Distribution of Fluorescently Labeled a-Actinin in Living and Fixed Fibroblasts

The distribution of fluorescently labeled a-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isothiocyanate-labeled protein (TMRITC-a-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% formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunofluorescence . Also, these patterns were found to coincide with the a-actinin revealed by immunofluorescence . These findings offer, for the first time, evidence indicating the validity of the immunofluorescence technique in the localization of a-actinin in cultured cells . With the combination of the injection procedure and the immunofluorescence 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 a-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 a-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci, whereas the microinjected a-actinin was incorporated into the foci of the networks . Thus, the microinjected fluorescent derivative of a-actinin appears to be incorporated into the functional pools of a-actinin within the living cell and to be utilized by the cell with fidelity . To study the dynamics of structural proteins within living cells, techniques amenable to studies of living cells must supplant those of immunofluorescence or electron microscopy, which require fixation of the cells . An obvious approach for overcoming the problem of fixation is the microinjection into living cells of "native" structural proteins that have been fluorescently labeled . The distribution of fluorescently labeled actin in living protozoans has been observed by this technique (24, 25) . The large size and thickness ofthese cells, though, makes it difficult to visualize the injected fluorescent proteins or the distribution of the endogenous proteins by immunofluorescence with fluorescence microscopy . Recently, this type of approach has been applied to living fibroblasts (i .e ., cells that are ideally suited for fluorescence microscopy) into which fluorescently labeled aactinin (8), a 130,000-dalton protein from smooth muscle (3), or actin (l6) was microinjected. Microinjection of mammalian cells (5, 7, 15), combined with the use of fluorescently labeled proteins, promises to have many useful applications for the study of the dynamic behavior of structural proteins in living cells . In the present study we have addressed several questions concerning this approach : (a) What is the relative distribution ofmicroinjected fluorescent a-actinin in relation to other structural proteins in the cell? (b) Does microinjected fluorescent a-actinin fail to incorporate into any particular area(s) in the cell that contains endogenous a-actinin? (c) Is the distribution ofinjected protein within the living cell significantly altered by the treatments normally used for the preparation of cells for immunofluorescence (i .e., fixation and extraction)? With the microinjection technique combined with immunofluorescence and double-label fluorescence microscopy we have found that microinjected fluorescent a-actinin coincides with the a-actinin revealed by immunofluorescence, is distributed in a periodic pattern along the actin stress fibers which is antiperiodic to tropomyosin, and is localized in the foci of the actin polygonal microfilament networks that are exclusive of tropomyosin . Furthermore, we have determined that fixation and extraction have little effect on the distribution of the injected protein . These findings suggest that the microinjected, fluorescently labeled a-actinin faithfully reflects the distribution of a-actinin Tu[ JOURNAL OF CE[[ Í310[0(N VOLUM[ 86 Aucusi 1980 608-615 © The Rockefeller University Press 0021-9525/80/08/0608/08 $1 .00 on D ecem er 4, 2017 jcb.rress.org D ow nladed fom in the living cells and offer, for the first time, some evidence indicating the validity of the immunofluorescence technique in the localization of a-actinin in cultured cells . MATERIALS AND METHODS