Microtubule Growth Is Slowed through Transient Exposure of GDP Tubulin

Abstract

Microtubule plus-end growth dynamics are determined by a kinetic race dictated by tubulin on- and off-rates at the microtubule tip. The apparent tubulin on- and off-rates can be estimated by measuring the mean plus-end growth rate across a range of free tubulin concentrations. However, when growth phases are studied at higher temporal resolution, fluctuations around the average growth rate are observed, implying more complex dynamics. To understand growth dynamics at the microtubule plus-end, we used Interference Reflection Microscopy (IRM) to quantify time-dependent variations in microtubule growth using GMPCPP, a non-hydrolyzable GTP analog. We found an expected linear dependence of the average growth rate on tubulin concentration. However, there was also a linear relationship between growth variance and tubulin concentration. Utilizing these measured values, we were able to constrain a biochemical model of microtubule growth with an on-rate of 0.7/ μM/pf/s. To determine the degree to which growth in GMPCPP mimics the GTP cap, we repeated the measurements in GTP. We again found a linear dependence of the average growth rate on tubulin; but found a much higher tubulin-dependent growth variance than expected from the GMPCPP experiments. When the growth variance was normalized to growth rate for each condition, there was a 5-fold increase in the relative growth variance in GTP compared to GMPCPP. This result suggests that GTP hydrolysis impacts tubulin binding kinetics at the microtubule plus-end. Consistent with this hypothesis, we then artificially varied the hydrolysis rates from 0 to 0.15 s−1 in the biochemical model. Increasing the hydrolysis rate increased the growth variance, and transient suppression of growth coincided with increasing numbers of exposed GDP tubulin at the plus-end. This work suggests that during growth, exposure of terminal GDP tubulin slows growth until a terminal GTP is restored.