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

Abstract

Cells were microinjected with 4 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 (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 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 EM, 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]Met of microinjected cells revealed that total protein synthesis was the same as that observed in uninjected cells. The microinjected antibody apparently did not produce deleterious effects on cellular metabolism. Through a direct interaction of antibodies with either .alpha.- or .beta.-tubulin subunits, 10-nm filaments were evidently dissociated from their normal distribution. The antibodies disrupted postulated 10-nm filament-microtubule interactions.