Abstract
In the presence of GTP, purified dimers of α- and β-tubulin will interact longitudinally and laterally to self-assemble into microtubules (MTs). This property provides a powerful in vitro experimental system to describe MT dynamic behavior at the micrometer scale and to study effects and functioning of a large variety of microtubule associated proteins (MAPs). Despite the plethora of such data produced, the molecular mechanisms of MT assembly remain disputed. Electron microscopy (EM) studies suggested that tubulin dimers interact longitudinally to form short oligomers which form a tube by lateral interaction and which contribute to MT elongation. This idea is however challenged: Based on estimated association constants it was proposed that single dimers represent the major fraction of free tubulin. This view was recently supported by measurements suggesting that MTs elongate by addition of single tubulin dimers. To solve this discrepancy, we performed a direct measurement of the longitudinal interaction energy for tubulin dimers. We quantified the size distribution of tubulin oligomers using EM and fluorescence correlation spectroscopy (FCS). From the distribution we derived the longitudinal interaction energy in the presence of GDP and the non-hydrolysable GTP analog GMPCPP. Our data suggest that MT elongation and nucleation involves interactions of short tubulin oligomers rather than dimers. Our approach provides a solid experimental framework to better understand the role of MAPs in MT nucleation and growth.
Highlights
Microtubules (MTs) are tubular structures resulting from the assembly of a- and b-tubulin-dimers
The first set of results showed that pre-formed tubulin oligomers where participating in MT elongation [6] whereas the later one argues that only single dimers are incorporated at the MT tip [7]
In this last paper the authors use the previously estimated free-energy of tubulin/tubulin interaction to predict that at 5 mM tubulin concentration only 5% of the tubulin dimers would be in an oligomeric form, whereas 95% of the tubulin should be in the form of individual dimers [7]
Summary
Microtubules (MTs) are tubular structures resulting from the assembly of a- and b-tubulin-dimers. One posits that tubulin-dimers first interact longitudinally to form protofilaments that subsequently interact laterally to form a tube. Some authors posit that tubulin dimers are forming short longitudinal oligomers that are subsequently incorporated at the MT ends [6] whereas others claim that MTs are elongating through incorporation of single dimers [7]. In order to obtain a firmer ground on which to understand how microtubule assembly occurs at the molecular scale, we set out to determine the equilibrium size of tubulin oligomers at very low tubulin concentration. This was achieved using two independent methods: quantitative analysis of EM-resolved tubulin oligomers and FC(C)S analysis of a two-color labeled tubulin mix
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