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Journal of Cell Science, Vol 94, 479-488, Copyright © 1989 by Company of Biologists
Submitted on April 6, 1989
Accepted on July 22, 1989
1 National Institute for Medical Research, Mill Hill, London NW7 IAA, UK; E.M.B.L., Meyerhofstrasse 1, D-6900 Heidelberg, Federal Republic of Germany
2 National Institute for Medical Research, Mill Hill, London NW7 IAA, UK
Author for correspondence
Polymorphism in the self-assembly of tubulin dimer and microtubule protein (tubulin plus the microtubule-associated proteins) has been investigated as a function of systematic variation of solution composition (i.e. buffer ion, [glycerol] and [Mg2+]). The nature of the assembly product was examined using negative staining and thin sectioning electron microscopy.
The morphology of the product of assembly of tubulin dimer was found to be strongly influenced by the concentration of glycerol and Mg2+ in Pipes and Mes buffers; the effects are less marked in phosphate buffer. Formation of bona fide microtubules in O.l M-Pipes occurs for a limited range of solution conditions (e.g. with [glycerol] <2 M and [Mg2+]<1mM). Conditions of elevated [glycerol] and [Mg2+],which enhance the rate and extent of assembly, have the adverse effect of strongly promoting the formation of polymorphic forms in addition to, and in place of, the normal microtubule morphology.
In both Pipes and Mes buffers, increasing [glycerol] from 1 to 3 M favours the formation of extended multiply curved sheets, apparently made up from a basic structure with an S-like crosssection. By contrast, increasing [Mg2+] promotes the formation of junctions between microtubule walls, giving products whose cross-section shows multiple hook-like appendages, attached to closed microtubules. The assembly of tubulin dimer in a typical ‘dimer assembly buffer’ (e.g. 0.05-0.1 MMes, with 1-3.4M-glycerol and 2-7mM-Mg2+), invariably produces substantial proportions of nonmicrotubule structures such as open sheets, ribbons, and hooked structures.
We conclude that the self-assembly of tubulin dimer exclusively into bona fide microtubules occurs over a very restricted range of solution conditions in the normally used Pipes- and Mes-based buffers. Deviation from these conditions readily promotes the formation of mixtures of polymorphic forms. Many buffer systems used for the assembly and disassembly of microtubules composed of tubulin dimer appear likely to promote the formation of structures related to, but significantly different from, normal microtubules. This represents a cautionary factor in the interpretation of in vitro assembly and disassembly properties of microtubules
Key words: tublin, microtubules, assembley, morphology, negative staining, thin section, electron microscopy
Submitted on April 6, 1989
Accepted on July 22, 1989
