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© The Rockefeller University Press, 0021-9525/2001//1427 $5.00
The Journal of Cell Biology, Volume 153, Number 7, , 2001 1427-1440

Differential Dynamics of 5 Integrin, Paxillin, and -Actinin during Formation and Disassembly of Adhesions in Migrating Cells

^a Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801
^b Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908
Dept. of Cell Biology, UVA School of Medicine, P.O. Box 800732, Charlottesville, VA 22908-0732 (for express mail add 1300 Jefferson Park Ave.).(804) 982-3912(804) 243-6813

horwitz{at}virginia.edu

To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed 5 integrin, -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. 5-GFP also rescued the adhesive defects in CHO B2 cells, which are 5 integrin deficient. In ruffling cells, 5-GFP and -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 appeared to remodel from older to newer adhesions at the leading edge. -Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of -actinin–GFP and paxillin-GFP, which grew in size. Subsequently, 5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. 5-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, 5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The 5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, 5-GFP was found in fibrous structures behind the cell, whereas -actinin–GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.

Key Words: cell adhesion • endocytosis • cytoskeleton • membrane protrusions • vesicle trafficking

© 2001 The Rockefeller University Press

C.M. Laukaitis and D.J. Webb contributed equally to this work.

Abbreviations used in this paper: CFP, cyan fluorescent protein; ECFP, enhanced CFP; ECM, extracellular matrix; EGFP, enhanced GFP; EYFP, enhanced YFP; Fn, fibronectin; GFP, green fluorescent protein; YFP, yellow fluorescent protein.

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