Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.

摘要

Whether polarized treadmilling is an intrinsic property of microtubules assembled from pure tubulin has been controversial. We have tested this possibility by imaging the polymerization dynamics of individual microtubules in samples assembled to steady-state in vitro from porcine brain tubulin, using a 2% glycerol buffer to reduce dynamic instability. Fluorescence speckled microtubules were bound to the cover-glass surface by kinesin motors, and the assembly dynamics of plus and minus ends were recorded with a spinning-disk confocal fluorescence microscopy system. At steady-state assembly, 19% of the observed microtubules (n = 89) achieved treadmilling in a plus-to-minus direction, 34% in a minus-to-plus direction, 37% grew at both ends, and 10% just shortened. For the population of measured microtubules, the distribution of lengths remained unchanged while a 20% loss of original and 27% gain of new polymer occurred over the 20-min period of observation. The lack of polarity in the observed treadmilling indicates that stochastic differences in dynamic instability between plus and minus ends are responsible for polymer turnover at steady-state assembly, not unidirectional treadmilling. A Monte Carlo simulation of plus and minus end dynamics using measured dynamic instability parameters reproduces our experimental results and the amount of steady-state polymer turnover reported by previous biochemical assays.