INTRACELLULAR PROCESSING OF TALIN OCCURS WITHIN FOCAL ADHESIONS

CHO cells spread out on fibronectin-coated plastic were perforated wit h the bacterial toxin alveolysin. This treatment preserves the integri ty of the cells but opens large and stable hydrophilic pores on the pl asma membrane. With these semi-intact cells, it has been possible to h ave a direct access to adhesion plaques. Furthermore, with this proced ure one can determine the distribution of a single intracellular prote in between membrane-associated and cytosolic pools. The introduction w ithin the perforated cells of polyclonal antibodies raised against tal in induced the detachment of the cells. This provides direct evidence that talin is required to stabilize the adhesion plaques. Furthermore, immunoprecipitation of talin in the membrane-associated and cytosolic fractions indicated that the membrane-associated talin was cleaved an d reduced to a stable 200-kDa fragment. Conversely, soluble talin rema ined intact. This 200-kDa fragment was similar to the proteolytic frag ment produced from talin by calpain II. Analysis of whole cell extract s and pulse chase experiments indicated that this proteolysis also occ ured in vivo, although to a smaller extent. The kinetics of the limite d proteolytic cleavage of talin suggested that the 200-kDa fragment wa s first produced within focal adhesion, and secondarily released into the cytosol. (C) 1995 Academic Press, Inc.