Differential dynamics of α5 integrin, paxillin, and α-actinin during formation and disassembly of adhesions in migrating cells

To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed alpha5 integrin, alpha -actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha5 integrin deficient. In ruffling cells, alpha5-GFP and alpha -actinin-GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appear ed to remodel from older to newer adhesions at the leading edge. ol-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of alpha -actinin-CFP and paxillin-GFP, which grew in size. Subsequently, alpha5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions, alpha5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body As cells migrated, alpha5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The alpha5 vesicles colocalized with transferrin receptor and FW 4-64 dye. After adhesions broke down in the rear, alpha5-GFP was found in fibrous structures behind the cell, whereas alpha -actinin-GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.