Mechanism of microtubule formation and the role of γ-tubulin complexes as nucleators

The spatial and temporal regulation of tubulin polymerization into microtubules (MTs) is a central question in cell biology. Our goal is to understand, in atomic detail, the molecular mechanisms underlying dynamic MT behavior and MT nucleation.

Of key importance is the structural and functional analysis of γ-tubulin complexes, which act in vivo to nucleate MT growth. Although central to all MT nucleation, the γ-tubulin small complex (γTuSC) is a surprisingly poor MT nucleator. We have determined the structure of the isolated yeast γTuSC by single-particle EM (electron microscopy). We can assemble γTuSCs into either ring complexes or filaments, and we have recently determined the cryo-EM structure of the filament at ~8-Å resolution. These structures have 6.5 γTuSCs/turn, resulting in the display of 13 γ-tubulins explaining how pairs of γ-tubulins present within γTuSCs can nucleate 13 protofilament MTs.

Although yeast γTuSC assemblies can form spontaneously, they are only stable at low pH. However, Spc110p, which links γTuSC to the spindle pole body (SPB), stabilizes the assemblies to physiological conditions. Thus, γTuSC assemblies are only formed at the SPB, ensuring high fidelity of MT nucleation. The cryoEM structure also reveals that the γ-tubulins within each γTuSC are too far apart to efficiently nucleate MTs, indicating an allosteric activation required for potent MT nucleation. By engineering a closed form, the EM resolution can be extended to 6.5Å, allowing a detailed model to be built from a distantly related human accessory protein. This is being used to help discover the allosteric regulator and to understand the role of post-translational modifications.As part of our efforts to understand the molecular basis of MT nucleation, we solved the atomic structures of human γ-tubulin complexed with GTP and GDP, providing the first eukaryotic GTP/GDP pair. These structures provided two key insights: γ-tubulin forms MT-like lateral interactions independent of nucleotide, and γ-tubulin remains in a curved conformation independent of nucleotide, contrasting sharply with the prevailing allosteric hypothesis for activation of tubulin assembly by GTP. Solution studies (conformation-specific ligand binding and small-angle x-ray scattering [SAXS]) on αβ-tubulin confirm that it too remains in a curved conformation independent of nucleotide. As a major paradigm shift, we proposed that the lattice and not the nucleotide is the allosteric effector. In this lattice model, GTP acts only to tune the longitudinal affinity. Lattice metastability is determined not by GTP hydrolysis but by the mechanical spring constant for straightening. This new view has a dramatic impact on understanding MT formation. To explore this we have developed new first principle kinetic models of MT assembly and show how a spring penalty is required to match observed behavior.


Relevant Publications

"Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation," Guillet, Valerie; Knibiehler, Martine; Gregory-Pauron, Lynn; Remy, Marie-Helene; Chemin, Cecile; Raynaud-Messina, Brigitte; Bon, Cecile; Kollman, Justin M.; Agard, David A.; Merdes, Andreas; et al, Nature Structural & Molecular Biology (2011), 18(8), 915-919. (pdf)

"Microtubule nucleation by γ-tubulin complexes," Kollman, Justin M.; Merdes, Andreas; Mourey, Lionel; Agard, David A., Nature Reviews Molecular Cell Biology (2011), 12(11), 709-721. (htmlpdf)

Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA, "Microtubule nucleating γ-TuSC assembles structures with 13-fold microtubule-like symmetry," Nature (2010), (htmlpdf)

Choy RM, Kollman JM, Zelter A, Davis TN, Agard DA, "Localization and orientation of the γ-Tubulin Small Complex components using protein tags as labels for single particle EM," J Struct Biol. 2009 Dec, 168(3):571-4. (htmlpdf)

Rice, Luke M., Montabana, Elizabeth A., Agard, David A., "The lattice as allosteric effector: structural studies of αβ- and γ-tubulin clarify the role of GTP in microtubule assembly." Proceedings of the National Academy of Sciences of the United States of America (2008), 105(14), 5378-5383. (pdf)

Kollman JM, Zelter A, Muller EG, Fox B, Rice LM, Davis TN, Agard DA., "The Structure of the γ-Tubulin Small Complex: Implications of Its Architecture and Flexibility for Microtubule Nucleation", Mol Biol Cell. 2008, 19(1), 207-15 (pdf)

Aldaz H, Rice LM, Stearns T, Agard DA. "Insights into microtubule nucleation from the crystal structure of human γ-tubulin." Nature. 2005 May 26;435(7041):523-7. (html or pdf).

Rice, L. and Agard, D. (2002). Centriole duplication: centrin in on answers? Curr Biol 2002 Sep 17;12(18), R618-9. (pdf).

Murphy, S.M, Preble, A.M., Patel, U.K., O'Connell, K.L., Dias, D.P., Moritz, M., Agard, D.A., Stults, J.T., and Stearns, T., GCP5 and GCP6: Two New Members of the Human γ-Tubulin Complex, Mol. Biol. Cell (2001)12 3340-3352, (html or pdf).

Moritz, M., Agard, D.A. (2001) -Tubulin complexes and microtubule nucleation. Curr Opin Struct Biol. 11(2):174-81 (pdf or html).

Moritz, M., Braunfeld, M.B., Guenebaut, V., Heuser, J., Agard, D.A. (2000) Structure of the γ-tubulin ring complex: a template for microtubule nucleation. Nat Cell Biol. 2(6):365-70 (pdf).

Moritz, M., Braunfeld, M.B., Alberts, B.M., Agard, D.A. (1998) Reconstitution of centrosome microtubule nucleation in Drosophila. Methods Cell Biol.(1998) 67:141-8. (pdf).

Moritz, M., Braunfeld, M.B., Sedat, J.W., Alberts, B., and Agard, D.A. (1995). Microtubule nucleation by γ-tubulin-containing rings in the centrosome. Nature 378: 638-640. (pdf).

Moritz, M., Braunfeld, M.B., Fung, J.C., Sedat, J.W., Alberts, B.M., and Agard, D.A. (1995). Three-dimensional structural characterization of centrosomes from early Drosophila embryos. J. Cell Biol. 130: 1149-1159. (pdf).