SITES OF MONOMERIC ACTIN INCORPORATION IN LIVING PTK2 AND REF-52 CELLS

The purpose of this study was to analyze where monomeric actin first b ecomes incorporated into the sarcomeric units of the stress fibers. We microinjected fluorescently labeled actin monomers into two cell line s that differ in the sarcomeric spacings of oc-actinin and nonmuscle m yosin II along their stress fibers: REF-52, a fibroblast cell line, an d PtK2, an epithelial cell line. The cells were fixed at selected time s after microinjection (30 s and longer) and then stained with an alph a-actinin antibody. Localization of the labeled actin and alpha-actini n antibody were recorded with low level light cameras. In both cell ty pes, the initial sites of incorporation were in focal contacts, lamell ipodia and in punctate regions of the stress fibers that corresponded to the alpha-actinin rich dense bodies. The adherent junctions between the epithelial PtK2 cells were also initial sites of incorporation. A t longer times of incorporation, the actin fluorescence extended along the stress fibers and became almost uniform. We saw no difference in the pattern of incorporation in peripheral and perinuclear regions of the stress fibers. We propose that rapid incorporation of monomeric ac tin occurs at the cellular sites where the barbed ends of actin filame nts are concentrated: at the edges of lamellipodia, the adherens junct ions, the attachment plaques and in the dense bodies that mark out the sarcomeric subunits of the stress fibers. (C) 1998 Wiley-Liss, Inc.