2 PHASES OF PSEUDOPOD PROTRUSION IN TUMOR-CELLS REVEALED BY A MICROPIPETTE

Pseudopod protrusion at the leading edge is a characteristic of migrating tumor cells as they traverse vascular subendothelial basement membranes to establish metastases. A micropipette system has been developed to study the dynamics of pseudopod protrusion in individual human melanoma cells in response to type IV collagen, a component of basement membranes. Soluble type IV collagen stimulates chemotaxis of A2058 melanoma cells through a G protein-coupled receptor, and induces an early burst of intracellular calcium (Savarese et al., 1992, J. Biol. Chem.267, 21928-21935). A micropipette filled with type IV collagen solution (100 μg/ml) was positioned so that the tip was adjacent to a cell suspended in Dulbeceo’s modified Eagle’s medium. Within 10 min, tumor cells generated a pseudopod which entered the micropipette with an average velocity of 0.24 μm/min and proceeded to lengthen for 40 min. Pseudopods from individual cells ranged from 7.5-10 μm at this time and were characterized by an irregular shape which did not fill the lumen of the micropipette. Pretreatment of cells with pertussis toxin (0.5 μg/ml), which inhibits cell migration by ∼90% (but not the calcium burst), blocked formation of the irregular, extended pseudopod, while allowing a much smaller outpouching, or bleb, to form. Lengths of such blebs from individual PT-treated cells reached a plateau at ∼20 min and ranged from 2.2-4.0 μm at the end point. Treatment of cells with bis-(amino-phenoxy)ethane tetraacetic acid (75 μM), an intracellular Ca2+ chelator, blocked initial bleb formation and prevented extension. From these observations we hypothesize that tumor cell pseudopod protrusion induced by soluble type IV collagen takes place in distinct, separable phases: an initial convex, symmetrical outpouching, caused by localized Ca+2-activated actin depolymerization and osmotic flux, followed by an extension with an irregular shape, which requires G protein-mediated actin polymerization. © 1994 Academic Press.