Dynamic instability of microtubules assembled from microtubule-associated protein-free tubulin: neither variability of growth and shortening rates nor "rescue" requires microtubule-associated proteins.

Abstract

The growth and shortening of microtubules in dynamic instability is known to be modulated by microtubule-associated proteins (MAPs). A full understanding of the mechanism of dynamic instability requires that one distinguish which of its aspects are mediated by microtubule-associated proteins (even in small residual concentrations) and which are intrinsic properties of the tubulin lattice itself. This paper addresses two of those aspects: whether MAPs cause the rescue events of dynamic instability (i.e., the transitions from shortening to growth) and whether MAPs are responsible for the marked variability of the rates at which microtubules grow and shorten. Very pure tubulin was prepared by sequential chromatographies on phosphocellulose and DEAE-Sephadex. Analysis by electrophoresis and immunoblotting showed it to be essentially MAP-free; it contained fewer than one MAP molecule per 10000 tubulin dimers. When its dynamic instability was studied by video-DIC microscopy, rescues were found to occur at a mean frequency of one per 4 microns of shortening. Variability of rates of growth and shortening, which is observed on the length scale of a few micrometers, was not changed by removal of MAPs. Because the mean distance between bound MAP molecules was calculated to be greater than 14 microns in these experiments, it is concluded that they cannot cause either rescue or variability of rates.