Visualization of the dynamic instability of individual microtubules by dark-field microscopy

Abstract

It has previously been shown that two populations of microtubules coexist in a dynamically unstable manner in vitro: those in one population elongate while those in the other shorten and finally disappear. This conclusion was based on changes in the number and length distribution of microtubules after dilution of the microtubule solution. Here, we demonstrate directly that growing and shortening populations coexist in steady-state conditions, by visualization of the dynamic behaviour of individual microtubules in vitro by dark-field microscopy. Real-time video recording reveals that both ends of a microtubule exist in either the growing or the shortening phase and alternate quite frequently between the two phases in a stochastic manner. Moreover, growing and shortening ends can coexist on a single microtubule, one end continuing to grow simultaneously with shortening at the other end. We find no correlation in the phase conversion either among individual microtubules or between the two ends of a single microtubule. The two ends of any given microtubule have remarkably different characteristics; the active end grows faster, alternates in phase more frequently and fluctuates in length to a greater extent than the inactive end. Microtubule-associated proteins (MAPs) suppress the phase conversion and stabilize microtubules in the growing phase.