Centrosome Structure

The eukaryotic centrosome plays a critical role in organizing the mitotic spindle as well as much of the interphase cytoskeleton. The spatial and temporal regulation of tubulin polymerization into microtubules is a central question in cell biology. While the centrosome has long been known to play a role in microtubule (MT) nucleation, the underlying machinery is unknown. Centrosomes are large structures (~1 µm3) with a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). It is within this PCM that MT nucleation takes place. To explore this problem at both a structural and biochemical level, we formed a collaboration with the Alberts lab (Dr. Michelle Moritz, UCSF) who had been able to isolate functional centrosomes from Drosophila early embryos. A major problem in the understanding of centrosome function has been the amorphous nature of the PCM, making it refractory to conventional analysis. We used our newly developed EM Tomography methods to determine the structure of centrosomes both with and without MTs. Using our 3D reconstructions we could trace the paths of each of the MTs back to their origins (minus ends) and look for organized structures. We found that MT nucleation sites are randomly organized and oriented throughout the PCM. Further, the MTs end abruptly, revealing no large nucleation machinery. Knowing that the centrosome-specific tubulin variant (g-tubulin) is involved in MT nucleation, we combined immunolocalization with 3D tomography to ascertain its location. In the absence of MTs, we found that the PCM contained a large number of randomly positioned and oriented g-tubulin-containing rings - each the approximate diameter of a MT. Furthermore, with MTs regrown, g-tubulin was localized specifically to the minus ends of the microtubules. Thus these g-tubulin rings were proposed to be responsible for MT nucleation. In keeping with this, the Alberts' lab was also able to isolate soluble g-tubulin ring complexes (g-TuRc) from either Xenopus or Drosophila extracts using immuno-affinity chromatography.

For the past year we have been using single particle reconstruction methods to examine the 3D structure of g-TuRc and to determine how it acts to nucleate MT assembly. In addition, Dr. Michelle Moritz has joined my group so that together we can obtain a biochemical and structural understanding of centrosome structure and function. High salt washes of the centrosome remove its ability to nucleate MTs. Simply adding back purified g-TuRc is insufficient. However, she can reconstitute MT nucleation using extracts. This paves the way for purification of the missing component(s) by complementation.

Relevant Publications

Aldaz H, Rice LM, Stearns T, Agard DA. "Insights into microtubule nucleation from the crystal structure of human gamma-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 gamma -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 gamma-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 gamma-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).

γ-Tubulin Ring Complex: 3D Tomographic reconstructions
negative stain (γTuSC outlined)