Figure 5.

Carba1 is a microtubule-depolymerizing agent that competes with colchicine for tubulin binding. (A) Immunofluorescence analysis of the Carba1 effect on interphase and mitotic microtubules (MTs). MTs in interphase (left panels) or in mitosis (right panels) were stained using an anti-tubulin antibody, as described in the methods section. DNA was stained using Hoechst reagent. Bars = 10 μm. (B) Time course of tubulin polymerization at 37 °C in the presence of vehicle (DMSO, black line) and Carba1 at different concentrations (colored lines) as indicated, measured by turbidimetry at 350 nm. Purified tubulin: 30 µM in BRB80 buffer with 1 mM GTP. Each turbidity value represents the mean ± SEM from three independent experiments. (C) Effect of Carba1 on the binding of [³H]-colchicine to tubulin. Carba1 (100 µM) was used to compete with [³H]-colchicine (50 nM) as described in the methods section. Each value represents the mean ± SEM of three independent experiments. Colchicine and nocodazole were used as positive and vinblastine as negative control. (D) Displacement of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) from the colchicine site. Fluorescence emission spectra of 10 μM MTC and 10 μM tubulin in 10 mM phosphate-0.1 mM GTP buffer pH 7.0, in the absence or presence of increasing concentrations of Carba1. (E) Displacement isotherm at 25 °C of the fluorescent probe MTC (10 μM) bound to tubulin (10 μM) by Carba1 (black line and circles). The solid line is the best-fit value of the binding equilibrium constant of the competitors, assuming a one-to-one binding to the same site.