Cytoskeleton changes in fibroblast adhesion and detachment

Abstract

The organization of the cytoskeleton in several anchorage-dependent fibroblast types has been compared with the pattern of adhesions to a glass substratum which will support either their growth or just their spreading. Components were stained separately for immunofluorescence microscopy using specific antisera against actin, tubulin, and gizzard 10-nm filament protein, and the adhesions were visualized by interference-reflexion microscopy. Of the cytoskeleton features, only stress fibres could be related to the pattern of focal adhesions; as shown before, each focal adhesion lies directly beneath a stress fibre, often near the terminus. Cells spread on fibronectin-treated glass in serum-free medium to arrest the development of focal adhesions, show correspondingly underdeveloped stress fibres. Actin geodesic domes, microtubules, and 10-nm filaments showed no relations with the adhesion pattern. During cell rounding leading to detachment with either EGTA or trypsin, stress fibres begin to disperse in advance of shape change whereas microtubules and 10-nm filaments seem to alter their distribution as a consequence of shape change. We therefore confirm that stress fibres are the cytoskeleton features most directly related to focal adhesions and are cytoskeleton targets for 2 agents which cause rounding and hence detachment. The sequences of events in dispersal of stress fibres by EGTA and by trypsin showed significant differences in detail. With trypsin, fibres higher in the cell and terminating at the cell edge were more sensitive than most basal fibres and, during disintegration, all types of fibre went through an intermediate ‘beaded’ structure. With EGTA, all stress fibres seemed to be similarly susceptible and the beaded stage was not seen. The implications of these differences for our understanding of the mechanisms of dispersal of stress fibres are discussed.