A rat monoclonal antibody against yeast alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) that reacts specifically with the tyrosylated form of alpha-tubulin and readily binds to tubulin in microtubules when injected into cultured cells (see Wehland, J., M. C. Willingham, and I. V. Sandoval, 1983, J. Cell Biol., 97:1467-1475) was used to study microtubule organization and function in living cells. Depending on the concentration of YL 1/2 that was injected the following striking effects were observed: (a) When injected at a low concentration (2 mg IgG/ml in the injection solution), where microtubules were decorated without changing their distribution, intracellular movement of cell organelles (saltatory movement) and cell translocation were not affected. Intermediate concentrations (6 mg IgG/ml) that induced bundling but no perinuclear aggregation of microtubules abolished saltatory movement and cell translocation, and high concentrations (greater than 12 mg IgG/ml) that induced perinuclear aggregation of microtubules showed the same effect. (b) YL 1/2, when injected at intermediate and high concentrations, arrested cells in mitosis. Such cells showed no normal spindle structures. (c) Injection of an intermediate concentration of YL 1/2 that stopped saltatory movement caused little or no aggregation of intermediate filaments and no dispersion of the Golgi complex. After injection of high concentrations, resulting in perinuclear aggregation of microtubules, intermediate filaments formed perinuclear bundles and the Golgi complex became dispersed analogous to results obtained after treatment of cells with colcemid. (d) When rhodamine-conjugated YL 1/2 was injected at concentrations that stopped saltatory movement and arrested cells in mitosis, microtubule structures could be visualized and followed for several hours in living cells by video image intensification microscopy. They showed little or no change in distribution and organization during observation, even though these microtubule structures appeared not to be stabilized by injected YL 1/2 since they were readily depolymerized by colcemid or cold treatment and repolymerized upon drug removal or rewarming to 37 degrees C, respectively. These results are discussed in terms of the participation of microtubules in cellular activities such as cell movement and cytoplasmic organization and in terms of the specificity of YL 1/2 for the tyrosylated form of alpha-tubulin.
Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers
