Temperature-jump studies of microtubule dynamic instability.

Evidence for a slowly dissociating tubulin-GTP cap at microtubule ends was derived from observation of a delay for attaining a maximum disassembly rate, after the temperature of steady state microtubules was rapidly decreased from 36 to 34 degrees C. The possibility that the microtubules were capped by a single tubulin-GTP subunit on each subhelix was ruled out, by comparison of the disassembly kinetics following a temperature decrease and dilution. The existence of a subpopulation of microtubules that underwent irreversible or near irreversible disassembly was demonstrated by a 30-s lag for attainment of a maximum assembly rate, after steady state microtubules were shifted from 34 to 36 degrees C. A dynamic instability model predicts that a maximum assembly rate will be delayed until disappearance of a subpopulation of microtubules that disassemble before being recapped. Analysis indicates that the 30-s lag resulted because approximately 2% of the mass in the steady state microtubule population was uncapped and disassembling and not readily recapped. The half-time for recapping of disassembling microtubules, by addition of tubulin-GTP subunits to ends, was equal to or greater than 20 s. Since tubulin-GDP dissociated from microtubules at a rate of about 4500 s-1, slow recapping resulted in dramatic shortening of disassembling microtubules.