Previous studies (Holmes, K. V., and P. W. Choppin. J. showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy . During cell fusion, microtubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei . No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei . Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 NLg/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 leg/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 p,g/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required . The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.KEY WORDS cell fusion immunofluorescence microscopy microfilaments -simian virus 5 cytochalasin B Previous studies from this laboratory showed that infection of monolayers of baby hamster kidney (BHK21-F) cells with the parainfluenza virus, sim-J. CELL BIOLOGY

Five different fibroblast strains derived from donors of a wide range of ages were used for investigation of senescence-associated changes in the organization of intermediate filaments (IFs) and the activity of cell locomotion. Results of immunofluorescence microscopy demonstrate that, in large and flat in vitro aged fibroblasts, vimentin-containing IFs are distributed as unusually organized large bundles. Electron microscopic examination shows that these large bundles are indeed composed of filaments of 8-10 nm. Such a profile of large bundles is rarely seen in young fibroblasts whose IFs are usually interdispersed among microtubules. Within the large filament bundles of senescent fibroblasts, cross-bridge-like extensions are frequently observed along the individual IFs. Immunogold labeling with antibody to one of the cross-bridging proteins, p50, further illustrates the abundance of interfilament links within the IF bundles. The senescence-related increase in interfilament association was also supported by the results of co-precipitation between vimentin and an associated protein of 50,000 D. Time-lapse cinematographic studies of cell locomotion reveal that accompanying aging, fibroblasts have a significantly reduced ability to translocate across a solid substratum. These results led me to suggest that the increased interfilament links via cross-bridges may in part contribute to the mechanism that orchestrates the formation of large filament bundles. The presence of enormous bundles in the cytoplasm may physically impede the efficiency of locomotion for these nondividing cells.The finding that replication of fibroblasts in culture is restricted to a defined time span introduces the establishment of a methodology for in vitro observation of cellular senescence. Not only have the initial reports of Swim and Parker (26) and Hayflick and Moorehead (8) been repeatedly confirmed in fibroblasts different from the original WI-38, but also this may be observed in cells of nonmesenchymal origin, such as endothelial and smooth muscle cells (see review in reference 20). In contrast to Hayflick's interpretation that loss of proliferative potential by human diploid fibroblasts in culture is a manifestation of aging, Martin et al. (15) and Bell et al. (1) suggest that the cessation of replication can be interpreted as the final event in differentiation.An increase in cell size and the presence of large rigid filamentous structures are two of the frequently observed phenotypic properties associated with the process of fibroblast senescence (3,28,33). Our recent results demonstrate that the filamentous structures commonly seen in the large, nonproliferating senescent cells are composed of numerous actincontaining microfilaments, intermediate filaments (IFs) 1, and an elaborate network of microtubules (29). These cytoskeletal components form a complex three-dimensional structure that occupies most of the cytoplasm.In apparently normal fibroblasts of a proliferating monolayer culture, individual IFs are in general in...