The Fygenson GroupUCSB Physics Dept..



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Microtubule Dynamic Instability

Suggestions of a Bio-Polymer Brush

D. Kuchnir Fygenson
Physics Department, UC Santa Barbara




ABSTRACT   Microtubules are hollow, crystalline aggregates of the protein tubulin that date back to the last great common ancestor between man and microbe. Despite their "primitive" nature, they exhibit a sophisticated dynamics, assembling and disassembling over long lengths under otherwise constant chemical conditions. Much is known about the details of this behavior, but an understanding of the underlying mechanism remains elusive. I suggest a mechanism in which a portion of the protein "unfolds", creating an asymmetrically distributed polymer brush in the hollow of the microtubule, and describe a number of experiments (some of which are underway) which will test this hypothesis.




A microtubule is...




... a hollow, cylindrical aggregate of the protein tubulin. Tubulin is a hetero-dimer of nearly identical polypeptide chains (a-tubulin and b-tubulin) that aggregate head-to-tail (abab) into protofilaments. Protofilaments aggregate in parallel (aa, bb) to form the microtubule wall. Lateral bonds are slightly tilted and angled inwards, so that 13 protofilaments form a tube whose axis is parallel to the protofilament axis and whose surface lattice is described by a left-handed 3-start helix, with a seam (where the tube closed) along which monomers of one type interface laterally with monomers of the other type. The alignment of the protofilaments preserves the asymmetry of the dimer, so that the microtubule ends are different, one exposes a-tubulin to solution (the minus end) and the other, b-tubulin (the plus end).



Dynamic Instability Is...

... a surprising, non-equilibrium phenomenon exhibited by microtubules both in vivo and in vitro. Individual microtubules spontaneously switch between extended periods of growth (assembling) and shortening (dissolving) at their ends. Chemical requirements for dynamic instability are minimal. An aqueous environment with roughly equal concentrations of Mg++ and GTP (1 - 5 mM) and a slightly acidic pH (~6.8), held at temperatures ranging from ambient to physiological (15 - 37 °C) is all that is needed to support microtubule dynamics in a wide range of tubulin concentrations (5 - 50 µM).
The behavior at the two ends of the microtubule is qualitatively the same, but the quantitative dynamic parameters differ. Dynamic parameters include: rate of growth (V+), rate of shortening (V-), frequency of catastrophe (growth Æ shortening) (ƒ+-), and frequency of rescue (shortening Æ growth) (ƒ-+).
How can the stablity of a microtubule keep changing under otherwise constant chemical conditions?


The Key Is..

... GTP (guanosine tri-phosphate). a- and b-tubulin each bind one molecule of GTP, but a-bound GTP is buried at the interface between the monomers while b-bound GTP is exposed at the opposite pole. In the dimer, a-bound GTP is effectively sequestered while b-bound GTP can exchange with GTP in solution.
As dimers aggregate into protofilaments, b-bound GTP becomes buried dimers, and is immediately hydrolyzed. (a-bound GTP is never hydrolyzed.) The hydrolysis reaction releases about 10 kBT of free energy (and a phosphate ion), some of which is stored in the microtubule. This energy is essential for dynamic instability. When GTP is replaced by an analogous molecule which is not hydrolyzed (e.g., GMPCPP), microtubules assemble but do not exhibit dynamic instability.


The Question Is...

... how is the free energy of GTP hydrolysis stored in the microtubule? What triggers its sudden release, causing the microtubule to rapidly and extensively disassemble? Why does a disassembling microtubule sometimes stop and suddenly enter another phase of steady growth? In sum, what is the mechanism behind dynamic instability?



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