Mechano-chemical signaling mechanisms in fish epithelial keratocytes

Date of Completion

January 2004


Biology, Cell




The process of cell motility involves repeated cycles of protrusion and adhesion formation at the front, cell body translocation, and retraction at the rear. The regulation of these events with each other is essential for maintaining cell movement. In the fish epithelial keratocyte when protrusion at the front of the cell occurs without retraction at the rear, forward movement is impeded and slows motility rate. Thus, retraction becomes the rate-limiting step of keratocyte movement. Keratocyte, however, possess an interesting mechano-chemical signaling mechanism that involves the activation of stretch-activated calcium channels (SACS) in response to increasing cytoskeletal tension that occurs when retraction fails. The subsequent influx of calcium leads to a transient elevate of [Ca2+]i that triggers retraction at the trailing edge. Thus, SAC's maintain a rapid continuous mode of movement in the keratocyte. However, it remains unknown what changes in contractile force production accompany SAC-mediated calcium transients or what Ca 2+-dependent signaling mechanisms are involved in inducing retraction at the rear. Here, using a new gelatin-based traction force assay in combination with high-resolution calcium imaging we describe the spatio-temporal changes in force production associated with SAC-mediated calcium transients. We found that SAC-mediated calcium transients act to up-regulate contractility (increase traction stress) until retraction occurs causing a precipitous drop in traction stress. The elevation in traction stress has a distinct spatio-temporal appearance, rising initially at points of highest tension and propagating toward the leading edge. Calcium transients are associated with kinetic changes in traction stress together with cell speed and cell shape, which we arise form alternating periods of increased and decreased adhesiveness. Inhibition of calcium signaling led to increased cell adhesiveness as shown by increased traction stress, cell elongation, reduced cell speed and clustering of adhesion proteins, indicating the importance of SAC-mediated calcium transients in regulating cell retraction. Furthermore, altering myosin light-chain kinase (MLCK) activity greatly reduced cell speed and the ability to generate traction stress suggesting its importance in retraction. However, inhibition of Ca2+-independent contractility through Rho kinase led to a slow decrease in retraction rate and cell speed, consistent with it having a minor role in cell motility. Inhibition of calpain, a Ca2+-dependent protease, showed no significant initial affect on cell motility. Taken together, we conclude that in the keratocyte mechano-chemical regulation of retraction through SACs involves primarily the activation of MLCK to increase cellular contractility and detach the rear edge, while calcium signaling may act to up-regulate calpain and other Ca2+-sensitive pathways to disassemble adhesions when contractile force alone is not enough to rupture adhesions. ^