Regulation of integrin mobility and cytoskeletal association in normal and RSV-transformed chick embryo fibroblasts

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Abstract
The mobility of the integrin receptor in trypsinised chick embryo fibroblasts (CEF) was investigated using the CSAT monoclonal antibody. The binding of CSAT to trypsinised CEF followed by incubation at 37°C resulted in patching and then capping of the receptor. This capping was dependent on cellular metabolism, since agents such as sodium azide or 2-deoxyglucose inhibited the process. Whereas about 95% of unclustered integrin was soluble in Nonidet P40-containing buffers, after capping more than 25% of surface integrin became detergent-insoluble, indicating a physical association with cytoskeletal elements. Thus the crosslinking of integrin via its βsubunit is sufficient, to induce cytoskeletal association. Unusually, the microfilament-disrupting drugs cytochalasins B and D potentiated CSAT-induced capping in terms of both cell number and the conformation of caps on individual cells. Double immunofluorescent staining demonstrated that in cytochalasin-treated cells both F-actin and talin colocalised with surface CSAT-integrin clusters. The co-distribution of these cytoskeletal components with surface integrin was retained in cytoskeletal preparations, although there was no quantitative increase of either talin or vinculin in the cytoskeletons. The cocapping of talin with integrin clusters on CEF could also be observed in the absence of cytochalasins. No differences were found in capping efficiency, talin and actin co-localisation or cytoskeletal association of surface-modulated integrin in Rous sarcoma virus (RSV)-transformed cells compared with untransformed counterparts, although differences in the response to cytochalasins were observed. These results provide novel evidence for a physiologically relevant association of integrin with cytoskeletal components and its regulation by surface configuration. The importance of this regulation, and the influence of cytoskeleton-specific agents and transformation by RSV are discussed.