Integrin-mediated Adhesion Regulates Cell Polarity and Membrane Protrusion through the Rho Family of GTPasesV⃞

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through α5β1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.

Protrusive growth from giant liposomes driven by actin polymerization

Development of protrusions in the cell is indispensable in the process of cell motility. Membrane protrusion has long been suggested to occur as a result of actin polymerization immediately beneath the cell membrane at the leading edge, but elucidation of the mechanism is insufficient because of the complexity of the cell. To study the mechanism, we prepared giant liposomes containing monomeric actin (100 or 200 μM) and introduced KCl into individual liposomes by an electroporation technique. On the electroporation, the giant liposomes deformed. Most importantly, protrusive structure grew from the liposomes containing 200 μM actin at rates (ranging from 0.3 to 0.7 μm/s) similar to those obtained in the cell. The deformation occurred in a time range (30 ∼ 100 s) similar to that of actin polymerization monitored in a cuvette (ca. 50 s). Concomitant with deformation, Brownian motion of micron-sized particles entrapped in the liposomes almost ceased. From these observations, we conclude that actin polymerization in the liposomes caused the protrusive formation.

DdLIM Is a Cytoskeleton-associated Protein Involved in the Protrusion of Lamellipodia in Dictyostelium

DdLim, a multi-domain member of the cysteine-rich family of LIM domain proteins, was isolated from Dictyostelium cells where it localizes in lamellipodia and at sites of membrane ruffling. The transcription and expression of DdLim are developmentally regulated, and the timing of its increased association with the actin cytoskeleton coincides with the acquisition in starved cells of a motile, chemotactic behavior. Vegetative cells that overexpress DdLim contain large lamella and exhibit ruffling at the cortex. The high frequency of large, multinucleated mutant cells found in suspension culture suggests that excess DdLim interferes with cytokinesis. DdLim was also identified as a protein in a Dictyostelium cell lysate that associated indirectly, but in a guanosine triphosphate-dependent manner, with a GST-rac1 fusion protein. The data presented suggest that DdLim acts as an adapter protein at the cytoskeleton-membrane interface where it is involved in a receptor-mediated rac1-signaling pathway that leads to actin polymerization in lamellipodia and ultimately cell motility.