Distribution of fluorescently labeled alpha-actinin in living and fixed fibroblasts

The distribution of flourescently labeled alpha-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isthiocyanate-labeled protein (TMRITC-alpha-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:3967-3971), was virtually unaffected by the fixation (3.5 percent formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunoflourescence. Also, these patterns were found to coincide with the alpha-actinin revealed by immunoflourescence. Also, these patterns were found to coincide with the alpha-actinin revealed by immunoflourescence. These findings offer, for the first time, evidence indicating the validity of the immunoflourescence technique in the localization of alpha-actinin in cultured cells. With the combination of the injection procedure and the immunoflourescence localization of endogenous structural proteins, it was determined that nearly all of the actin stress fibers were decorated in a periodic manner with the injected alpha-actinin. Endogenous tropomyosin in the injected cells was found to be 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, whereas the microinjected alpha-actinin was incorporated into the foci of the networks. Thus, 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 utilized by the cell with fidelity.