Talin dynamics in living microinjected nonmuscle cells.
To investigate the role of talin in the anchoring of actin-containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G-25 and DEAE-cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone-permeabilized nonmuscle cells. In cells that were double-stained with fluorescent talin and an affinity-purified anti-talin antibody, a one-to-one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer-enhanced video microscopy was used to document adhesion plaque dynamics such as 1) changes in plaque shape, 2) alterations in plaque positions, and 3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.
Hock
R S
RS
Department of Anatomy, University of Pennsylvania School of Medicine, Philadelphia 19104-6058.
Sanger
J M
JM
Sanger
J W
JW
D000818:Animals; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002645:Chickens; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003599:Cytoskeleton / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D008845:Microinjections; D011485:Protein Binding; D012235:Rhodamines; D016608:Talin; D016596:Vinculin
D003598:Cytoskeletal Proteins; D012235:Rhodamines; D016608:Talin; D016596:Vinculin; C027305:Lissamine Rhodamine B sulfonyl chloride