Proposed is a plan of research designed to determine the mechanism of microtubule nucleation by the centrosome in molecular detail. A combination of hierarchical structural approaches (EM Tomography, EM single particle reconstruction, and x-ray crystallography) and biochemical dissection and reconstitution methods will be utilized to determine the structures of gamma-tubulin and its complexes in vivo and in situ. Spanning size scales from the atomic to the entire organelle, our goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between tubulin, gamma-tubulin complexes, and the centrosomal matrix. This is a continuation and expansion of a well-established research program that has made dramatic progress in the analysis of large, complex, supramolecular assemblies through a combination of light and electron microscopies. In addition, we plan to continue our efforts to determine the three-dimensional fine structure of mitotic chromosomes in a variety of biologically well-defined functional states. The long range goal is to understand the structural complexities that underlie DNA condensation and its organization into higher-order structures that can support as well as modulate transcriptional activity, and that change throughout the cell cycle. Current efforts focus on the structural analysis of Xenopus sperm chromatic condensed in vitro using Xenopus extracts as pioneered by Professor T. Mitchison (Harvard). By providing a reproducible and well controlled sampling of haploid chromosome condensation states, this will allow us to understand the condensation process as well as to determine the 3D organization of key protein components being discovered by other laboratories using this system to understand function. Furthermore, the structural consequences of immuno-depletion of such key protein components will be determined. In addition, we continue to pursue structural studies on HeLa telophase chromosomes. While ultimately a less powerful system than the Xenopus vitro condensation approach, for now it offers several important technical advances for structural analysis that makes it worthwhile. Key will be the use of High Pressure Freezing, cyro embedding, DNA-specific staining and cryo, low dose automated Intermediate Voltage Electron Microscope Tomography (IVEM-T) to allow tracing the 3D chromatin paths.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031627-18
Application #
6385476
Study Section
Special Emphasis Panel (ZRG1-SSS-I (03))
Program Officer
Deatherage, James F
Project Start
1983-03-01
Project End
2004-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
18
Fiscal Year
2001
Total Cost
$279,734
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Greenan, Garrett A; Keszthelyi, Bettina; Vale, Ronald D et al. (2018) Insights into centriole geometry revealed by cryotomography of doublet and triplet centrioles. Elife 7:
Chaikeeratisak, Vorrapon; Nguyen, Katrina; Khanna, Kanika et al. (2017) Assembly of a nucleus-like structure during viral replication in bacteria. Science 355:194-197
Zheng, Shawn Q; Palovcak, Eugene; Armache, Jean-Paul et al. (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14:331-332
Greenberg, Charles H; Kollman, Justin; Zelter, Alex et al. (2016) Structure of ?-tubulin small complex based on a cryo-EM map, chemical cross-links, and a remotely related structure. J Struct Biol 194:303-10
Lyon, Andrew S; Morin, Geneviève; Moritz, Michelle et al. (2016) Higher-order oligomerization of Spc110p drives ?-tubulin ring complex assembly. Mol Biol Cell 27:2245-58
Chiu, Po-Lin; Li, Xueming; Li, Zongli et al. (2015) Evaluation of super-resolution performance of the K2 electron-counting camera using 2D crystals of aquaporin-0. J Struct Biol 192:163-73
Kollman, Justin M; Greenberg, Charles H; Li, Sam et al. (2015) Ring closure activates yeast ?TuRC for species-specific microtubule nucleation. Nat Struct Mol Biol 22:132-7
Peng, Yutian; Moritz, Michelle; Han, Xuemei et al. (2015) Interaction of CK1? with ?TuSC ensures proper microtubule assembly and spindle positioning. Mol Biol Cell 26:2505-18
Erb, Marcella L; Kraemer, James A; Coker, Joanna K C et al. (2014) A bacteriophage tubulin harnesses dynamic instability to center DNA in infected cells. Elife 3:
Mennella, Vito; Agard, David A; Huang, Bo et al. (2014) Amorphous no more: subdiffraction view of the pericentriolar material architecture. Trends Cell Biol 24:188-97

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