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The centrosome and the mechanism of microtubule nucleation
Author(s) -
Rice Luke,
Lyle John,
Moritz Michelle,
Agard David A.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a35
Subject(s) - microtubule , tubulin , centrosome , microtubule nucleation , biophysics , gtpase , chemistry , cytoskeleton , microbiology and biotechnology , biology , biochemistry , cell , cell cycle
Microtubules are hollow polymers of α β‐tubulin that exhibit GTP‐dependent assembly dynamics and comprise a critical part of the eukaryotic cytoskeleton. Initiation of new microtubules in vivo requires γ‐tubulin, organized as an oligomer within the γ‐Tubulin Ring Complex (γ‐TuRC) of higher eukaryotes. Structural insight into γ‐tubulin, its oligomerization, and how it promotes microtubule assembly remains lacking. We have determined the 2.7Å crystal structure of human γ‐tubulin bound to GTPγ S. Unexpectedly, γ‐tubulin:GTPγ S adopts a curved conformation very similar to that seen in GDP‐bound microtubule depolymerization products. This suggests that, unlike signaling GTPases, tubulins may not undergo nucleotide‐dependent conformational switching. We support this with recent SAXS data and conformationally‐sensitive binding experiments. Together, these results have important implications for α β‐tubulin assembly; in particular, they suggest that there are unanticipated barriers to de novo microtubule assembly. We propose a model for microtubule assembly in which guanine nucleotides do not regulate curved‐to‐straight transitions, but instead serve to modulate the strength of longitudinal interactions within the microtubule lattice. To gain structural and functional sights into the mechanism of nucleation, we are using cryo electron microscopic tomography to directly visualize them at the minus ends of microtubules within intact frozen hydrated centrosomes at about 5nm resolution. In an effort to better define the nucleation site, we have developed software designed to find, align and average the minus end density, with a goal of reaching 2.5nm resolution. Our current reconstructions clearly show the γ‐TuRC cap and also reveal the fibrous nature of the pericentriolar material (PCM). Using isolated γ TuRCs and similar reconstruction approaches, we are working towards a 2.5nm reconstruction revealing the lock washer shape of the γ TuRC, likely made up from the lateral association of γ –tubulin small complexes (γ TuSC) and the existence of an additional cap structure that may act to stabilize the ring of γ TuSCs.