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-...

Abstract. Filamentous actin in living cultured cells was labeled by microinjecting trace amounts of rhodamine-phalloidin (rh-pha) as a specific, highaffinity probe. The microinjection caused no detectable effect on cell morphology or cell division. The distribution of rh-pha-labeled filaments was then examined in dividing cells using image-intensified fluorescence microscopy, and the exchangeability of labeled illaments along stress fibers was studied during interphase using fluorescence recovery after photobleaching. rh-pha showed a rapid concentration at the contractile ring during cell division. In addition, recovery of fluorescence after photobleaching occurred along stress fibers with a halftime as short as 8 min. These observations suggest that at least some actin filaments undergo continuous movement and reorganization in living cells. This dynamic process may play an important role in various cellular functions.