A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. II. Effects on cell movement, organization of microtubules, and intermediate filaments, and arrangement of Golgi elements.

Abstract

A rat monoclonal antibody against yeast a-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 a-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 (>12 mg IgG/ml) that induced perinuclear aggregation of microtubules showed the same 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°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 a-tubulin.As a cytoskeletal component of interphase cells microtubules are involved in the determination of cell shape, in cell locomotion, intracellular movement, and cytoplasmic organization (for recent reviews see references 13, 2 l, 52). Evidence for the involvement of microtubules in these cellular activities has mainly derived from the application of drugs that specif-

Molecular Motor KIF1C Is Not Essential for Mouse Survival and Motor-Dependent Retrograde Golgi Apparatus-to-Endoplasmic Reticulum Transport

“…Vti1b is a putative mammalian SNARE protein that is required for vesicle fusion and docking at the Golgi apparatus (1). p58 is located specifically at the cis/medial site of the Golgi apparatus (3,37). No significant difference in the Golgi shape was observed between kif1C ϩ/ϩ and kif1C Ϫ/Ϫ cells.…”

Characterization of KIF1C, a New Kinesin-like Protein Involved in Vesicle Transport from the Golgi Apparatus to the Endoplasmic Reticulum

“…Golgi to ER transport is MT-dependent and occurs toward the plus ends of microtubules (40). Since all proteins of the Unc104 subfamily are described to be MT plus end-directed motors (28,29), we wondered whether KIF1C functions as a vesicle-transporting motor protein in the membrane flow from the Golgi to the ER.…”

“…In a control experiment, the Golgi apparatus was detected using a monoclonal antibody against the protein 58K (Fig. 6C) which is described to be located specifically at the cis/medial site of the Golgi apparatus by binding simultaneously to microtubules and the Golgi membranes (39,40). To prove that KIF1C indeed colocalizes with the Golgi, we performed double staining of KIF1C and 58K.…”

Functions of intermediate filaments

“…74-75]. Depolymerization of microtubules, either by specific drugs or by the intracellular injection of monoclonal antitubulin antibodies [Wehland and Willingham, 1983;Blose et al, 19841, leads to the collapse of the extended vimentin filament system. In cultured mouse fibroblasts, the collapse of vimentin filament organization in response to the depolymerization of microtubules appears to involve an energy-dependent and cytochalasin-sensitive, presumably microfilament-based, process [Hollenbeck et al, 19891.…”