Two 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 micrograms/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 micron/min and proceeded to lengthen for 40 min. Pseudopods from individual cells ranged from 7.5-10 microns 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 microgram/ml), which inhibits cell migration by approximately 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 approximately 20 min and ranged from 2.2-4.0 microns at the end point. Treatment of cells with bis-(amino-phenoxy)ethane tetraacetic acid (75 microM), 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.
Dong
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Bioengineering Program, College of Engineering, Pennsylvania State University, University Park 16802.
Aznavoorian
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Liotta
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D002118:Calcium / Q000502:physiology; D002633:Chemotaxis / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology; D003584:Cytological Techniques / Q000295:instrumentation*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008545:Melanoma / Q000473:pathology*; D008846:Micromanipulation; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000648:ultrastructure*; D014407:Tumor Cells, Cultured
D004533:Egtazic Acid; D003094:Collagen; D019204:GTP-Binding Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium