10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin.

Abstract

Cells were microinjected with four mouse monoclonal antibodies that were directed against either alpha-or beta-tubulin subunits, one monoclonal with activity against both subunits, and a guinea pig polyclonal antibody with activity directed against both subunits, to determine the effects on the distribution of cytoplasmic microtubules and 10-nm filaments . The specificities of the antibodies were confirmed by Western blots, solid phase radioimmunoassay, and Western blot analysis of alpha-and beta-tubulin peptide maps. Two monoclonals DM1A and DM3B3, an anti-alpha-and anti-beta-tubulin respectively, and the guinea pig polyclonal anti-alpha/beta-tubulin antibody (GP1T4) caused the 10-nm filaments to collapse into large lateral aggregates collecting in the cell periphery or tight juxtanuclear caps; the other monoclonal antibodies had no effect when microinjected into cells . The filament collapsing was observed to be complete at 1 .5-2 h after injection . During the first 30 min after injection a few cytoplasmic microtubules near the cell periphery could be observed by fluorescence microscopy. This observation was confirmed by electron microscopy, which also demonstrated assembled microtubules in the juxtanuclear region . By 1 .5 h, when most of the 10-nm filaments were collapsed, the complete cytoplasmic array of microtubules was observed . Cells injected in prophase were able to assemble a mitotic spindle, suggesting that the antibody did not block microtubule assembly . Metabolic labeling with [35S]methionine of microinjected cells revealed that total protein synthesis was the same as that observed in uninfected cells. This indicated that the microinjected antibody apparently did not produce deleterious effects on cellular metabolism . These results suggest that through a direct interaction of antibodies with either alpha-or beta-tubulin subunits, 10-nm filaments were dissociated from their normal distribution . It is possible that the antibodies disrupted postulated 10-nm filament-microtubule interactions. l0-nm filament-microtubule interaction have been postulated based on studies by electron microscopy (4,17,26,32,53,55,58), immunofluorescence microscopy (8, 24), and biochemistry (43,54,55) in many eucaryotic cells. When cells are incubated in the presence of antimicrotubule drugs, such as colchicine, colcemid, or vinblastine, and cytoplasmic microtubules depolymerize (51) and the 10-nm filaments subsequently coil into juxtanuclear caps (9, 26) or collapse into large aggregates of filaments in the cytoplasm (12,28,33,35). Upon release from these drugs, the microtubules rapidly reassemble into their original cytoplasmic array (51) and the 10-nm filaments then uncoil and redistribute with the majority of microtubules (26). Although it is not known if these THE JOURNAL OF CELL BIOLOGY " VOLUME 9S MARCH 1984 847-858 ® The Rockefeller University Press " 0021-9525/84/03/0847/12 $1 .00 drugs directly affect the 10-nm filaments, it is thought that as a result of microtubule depolymerization, 10-nm filaments c...

Vimentin Dephosphorylation by Protein Phosphatase 2A Is Modulated by the Targeting Subunit B55

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