MICRO-INJECTION OF FLUORESCENTLY LABELED ALPHA-ACTININ INTO LIVING FIBROBLASTS

α-Actinin from chicken gizzard labeled with tetramethylrhodamine isothiocyanate has been incorporated into living fibroblast cells by microinjection. Fluorescent labeling of α-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 α-actinin in the living cells was strikingly similar to that found by indirect immunofluorescence localization of endogenous α-actinin in fixed samples of the same cell type. Control studies in which heat-treated (100°C, 2 min) fluorescent α-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 α-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.