Interaction of Fluorescently-labeled the Cytoskeleton in Cell Models Contractile Proteins with

TO determine if a living cell is necessary for the incorporation of actin, alphaactinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows. Actin and proteins that associate with actin such as tropomyosin, myosin, and alpha-actinin are distributed in nonmuscle cells in patterns that are characteristic for each protein (1 l, 20, 21, 22, 29, 38, 49). In a variety of different cell types, actin has been localized by the use of fluorescent staining agents in stress fibers, ruffles, attachment plaques, and cell junctions (22, 29, 31, 38). Some studies also show it to be concentrated in both cleavage furrows and mitotic spindles (6, 29, 30, 32, 36, 37), whereas others find actin in either the spindle (16, 48, 49) or the furrow (1-3, 14), but not in both. In stress fibers, actin usually appears to be distributed continuously along the length of the fibers (22, 29, 30), although in a few cases, its distribution is discontinuous (15, 31). Tropomyosin is localized in a striated pattern along stress fibers and appears to be absent from areas where alpha-actinin is present, i.e., stress fiber densities, ruffles, cell junctions, attachment plaques, and loci of polygonal networks (20, 38). Like tropomyosin, alpha-actinin is found in a striated pattern along stress fibers (21, 38). Colocalization studies indicate that the two proteins are in adjacent bands (15, 52). In experiments where fluorescently labeled actin, tropomyosin, or alpha-actinin have been injected into living cells, they become localized in precisely the areas where previous studies with antibodies or actin-binding agents have shown them to be concentrated (7, 18, 35, 37, 40, 45, 50). Observations of cells injected with labeled actin or alpha-actinin show that the injected proteins behave in the same way as endogenous actin and alpha-actinin when living cells are followed in time-lapse or experimentally manipulated (7, 17, 35, 40, 47, 50). Although injected tropomyosin appears to be localized along the whole length of stress fibers rather than in a striated pattern, it is excluded from ruffles and attachment plaques (50). If cells that have been injected with fluorescent tropomyosin are treated with phalloidin or cytochalasin-B, the fluorescent tropomyosin becomes localized in aggregates in the same way as the endogenous tropomyosin (50). These experiments all suggest that injected fluorescent proteins are selectively incorporated by living cells into structures and domains where their endogenous counterparts are localized and that once incorporated they respond in the same way as the cells' own proteins. In this study, we have exposed cell models to fluorescently labeled actin, alpha-actinin, and troTHE JOURNAL OF CELL BIOLOGY • VOLUME 99 SEPTEMBER 1984 918-928 918 © The Rockefeller University Press • 0021-9525184/09/0918/11 $1.00 on M ay 9, 2017 D ow nladed fom Published September 1, 1984