveolin-1 has been shown to positively affect angiogenesis and vascular remodeling in vivo via studies using knockout mice. In fact, defects in these two processes are among the major hallmarks of an otherwise benign caveolin-null phenotype. Current dogma on the function of caveolin-1 does not predict or account for these deficits. The overall objective of the following studies was to uncover the role of caveolin-1 in angiogenesis and vascular remodeling through study of the protein in cell-substratum remodeling during cell motility in vitro.; In the first study, caveolin-1 and its parent organelle, caveolae, conspicuously polarize to the rear of migrating human umbilical vein endothelial cells. Moreover, caveolin-1 localizated at the cell rear is mutually exclusive with focal adhesion staining and lamellipodial protrusion. Acute caveolin-1 knockdown by small, interfering RNA diminished the ability of endothelial cells to polarize and migrate toward a chemotactic stimulus.; In the second study, live cell imaging was used to study the dynamics between caveolin-1, focal adhesions, and the actin cytoskeleton. Caveolin-1 recruitment and transient association with focal adhesions at the trailing edge resulted in adhesion sliding and disassembly, concomitant with recoil of the trailing edge into the cell body proper. Moreover, association of caveolin-1 with actin stress fibers previously associated with adhesions in the collapsing trailing edge was observed. Mouse embryonic fibroblasts from caveolin-1 null mice demonstrated defects in trailing edge recoil compared to control cells with no decrease in cell contractility, suggesting a specific deficit in adhesion disassembly. Furthermore, caveolin-null cells displayed a decrease in overall chemokinetic motility and an increase in directional persistence, an indication that caveolin-1 contributes to movement plasticity via trailing edge focal adhesion disassembly.; In the final study, the interaction of polarized caveolin-1 with actin stress fibers at the cell rear was characterized. Caveolin-1 predictably associated with the cell perimeter depending on the direction of cell migration. Importantly, inhibition on non-muscle myosin by blebbistatin treatment abrogated initial polarization of caveolin-1, but did not affect caveolin-1 that had already polarized. Using live cell imaging in conjunction with photobleaching, actin-associated caveolin-1 was found to be extremely static upon polarization to the cell rear. In contrast, the initial polarization of caveolin-1 to retracting areas was highly dynamic. Furthermore, GM1 internalization at the cell rear was negligible, confirming that polarized caveolae are highly static. Forced disruption of the actin cytoskeleton by cytochalasin D treatment resulted in caveolin-1 depolarization and disaggregation into small puncta displaying frenetic, kiss-and-run movement. Furthermore, cytoskeletal remodeling in response to change in direction of a cell resulted in similar caveolin depolarization.; In summary, stress fibers associate with and exert traction on trailing edge focal adhesions during cell motility. This traction force is prerequisite for caveolin-1 recruitment. Arrival and transient association of caveolin-1 with focal adhesions results in adhesion disassembly and stable interaction of caveolin with actin stress fibers. Thus, a novel mechanism in cellular mechanotransduction can be described, whereby cells utilize caveolin-1 recruitment to relieve strain generated at the cell perimeter by the actin cytoskeleton during movement. This novel function of caveolin-1 may analogously occur in vivo, beyond the context of endothelial cell migration. The deficits in angiogenesis and vascular remodeling seen in caveolin-1 null mice might thus be explained by the role of caveolin-1 in cell-substratum remodeling in response to strain.