Distribution of flourescently labeled α-actinin in living and fixed fibroblasts

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Abstract
The distribution of fluorescently labeled-.alpha.-actinin after microinjection into [gerbil fibroma CCL146] fiborblasts was determined in both living and fixed cells. The distribution of the injected tetramethylrhodamine isothiocyanate-labeled protein (TMRITC-.alpha.-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles and polygonal microfilament networks, was virtually unaffected by the fixation (3.5% formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunofluorescence. Also, these patterns coincided with the .alpha.-actinin revealed by immunofluorescence. Evidence for the validity of the immunofluorescence technique in the localization of .alpha.-actinin in cultured cells is offered for the 1st time. With the combination of the injection procedure and the immunofluorescence localization of endogenous structural proteins. Nearly all actin stress fibers were decorated in a periodic manner with the injected .alpha.-actinin. Endogenous tropomyosin in the injected cells was distributed with a periodic pattern along the stress fibers that was antiperiodic to the pattern observed for the microinjected .alpha.-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci; the microinjected .alpha.-actinin was incorporated into the foci of the networks. The microinjected fluorescent derivative of .alpha.-actinin appears to be incorporated into the functional pools of .alpha.-actinin within the living cell and to be used by the cell with fidelity.