Functional studies of the domains of talin.
The protein talin has two domains of approximately 200 and 47 kD, which can be cleaved apart by a variety of proteases. To examine the function of these two structural domains of talin, we have digested purified talin with a calcium-dependent protease and separated the resulting fragments chromatographically. Both fragments were radioiodinated and used to probe Western blots of whole fibroblasts and chicken gizzard extracts. The large talin fragment bound to vinculin and metavinculin. The small fragment did not demonstrate any binding in this assay. The fragments were labeled fluorescently and microinjected into fibroblasts in tissue culture. The large talin fragment incorporated quickly into focal adhesions where it remained stable for at least 14 h. The small fragment associated with focal adhesions of fibroblasts but was also distributed diffusely in the cytoplasm and the nucleus. These experiments suggest that talin has at least two sites that contribute to its localization in focal adhesions. Intact talin microinjected into Madin-Darby bovine kidney epithelial cells localized to the focal adhesions but was excluded from the zonulae adherentes, despite the localization of vinculin to both of these sites. In contrast, the large talin fragment, when microinjected into these epithelial cells, incorporated into both focal adhesions and zonulae adherentes. The difference in localization between the large talin fragment and intact talin seems to be due to the removal of the small domain. This difference in localization suggests that talin binding sites in zonulae adherentes have limited accessibility.
Nuckolls
G H
GH
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599.
Turner
C E
CE
Burridge
K
K
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000032:analysis; D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000502:physiology; D008845:Microinjections; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000502:physiology; D012235:Rhodamines; D013329:Structure-Activity Relationship; D016608:Talin; D013861:Thiocyanates; D016596:Vinculin
D000199:Actins; D015820:Cadherins; D003598:Cytoskeletal Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D010455:Peptides; D012235:Rhodamines; D016608:Talin; D013861:Thiocyanates; D016596:Vinculin; C034996:tetramethylrhodamine iodoacetamide; D016650:Fluorescein-5-isothiocyanate