Probing the role of nonmuscle tropomyosin isoforms in intracellular granule movement by microinjection of monoclonal antibodies.

Abstract

Chicken embryo fibroblast (CEF) cells were microinjected with several different monoclonal antibodies that recognize certain nonmuscle isoforms of tropomyosin. Immediately after injection, cells were recorded with a time-lapse video imaging system; later analysis of the tapes revealed that particles in cells injected with one of these antibodies (CG1, specific for CEF tropomyosin isoforms 1 and 3) showed a dramatic decrease in instantaneous speed while moving, distance moved per saltation, and proportion of time spent in motion. Injection of Fab fragments of CG1 resulted in similar changes in the pattern of granule movement. This inhibition of granule movement by CG1 antibody was reversible; at 2.5 h after injection, granules in injected cells had already reached three-fourths of normal speed. The speed of granule movement in cells injected either with antibody specific for tropomyosin isoforms not present in CEF cells, or with CG1 antibody preabsorbed with tropomyosin, was not significantly different from the speed of granules in uninjected cells. When cells were injected with CG1 or Fab fragments of CG1, fixed, and counter-stained with rabbit antibodies to reveal the microtubule, microfilament, and intermediate filament systems, no obvious differences from the patterns normally seen in uninjected cells were observed. Examination of the ultrastructure of injected cells by EM confirmed the presence of apparently intact and normal microtubule, actin, and intermediate filament networks. These experiments suggest that tropomyosin may play an important role in the movement of vesicles and organelles in the cell cytoplasm. Also, we have shown previously that the CG1 determinant can undergo a motility-dependent change in reactivity, that may be important for the regulatory function of nonmuscle tropomyosin (Hegmann, T. E., J. L.-C. Lin, and J. J.-C. Lin. 1988. J. Cell Biol. 106:385-393). Therefore, in addition to postulated microtubule-based motors, microfilaments may play a critical role in regulating granule movement in nonmuscle cells.