Cellular invasion into matrix beads: Localization of β1 integrins and fibronectin to the invadopodia

Abstract

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, β1 integrins and FN occupied separate intracellular compartments during the early stage of spreading on FN-beads. Later, β1 integrins were largely localized at the ventral cell surface of both CEF and RSVCEF. Polyclonal anti-integrin antibody recognizing β1 and several a chains, however, labeled both ventral and dorsal cell surfaces. During invasion by RSVCEF, β1 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 β1 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 β1 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.