Chapter 5 Purification and Reassembly of Tubulin from Outer Doublet Microtubules

Abstract

The chapter discusses the purification and reassembly of tubulin from outer doublet microtubules. The most commonly used microtubule system is that of vertebrate brain. This system has the advantage of yielding large quantities of native tubulin; however, the tubulin derives from the total brain microtubule population, which shows considerable heterogeneity. The optimal in vitro assembly requirements of outer doublet and vertebrate brain tubulins are very similar, as are the properties of the assembled microtubules. This appears to be generally true for all tubulins so far examined, irrespective of the properties, functions, cellular location, or phylogeny of the microtubules from which the tubulin derives. The reconstituted microtubules are “labile” singlet microtubules, archetypical of which are reconstituted vertebrate brain microtubules. Structurally bonafide singlet microtubules can be formed in vitro from outer doublet and dogfish brain tubulins in the absence of associated proteins. Direct evidence that tubulin conformational changes alter the microtubule lattice is lacking. Studies on the mechanism of colchicine poisoning of microtubule assembly are consistent with this hypothesis. Colchicine poisons microtubule assembly by first complexing with tubulin dimers, which then copolymerize with uncomplexed tubulin into the microtubules and decrease the apparent rate of subsequent drug-free dimer addition.