Cellular invasion into matrix beads: localization of beta 1 integrins and fibronectin to the invadopodia.
We have examined the contribution of adhesion mechanisms to cell invasiveness by growing chicken embryo fibroblasts (CEF) or Rous sarcoma virus-transformed cells (RSVCEF) on fibronectin-coated crosslinked gelatin beads (FN-beads). RSVCEF attached more readily and spread more rapidly on FN-beads than CEF, suggesting an increase in the adhesion-related motility of the transformed cells. In addition, RSVCEF invaded the FN-beads, but CEF did not, by extending specialized cell surface protrusions called invadopodia at sites of cell invasion. FN removal by RSVCEF cultured on prelabeled fluorescent FN-beads (FL-FN) was evident at sites of invadopodia, and internalized FL-FN occurred in vacuoles near the ventral membrane of cells at sites of FN removal. The precise distribution of FN and integrins in cells invading FN-beads was determined by immunofluorescence and immunoelectron microscopy of frozen thin-sections. In both CEF and RSVCEF, beta 1 integrins and FN occupied separate intracellular compartments during the early stage of spreading on FN-beads. Later, beta 1 integrins were largely localized at the ventral cell surface of both CEF and RSVCEF. Polyclonal anti-integrin antibody recognizing beta 1 and several alpha chains, however, labeled both ventral and dorsal cell surfaces. During invasion by RSVCEF, beta 1 integrins were concentrated at extended invadopodia and also colocalized with internalized FL-FN material in phagocytic vesicles. Furthermore, secreted FN was deposited by RSVCEF at the base of invadopodia colocalizing with beta 1 integrin. Both FL-FN matrix removal and formation of the invadopodia were found to be resistant to treatment with GRGDS at concentrations that inhibit the interaction between cells and FN-beads. Thus, the localization of beta 1 integrins to the plasma membrane contacting immobilized FN results in an extremely tight cellular adherence to the matrix bead, that stabilizes invadopodia and also mediates endocytic clearance of degraded FN-matrix material.
Mueller
S C
SC
Department of Anatomy and Cell Biology, Georgetown University School of Medicine, Washington, DC 20007.
Chen
W T
WT
D000818:Animals; D002448:Cell Adhesion; D002455:Cell Division; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts / Q000166:cytology; D005353:Fibronectins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D016023:Integrins / Q000378:metabolism*; D008863:Microspheres; D010587:Phagocytosis
D005353:Fibronectins; D016023:Integrins