MONOCLONAL-ANTIBODIES RECOGNIZING THE N-TERMINAL AND C-TERMINAL REGIONS OF TALIN DISRUPT ACTIN STRESS FIBERS WHEN MICROINJECTED INTO HUMAN FIBROBLASTS

We have characterized a panel of 6 monoclonal antibodies raised agains t human platelet talin by Western blotting, immune precipitation, and immunofluorescence, and shown that antibodies TA205 and TD77 disrupt a ctin stress fibers and focal adhesions, and inhibit cell motility when microinjected into human fibroblasts. Using a series of chick talin f usion proteins spanning the entire length of the molecule, we have map ped the epitopes recognized by these antibodies to the conserved N- an d C-terminal regions of the protein. TA205 bound to an epitope contain ed within residues 139-433, a region which overlaps an F-actin binding site, and which shows homology with the ezrin/radixin/moesin family o f cytoskeletal proteins. The epitope recognized by TD77 was located wi thin the C-terminal region of the protein (residues 2269-2541) which a lso contains an F-actin binding site homologous to that in the yeast a ctin-binding protein SIa2p. To investigate the possibility that TD77 d isrupts actin stress fibers by binding directly to the C-terminal acti n binding site, additional talin fusion proteins were generated and an alyzed for TD77 and actin binding. Fusion proteins containing residues 2269-2541, 2304-2541, and 2304-2463 all cosedimented with F-actin, wh ereas TD77 did not recognize the latter fusion protein. These results show that the C-terminal actin-binding site is distinct from the regio n recognized by the anti-functional antibody TD77, raising the possibi lity that it binds to a novel functionally important ligand-binding si te in the talin molecule. (C) 1997 Wiley-Liss, Inc.