Abstract

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.

Highlights

  • Evidence for aslowly dissociating tubulin-GTP cap minal subunits andfor dissociation of tubulin-GTP subunits at microtubule endws as derived from observation of afrom ends

  • Microtubules formed with pure tubulindimer in the absence of stabilizing agents such as glycerol exist in two populations, one growing, theother shrinking; individual microtubules infrequently shift from one population to the other(1)

  • Beef brain microtubule protein, purified as described previously (9),was made free of microtubule-associated proteins using phosphocellulose (10).The protein was stored in 50 mM Pipes,' 2 mM EGTA, 1 mM MgSO, pH 6.9;for assembly,the M$+ and GTPconcentrations were increased to 7.5 and 1.5 mM, respectively

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Summary

RESULTS

Beef brain microtubule protein, purified as described previously (9),was made free of microtubule-associated proteins using phosphocellulose (10).The protein was stored in 50 mM Pipes,' 2 mM EGTA, 1 mM MgSO,, pH 6.9;for assembly,the M$+ and GTPconcentrations were increased to 7.5 and 1.5 mM, respectively. A dual wavelength spectrophotometer was used, it was not possible to measure simultaneously microtubule turbidity (350 nm) and the absorbance of the temperature-sensitive buffer-dye mixture (572 nm), and thereby determine the temperature within the solution intersected by the light beam in the cuvette during thermally induced microtubule assembly and disassembly. To estimate this temperature, measurements were made with a thermistor during the temperature-induced perturbations of the microtubules and again immediately after, during the same temperature transitions with the dye.

Time initiated
Noncongruence of Microtubule Assembly and Disassembly
DISCUSSION
This is done by inclusion of a term thadt escribes disassembly
Full Text
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