Reorganization of Actin in Living Fibroblasts Filament Bundles

We investigated how actin bundles assemble, disassemble, and reorganize during cell movement. Living chick embryonic fibroblasts were microinjected with actin molecules that had been fluorescently labeled with tetramethylrhodamine. We found that the fluorescent analogue of actin can be used successfully by both existing and newly formed cellular structures. Using time-lapse photography coupled to image-intensified fluorescence microscopy, we were able to detect various patterns of reorganization in motile cells~ Assembly of stress fibers occurred near both the leading and the trailing ends of the cell. The initial structure appeared as discrete spots that subsequently extended into stress fibers. The extension occurred unidirectionally. The site of initiation near the leading edge remained stationary relative to the substrate during subsequent cell advancement. However, the orientation of the fiber could change according to the direction of cell movement. In addition, existing stress fibers could merge or fragment. The shortening of stress fibers can occur from either end of the fiber. Shortening from the proximal end (centrifugal shortening) was accompanied by a decrease in fluorescence intensity, as if the bundle were disassembling, and usually led to the total disappearance of the bundle. Shortening from the distal end (centripetal shortening), on the other hand, is usually accompanied by an increase in fluorescence intensity at the distal end of the bundle, as if this end had pulled loose from its attachment and retracted toward the center of the cell. Besides stress fibers, arc-like actin bundles have also been detected in spreading cells. These observations can explain how the organization of actin bundles coordinates with cell movement, and how stress fibers reach a highly regular pattern in static cells. Light and electron microscopic studies have clearly demonstrated the presence of actin bundles in nonmuscle cells (2). The most prominent class, stress fibers, contain a number of accessory proteins such as alpha-actinin, tropomyosin, and myosin, arranged in a regular pattern somewhat similar to that in myofibrils (7, 14, 18). At least one end of the stress fiber is usually attached to the membrane, opposite to the site of adhesion plaque on the external surface (10, 12). Besides stress fibers, actin bundles have also been identified in the "arcs" of spreading cells (9, 20) and in microvilli (26). Although these different types of bundles probably represent different structures and probably serve different functions, they share a common characteristic: the ability to reorganize. For example, "arcs" form near the leading edge of the cell, then move toward the nucleus and disappear (9, 20). Stress fibers exhibit a drastic reorientation when cells are placed in an electric field (16). Even in relatively static cells, changes in the organization of actin bundles have been detected (29). This is not surprising in view of the highly dynamic nature of the actin filaments, which are capable of polymerization-depolymerization, and of interaction with molecules such as myosin and gelsolin (for reviews, see references 5, 17). However, little is known about exactly how the relatively large bundle structures can be effectively reorganized in a cell. For example, how are actin bundles "translocated" during cell movement? Do cells constantly assemble new bundles at the leading edge and disassemble bundles at the trailing end? Or do existing bundles somehow move during cell locomotion? In this study, we employed fluorescent analogue cytochemTHE JOURNAL OF CELL BIOLOGY • VOLUME 99 OCTOBER 1984 1478-1485 1478 © The Rockefeller University Press • 0021 9525[84/10/1478/08 $1.00 on N ovem er 2, 2017 jcb.rress. org D ow nladed fom istry to follow directly changes in the organization of actin bundles in living chick embryonic fibroblasts. The approach involves preparation of functional fluorescent conjugates of cellular components, followed by microinjection of the fluorescent analogue into living cells. Since the introduction of the technique (22), a large number of fluorescently labeled cytoskeletal components, including actin, alpha-actinin, tropomysin, vinculin, and tubulin, have been successfully injected into living cells and used by normal cellular structures (for reviews, see references 13, 24, 27). Thus this approach offers a unique opportunity to study the reorganization of specific molecules in living cells. One common problem encountered in this approach is the use of intense excitation light, which causes photobleaching and possible cellular damage, during the detection of weak fluorescence emitted from a single cell (discussed in reference 28). As a result, each injected cell can be studied only for limited period of time. In this study, we have minimized the problem by using a highly sensitive image intensifier coupled to a sophisticated time-lapse recording technique. The sequences of images taken at short t ime intervals revealed various ways through which actin filament bundles reorganize in living, motile cells. MATERIALS A N D M E T H O D S Preparation of Fluorescent Analogue: Actin was purified from rabbit back and leg muscles according to Spudich and Watt (21). Iodoacetamidotetramethylrhodamine (Research Organics, Cleveland, OH) was dissolved in a buffet containing 2 mM Tris, 0.2 mM CaCI2. After titrating the pH to 9.0, the dye solution was mixed 1:1 with a solution of 5 mg/ml G-actin in 2 mM Tris, 0.2 mM CaCI, 0.5 mM ATP, 0.05 mM dithiothreitoi, pH 8.0, to obtain a final concentration of 0.3 nag dye/rag actin. The solution was centrifuged at 100,000 g for 2 h to remove undissolved dye aggregates. The supernatant was incubated at 0"C for 4 h, followed by addition of 5 mM dithiothreitol to stop the reaction. The solution was then dialyzed overnight against 2 mM Tris, 0.2 mM CaCl2, 0.5 mM ATP, 0.2 mM dithiothreitol, pH 7.8. Residual quenched dye molecules were removed by desalting through a G-25 column. Fluorescent fractions in the void volume were collected and carried through two cycles of polymerization and depolymerization. The conjugate, at a conFIGURE 1 Assembly of a stress fiber (arrows) at the leading edge of the cell. Ruffle region was marked (arrowhead). Photographs taken at t = 0 (a), 6 (b), 9 (c), and 15 min (d). Bar, 5/~m. x 1,925. WANG Actin Bundles in Fibroblasts 1479 on N ovem er 2, 2017 jcb.rress. org D ow nladed fom