Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell.

M Samuels,R M Ezzell,T J Cardozo,J L Coll,D R Critchley,E D Adamson

doi:10.1083/jcb.121.4.909

Abstract

A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell-substrate adhesion. We have previously shown that neither uvomorulin (E-cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F-actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins.

Highlights

A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cellcell adhesion and in cell-substrate adhesion
In wild-type ceils, F-actin is organized into stress fibers that terminate at focal contacts containing vinculin (Fig. 2, C and D). 5.51 cells do not have stress fibers and do not stain with vinculin antibodies (Fig. 2, E and F)
Wild-type F9 cells observed 15 min after seeding have already begun to attach to a gelatin-coated substrate and while still rounded in shape are able to organize vinculin into short nascent focal adhesion points (Fig. 2 A) even though actin stress fibers are not apparent (Fig. 2 B). These findings suggest that vinculin is required for attachment and spreading as well as for the formation of stress fibers

Summary

Introduction

A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cellcell adhesion (compaction) and in cell-substrate adhesion. 5.51Vin and Vin, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques Both clones adhered well to substrates and both exhibited F-actin stress fibers with vinculin localized at stress fiber tips in focal contacts. ARLIERstudies have established that the cytoplasmic domains of two transmembrane glycoproteins, uvomorulin (Nagafuchi and Takeichi, 1988) and/31 integrins (Hayashlet al., 1990; Marcantonio et al, 1990; Reszka et al, 1992) are essential for transmembrane linkage to the cytoskeleton and for cell adhesion The former glycoprotein links cells together in tight aggregates and epithelial sheets, the latter attaches cells to the extraceUular matrix. RA treatment of 5.51 cells results in only parietal-type endoderm differentiation; epithelial layers of polarized visceral endoderm cells are not seen (Grover et al, 1987)

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Conclusion