Antibody mapping of functional domains in vinculin.
We have analyzed the functional domain structure of vinculin, a protein involved in linking microfilaments to the cytoplasmic face of cell membranes in animal cells. For this purpose, we used several monoclonal antibodies raised against chicken gizzard vinculin whose epitopes could be assigned to discrete regions in the vinculin sequence by immunoblotting of proteolytic fragments combined with N-terminal amino acid sequencing. Two of these antibodies induced the disruption of stress fibers and changed the number of morphology of focal contacts after microinjection in chicken embryo fibroblasts. Based on the location of its epitope in comparison with vinculin domains previously identified by other groups, we propose that one of these antibodies (15B7) interferes with the binding of vinculin to talin, the most peripheral of the microfilament proteins. The second antibody (14C10) binds within a region comprising three internal repeats and might therefore distort the inner architecture of vinculin. A third antibody (As3) inhibited the binding of F-actin to vinculin in an in vitro assay but had no effect on the microfilament system in cells. These data emphasize the role of vinculin as a key protein in microfilament-membrane linkage and support previous work on a direct interaction between vinculin and actin.
Developmental Biology Unit, University of Bielefeld, FRG.
A
A
Westmeyer
K
K
Ruhnau
A
A
Wegner
B M
BM
Jockusch
D000199:Actins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D002642:Chick Embryo; D002645:Chickens; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005347:Fibroblasts / Q000378:metabolism; D005895:Gizzard, Avian / Q000378:metabolism; D015151:Immunoblotting; D007074:Immunoglobulin G; D009130:Muscle, Smooth / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D015183:Restriction Mapping; D016596:Vinculin
D000199:Actins; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003598:Cytoskeletal Proteins; D000939:Epitopes; D007074:Immunoglobulin G; D010446:Peptide Fragments; D016596:Vinculin