A method is described for measuring the quantities of stable and dynamic microtubules in a population in vitro. The method exploits the tendency of dynamic microtubules to depolymerize rapidly after being sheared. Stable microtubules, such as those protected by microtubule-associated proteins (MAPs), are broken to a smaller size by shearing, but do not depolymerize into subunits. The usual difficulty with this procedure is that the tubulin released from the dynamic microtubules rapidly repolymerizes before the end point of depolymerization can be measured. This has been overcome by including a small quantity of tubulin–colchicine complex in the mixture to block the repolymerization. For a total of 24 μM tubulin in a polymerization mixture, 10 μM of the sample polymerized originally under the conditions used. When 1.05 μM tubulin–colchicine complex was added at the time of shearing, the dynamic microtubules depolymerized, but the tubulin was released was unable to repolymerize and a small fraction of stable microtubules that resisted shear-induced depolymerization could then be detected. When traces of MAPs (0.23–2.8% by mass) were included in the tubulin mixture, the fraction of stable microtubules increased from 5% in the absence of added MAPs to 41% in the presence of 2.8% MAPs. All the MAPs in the mixture were found in the stable fraction and this stable fraction forms early during microtubule assembly. Calculations on the extent of enrichment of MAPs in the stable fraction indicated that as little as 4% MAPs in a microtubule protected it from shear-induced disassembly. The results suggest that low levels of MAPs may distribute nonrandomly in the microtubule population.Key words: dynamics, microtubules, tubulin, microtubule-associated proteins, self-assembly.
Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single-cell tracking and traction force microscopy
