Fig. 1.

PIP₂ inhibits tubulin polymerization.A, Comparison of the effects of various phosphoinositides on microtubule polymerization. Where indicated, PIP₃, PIP₂, PIP, PI, PC, PE, and the inositol phosphate IP₃ (final molar concentration of 75 μm) were preincubated with tubulin (Tub) as described in Materials and Methods. Microtubule polymerization reactions were performed for 1 hr at 37°C. Pellets were resuspended in cold polymerization buffer and subjected to SDS-PAGE and immunoblotting with a monoclonal anti-α tubulin antibody. Values are means ± SEM of five independent experiments performed in triplicate. *Significantly different from the control (tubulin, subjected to polymerization without any addition);p < 0.05, one-way ANOVA. Colorimetric measurements of the protein content of depolymerized pellets (performed before SDS-PAGE) corroborated these findings. B, PIP₂ inhibition of microtubule formation is concentration-dependent. PIP₂, PC, mixed vesicles of PIP₂ and PC (at the ratio of 1:1), and vehicle were added to microtubule polymerization reactions. Polymerization was performed for 30 min as described in Materials and Methods. Absorbance at 350 nm was monitored. Values were obtained after 20 min, when the polymerization reactions were at equilibrium. Values are means ± SEM of three separate experiments done in triplicate. The net absorbance of microtubule polymerization reactions without added phosphoinositides was 1.09 ± 0.12 (control). *p < 0.05; **p < 0.01.