The Structure and Biochemistry of theγ-Tubulin Ring Complex

I am studying the mechanism of microtubule nucleation from the highlyconserved γ-tubulin ring complex (γTuRC) of the centrosome and other microtubule-organizing centers (MTOCs). I am focusing on the structure-function relationship of the arrangement of γ-tubulins within the γTuRC to microtubule-nucleating activity. I have found that a “closed” γTuRC conformation is more active than an “open” conformation, which suggests a point of regulation for microtubule nucleating activity (Kollman et al 2015, Nat StrucMolBiol 22:132). A promising candidate for a regulator is casein kinase 1δ (Hrr25p in S. cerevisiae), a member of the highly conserved casein kinase family. It associates with MTOCs in many organisms, but its specific targets and roles in MTOCs have yet to be defined. We found that Hrr25p localizes to the yeast centrosome/spindle pole body and phosphorylates the (γTuSC) in vivo and in vitro. Hrr25p kinase activity is important for normal microtubule function and spindle positioning in vivo. In vitro microtubule assembly experiments revealed that Hrr25p enhances the microtubule nucleating activity of the yeast γTuRC (Peng et al (2015) MBoC 26:2505). To better understand the interaction of Hrr25p and γTuSC on a molecular level, we are pursuing a structural study by single-particle cryo-electron microscopy.

I am also involved in the experimental side of our kinetic modeling of microtubule nucleation by collecting microtubule assembly data through turbidity, TIRF, and DIC microscopy under various conditions. Our aim is to test the effects of mutations in γ- and αβ-tubulin on the mechanism of nucleation and assembly, as well as to assess the contributions of regulatory molecules such as Hrr25p.

PEER REVIEWED PUBLICATIONS

  1. Lyon, A.S., G. Morin, M. Moritz, K.C.B. Yabut, T. Vojnar, A. Zelter, E. Muller, T.N. Davis, D.A. Agard. 2016. Higher-order oligomerization of Spc110p drives γ-tubulin ring complex assembly. Mol. Biol. Cell. 27: 2245-2258.
  2. Peng, Y., M. Moritz, X. Han, T.H. Giddings, A. Lyon, J. Kollman, M. Winey, J. Yates III, D.A. Agard, D.G. Drubin, G. Barnes. 2015. Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning. Mol. Biol. Cell. 26: 2505-2518.
  3. Kollman, J. M., C. H. Greenberg, S. Li, M. Moritz, A. Zelter, K. K. Fong, J. J. Fernandez, A. Sali, J. Kilmartin, T. N. Davis and D. A. Agard. 2015. Ring closure activates yeast γTuRC for species-specific microtubule nucleation. Nat StructMolBiol 22(2): 132-137.
  4. Murphy, S.M., A. M. Preble, U.K. Patel, K.L. O'Connell, D.P. Dias, M. Moritz, D.A. Agard, J.T. Stults, and T. Stearns. 2001. GCP5 and GCP6: Two new members of the human γ-tubulin complex. Molecular Biology of the Cell 12:3340-3352.
  5. Moritz, M., M.B. Braunfeld, V. Guenebaut, J. Heuser, D.A. Agard. 2000. Structure of the γ-tubulin-ring complex: a template for microtubule nucleation. Nature Cell Biology 2:365-370.
  6. Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. Recruitment of the γ-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. J. Cell Biol. 142:775-786.
  7. Moritz, M., M.B. Braunfeld, J.W. Sedat, B.M. Alberts, and D.A. Agard. 1995. Microtubule nucleation by γ-tubulin-containing rings in the centrosome. Nature. 378:638-40.
  8. Moritz, M., M.B. Braunfeld, J.C. Fung, J.W. Sedat, B.M. Alberts, and D.A. Agard. 1995. Three-dimensional structural characterization of centrosomes from early Drosophila embryos. J. Cell Biol. 130:1149-59.
  9. Moritz, M., B.A. Pulaski, and J.L. Woolford, Jr. 1991. Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive mutants defective in yeast ribosomal protein L16. Mol. Cell. Biol. 11:5681-5692.
  10. Moritz, M., A.G. Paulovich, Y.-F. Tsay, and J.L. Woolford, Jr. 1990. Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly. J. Cell Biol. 111:2261-2274.
  11. Rotenberg, M.O., M. Moritz, and J.L. Woolford, Jr. 1988. Depletion of Saccaharomycescerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes. Genes & Development 2:160-172.

REVIEW ARTICLES

  1. Moritz, M. and D.A. Agard. 2001. γ-Tubulin complexes and microtubule nucleation. Current Opinion in Structural Biology11(2):174-181.
  2. Kellogg, D.R., M. Moritz, and B.M. Alberts. 1994. The centrosome and cellular organization. Annu. Rev. Biochem.63:639-74.
  3. Moritz, M. 1993. Watching the tube. Current Biology3:387-390.

BOOKS CHAPTERS

  1. Moritz, M. 2007. Preparing cytoplasmic extracts from Drosophila embryos. In CSH Drosophila Protocols, Mar. 1, Sullivan, W., Ashburner, M, Hawley, R.S., editors, CSH Press pp. 571-575.
  2. Moritz, M., L.M. Rice, and D.A. Agard. 2004. Microtubule nucleation. In Centrosomes in Development and Disease.Nigg, E.A., editor, Wiley-VCH. pp. 27-41.
  3. Moritz, M., M.B. Braunfeld, B.M. Alberts, D.A. Agard. 2001. Reconstitution of centrosome nucleation in Drosophila. In Methods in Cell Biology 67:141-148.
  4. Moritz M. Drosophila Protocols. Sullivan W, Ashburner M, Hawley RS, editors. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2000. Chapter 33, Preparing Cytoplasmic Extracts from Drosophila Embryos; p.571-575.
  5. Moritz, M. and B.M. Alberts. 1998. Isolation of centrosomes from Drosophila embryos. Methods in Cell Biol. 61: 1-12.