Tubulin dimers oligomerize before their incorporation into microtubules.
In the presence of GTP, purified dimers of alpha- and beta-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.
Mozziconacci
Julien
J
Pierre et Marie Curie University, UMR 7600 LPTMC, Paris, France.
Sandblad
Linda
L
Wachsmuth
Malte
M
Brunner
Damian
D
Karsenti
Eric
E
D000818:Animals; D019281:Dimerization; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008854:Microscopy, Electron; D008870:Microtubules / Q000737:chemistry* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D013552:Swine; D014404:Tubulin / Q000378:metabolism* / Q000648:ultrastructure
D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate