PHYSICAL AND BIOCHEMICAL REGULATION OF INTEGRIN RELEASE DURING REAR DETACHMENT OF MIGRATING CELLS

Cell migration can be considered as a repeated cycle of membrane protr usion and attachment, cytoskeletal contraction and rear detachment, At intermediate and high levels of cell-substratum adhesiveness, cell sp eed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (EC M) linkages. Often, cytoskeletal linkages fracture to release integrin adhesion receptors from the cell. Cell-extracellular matrix bonds may also dissociate, allowing the integrins to remain with the cell, To i nvestigate molecular mechanisms involved in fracturing these linkages and regulating cell speed? we have developed an experimental system to track integrins during the process of rear retraction in Chinese hams ter ovary (CHO) cells, Integrin expression level was varied by transfe cting CHO B2 cells, which express very little endogenous alpha 5 integ rin, with a plasmid containing human alpha 5 integrin cDNA and sorting the cells into three populations with different alpha 5 expression le vels, Receptor/ligand affinity was varied using CHO cells transfected with either alpha IIb beta 3 or alpha IIb beta 3(beta 1-2), a high aff inity variant. alpha IIb beta 3(beta 1-2) is activated to a higher aff inity state with an anti-LIBS2 antibody. Fluorescent probes were conju gated to non-adhesion perturbing antiintegrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip, T he rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence mic roscopy and a cooled CCD camera. We find that rear retraction rate app ears to limit cell speed at intermediate and high adhesiveness, but no t at low adhesiveness. Upon rear retraction, the amount of integrin re leased from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase, In fact, integri n release is a constant function of cell-substratum adhesiveness and t he number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an invers e relationship to the amount of integrin released at the rear of the c ell, At high cell-substratum adhesiveness, calpain, a Ca2+-dependent p rotease, is also involved in release of cytoskeletal linkages during r ear retraction. Inhibition of calpain results in decreased integrin re lease from the cell membrane, and consequently a decrease in cell spee d, during migration, These observations suggest a model for rear retra ction in which applied tension and calpain-mediated cytoskeletal linka ge cleavage are required at high adhesiveness, but only applied tensio n is required at low adhesiveness.