Physical and biochemical regulation of integrin release during rear detachment of migrating cells.

Cell migration can be considered as a repeated cycle of membrane protrusion and attachment, cytoskeletal contraction and rear detachment. At intermediate and high levels of cell-substratum adhesiveness, cell speed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (ECM) 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 investigate 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 hamster ovary (CHO) cells. Integrin expression level was varied by transfecting CHO B2 cells, which express very little endogenous alpha5 integrin, with a plasmid containing human alpha5 integrin cDNA and sorting the cells into three populations with different alpha5 expression levels. Receptor/ligand affinity was varied using CHO cells transfected with either alphaIIbbeta3 or alphaIIbbeta3(beta1-2), a high affinity variant. alphaIIbbeta3(beta1-2) is activated to a higher affinity state with an anti-LIBS2 antibody. Fluorescent probes were conjugated to non-adhesion perturbing anti-integrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip. The rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence microscopy and a cooled CCD camera. We find that rear retraction rate appears to limit cell speed at intermediate and high adhesiveness, but not at low adhesiveness. Upon rear retraction, the amount of integrin released from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase. In fact, integrin release is a constant function of cell-substratum adhesiveness and the number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an inverse relationship to the amount of integrin released at the rear of the cell. At high cell-substratum adhesiveness, calpain, a Ca2+-dependent protease, is also involved in release of cytoskeletal linkages during rear retraction. Inhibition of calpain results in decreased integrin release from the cell membrane, and consequently a decrease in cell speed, during migration. These observations suggest a model for rear retraction in which applied tension and calpain-mediated cytoskeletal linkage cleavage are required at high adhesiveness, but only applied tension is required at low adhesiveness.