The post-hydrolysis nucleotide state is required for CLASP1-mediated microtubule depolymerization. (A) Representative kymograph of a GMPCPP-stabilized microtubule incubated sequentially with no nucleotide, 1 mM GTP, and 1 mM GMPCPP. The white horizontal lines indicate the times of solution exchange; the purple line is a manual trace of the microtubule end position overlaid onto the kymograph. (B) Representative kymographs of GMPCPP-stabilized microtubules incubated with buffer or 200 nM CLASP1 in the presence of 1 mM GDP, 1 mM GMPCPP, and 1 mM GTPγS. (C) Quantification of microtubule depolymerization rates for the conditions in the presence of different nucleotides. The mean depolymerization rates were 3.8 ± 0.3 nm/s; (SE, N = 60) with GTP, 3.1 ± 0.7 nm/s; (SE, N = 40) with GDP, 0.115 ± 0.008 nm/s; (SE, N = 60) with GMPCPP, and 0.61 ± 0.06 nm/s; (SE, N = 60) with GTPγS. Data were obtained from at least two independent experimental days and individual data points from different experiments are plotted in different shades. The means for each experimental repeat are plotted as larger points in the same color. The squares indicate the average of the experimental means, and the vertical bars are the standard errors of the means. A one-way ANOVA followed by Tukey’s HSD test for multiple comparisons revealed that there was a statistically significant difference in the microtubule depolymerization rate between GTP and GMPCPP (P < 0.001), GDP and GMPCPP (P < 0.001), and GTPγS and GMPCPP (P < 0.001). The corresponding P values are also indicated on the graph.