The amoeboid migration of tumor cells is characterized by protrusion of pseudopodia. These specialized regions of the cell cortex thus constitute the basic machinery of cell locomotion. Type IV collagen stimulates chemotaxis of human melanoma cells through a G protein-coupled receptor; however, the downstream effectors of G protein activation which ultimately cause pseudopodial protrusion and cell translocation are unknown. We have isolated chemoattractant-induced pseudopodia from 1 micromole-pore chemotaxis filters after mechanical shearing from the tumor cell bodies. Analysis by Western immunoblot has revealed that the pseudopodial extracts have an increased ratio of Gia2 to Giao, relative to lysates from unstimulated whole cells. Thus, it appears that Gao is sequestered out of pseudopods, and may not be involved in transducing motility signals. Western immunoblotting has confirmed the presence of actin and the 280 kD actin binding protein in pseudopodial extracts, supporting the premise that pseudopodia contain actin cross-linked into a three dimensional, orthogonal network. A micropipette system was used to follow the dynamics of pseudopodial protrusion in an individual tumor cell stimulated by type IV collagen, with and without an inhibitor of signal transduction (PT). In untreated cells, type IV collagen induced extension of irregularly- shaped pseudopodia. Treatment of cells with PT, which inhibits G protein- linked signals but not the type IV collagen-induced Ca+2 burst, inhibited extension of the pseudopod while allowing formation of a much smaller, symmetrical membrane bleb. Conceivably this is due to Ca+2-activated actin disassembly and osmotic fluid flux. Thus, two stages of pseudopod protrusion are revealed which are mediated by separate intracellular signals. Goals of future studies are to identify the signals and effector proteins regulating pseudopodial protrusion and cell motility.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01CB009185-05
Application #
3752086
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Division of Cancer Biology and Diagnosis
Department
Type
DUNS #
City
State
Country
United States
Zip Code