DOWN-REGULATION OF TALIN ALTERS CELL-ADHESION AND THE PROCESSING OF THE ALPHA-5-BETA-1 INTEGRIN

The role of talin was addressed by down regulating its expression usin g an antisense RNA strategy, HeLa cells were transfected with a talin 5' cDNA fragment under the control of the inducible human metallothion ein promotor, Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slo wed down the kinetics of cell spreading. Mock-transfected cells, sprea d out onto fibronectin, exhibited large peripheral adhesion plaques, I n contrast, cells with reduced talin expression showed smaller focal c ontacts localized all over the ventral face, and displayed a marked de crease in the number of stress fibers, Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor in dicated a shift in the mobility for both alpha 5 and beta 1 subunits. Surprisingly, beta 1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clo nes, Western blot analysis indicated the presence of two forms of beta 1. we analyzed the processing of beta 1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature beta 1, while the talin defici ent AT22 clone showed that the beta 1 precursor was slowly converted i nto a very low molecular mass product, Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts, In addition, down regulation of talin impairs the folding and processing of beta 1 integrins.