Michelle Moritz

Assistant Research Biochemist

Room S416
Department of Biochemistry & Biophysics
University of California, San Francisco
Mission Bay, Genentech Hall
600 16th St
San Francisco CA
94143-2240
(415) 502-2930 (Ph)
(415) 476-1902 (Fax)

The Structure and Biochemistry of the Centrosome and the γ-Tubulin Ring Complex

My focus has been to understand the structure-function relationship of microtubule nucleating sites in the centrosome, and in a more simplified form, the isolated γ-tubulin ring complex (γ-TuRC, Zheng et al. 1995. Nature . 378:578). We use a combination of biochemical and cell biological techniques, as well as cryo- and tomographic electron microscopy to explore this problem. Using centrosomes isolated from early Drosophila embryos and electron tomography, we have provided evidence that the microtubule nucleating sites consist of ring-shaped structures that contain γ-tubulin (Moritz et al, 1995a, b), an isoform of tubulin that has been strongly implicated in microtubule nucleation. We are currently using electron tomography and cryo-electron microscopy to explore these ring structures at higher resolution, both in the intact centrosome and as isolated γ-TuRCs. These studies are revealing information about the arrangement of the approximately seven different constituent proteins within the γ-TuRC, as well as information on the structural basis of microtubule nucleation by the γ-TuRC. EM studies of isolated γ-TuRCs in association with microtubules have revealed that the γ-TuRC forms a cap on the minus end of the microtubule (Moritz et al. 2000. Nat. Cell Biol.). This is most consistent with a model in which γ-TuRC acts as a template for microtubule nucleation (Moritz and Agard, Curr. Opin. Struc. Biol., In Press).

In addition, we have developed an in vitro complementation assay involving salt-inactivated isolated centrosomes which allowed us to show that the γ-TuRC, as well as an additional factor of ~220kD, are required for microtubule nucleation by centrosomes (Moritz et al, 1998a). We have been using electron tomography to examine the structure of the salt-stripped centrosome "scaffolds" to which γ-TuRCs attach. So far, the scaffolding appears to be a dense network of long fibers of approximately 10 nm in diameter, perhaps containing coiled regions. The protein composition of these scaffolds is complex, but no known centrosomal proteins remain in these structures. Hence, we are also attempting to identify the salt-insoluble proteins.

Publications

GCP5 and GCP6: Two New Members of the Human γ-Tubulin Complex, 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. Mol. Biol. Cell 2001;12 3340-3352, html or pdf.

M. Moritz, M.B. Braunfeld, B.M. Alberts, D.A. Agard, "Reconstitution of Centrosome Microtubule Nucleation in Drosophila," Methods in Cell Biology. (2001) 67:141-8.

M. Moritz and D.A. Agard, "γ-Tubulin Complexes and Microtubule Nucleation," Current Opinion in Structural Biology, 2001 Apr;11(2):174-81 (pdf or html).

M. Moritz, Braunfeld MB, Guenebaut V, Heuser J, Agard DA. "Structure of the g-tubulin ring complex: a template for microtubule nucleation," Nat Cell Biol. 2000 2(6), 365-70 (Abstract)

M. Moritz "Preparing cytoplasmic extracts from Drosophila embryos. In Drosophila: A laboratory manual," 2000, Ashburner, M. (ed.), Cold Spring Harbor Laboratory Press. 571-5.

M. Moritz and B.M. Alberts, "Isolation of centrosomes from Drosophila embryos," Methods in Cell Biol.,1998, 61, 1-12.

M. Moritz, Y. Zheng, B.M. Alberts, and K. Oegema, "Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds," J. Cell Biol.,1998, 142, 775-786.

M. Moritz, M.B. Braunfeld, J.W. Sedat, B.M. Alberts, and D.A. Agard, "Microtubule nucleation by -tubulin-containing rings in the centrosome," Nature, 1995, 378, 638-40.

M. Moritz, M.B. Braunfeld, J.C. Fung, J.W. Sedat, B.M. Alberts, and D.A. Agard, "Three-dimensional structural characterization of centrosomes from early Drosophila embryos," J. Cell Biol.,1995, 130, 1149-59 Abstract.

D.R. Kellogg, M. Moritz, and B.M. Alberts, "The centrosome and cellular organization," Annu. Rev. Biochem.,1994, 63, 639-74.

M. Moritz, "Watching the tube," Current Biology, 1993, 3, 387-390.

M. Moritz, B.A. Pulaski, and J.L. Woolford, Jr., "Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive mutants defective in yeast ribosomal protein L16," Mol. Cell. Biol., 1991, 11, 5681-5692.

M. Moritz,, A.G. Paulovich, Y.-F. Tsay, and J.L. Woolford, Jr., "Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly," J. Cell Biol., 1990, 111, 2261-2274 Abstract.

M.O. Rotenberg, M. Moritz, and J.L. Woolford, Jr., "Depletion of Saccaharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes," Genes & Development, 1988, 2, 160-172.