Microinjection of fluorescently labeled alpha-actinin into living fibroblasts.
alpha-Actinin from chicken gizzard labeled with tetramethylrhodamine isothiocyanate has been incorporated into living fibroblast cells by microinjection. Fluorescent labeling of alpha-actinin was carried out such that the conjugated protein was functional in vitro as shown by its ability to bind to F-actin. Within 1-2 hr after injection, diffuse fluorescence was observed throughout the cytoplasm and only faint fluorescence was apparently associated with the stress fibers. During the ensuing 2-15 hr, however, most of the fluorescence was seen as periodicities along the stress fibers and as foci of the microfilament polygonal networks. This distribution of alpha-actinin in the living cells was strikingly similar to that found by indirect immunofluorescence localization of endogenous alpha-actinin in fixed samples of the same cell type. Control studies in which heat-treated (100 degrees C, 2 min) fluorescent alpha-actinin or tetramethylrhodamine isothiocyanate alone was injected into the cells indicated that the stress fiber and polygonal network labeling was specific for "native" fluorescently labeled alpha-actinin. These results suggest that the dynamic properties of proteins and structures in cultured mammalian cells can be studied with the use of microinjection and fluorescence microscopic techniques.
D000185:Actinin / Q000378:metabolism*; D000818:Animals; D003599:Cytoskeleton / Q000648:ultrastructure*; D005347:Fibroblasts / Q000648:ultrastructure; D005849:Gerbillinae; D008856:Microscopy, Fluorescence / Q000379:methods; D009124:Muscle Proteins / Q000378:metabolism*; D011489:Protein Denaturation; D013861:Thiocyanates
D009124:Muscle Proteins; D013861:Thiocyanates; D000185:Actinin