This MIRA application is to consolidate my effort on three separate GM grants: our long standing centrosome structure and microtubule nucleation grant (GM031627-30), yeast spindle pole body program project grant (Winey 1P01GM105537) and a MPI grant on the newly discovered phage tubulin PhuZ (Joe Pogliano, GM104556). This work is exciting and has been well appreciated. Most importantly, the collaborative efforts have been invaluable, resulting in remarkable synergies and a great expansion of our reach and understanding (genetics, in vivo function, computational modeling, single molecule biophysics). Similarly, our unique combination of biophysics and structure has significantly enhanced what our collaborators could accomplish. Unfortunately under the new rules, I cannot continue my funded participation in these efforts. Tubulins play important and remarkably varied roles throughout eukaryotic and prokaryotic biology. In most eukaryotes, the microtubule (MT) cytoskeleton is responsible for chromosome segregation, nuclear positioning, vesicle trafficking and cell polarity. As the principle nucleator of MTs, the centrosome is of fundamental importance and harbors the centrioles, specialized structures required for cilia formation and contribute to spindle-pole positioning. The centrosome also has important roles beyond MT nucleation, including functions in cytokinesis, progression through the cell cycle, and in sequestering or positioning some proteins to control when and where they are active. Thus, the centrosome is important in embryonic development and human disease. While the analogous structure in the yeast S. cerevisiae (the spindle pole body or SPB) is morphologically distinct, a conserved set of ?-tubulin complexes is used in both systems to nucleate MT assembly. Our long-term goals are to synthesize an atomic resolution picture of the relevant structural and functional interactions between ??- and ?-tubulin complexes, and to elucidate the principles underlying assembly and regulation. We have determined the structure of the yeast ?TuSC ring to 6.5 resolution, revealing principles of MT nucleation and protofilament number determination, and have identified at least two unexpected levels of regulation: via assembly mediated by oligomerization of the Spc110p attachment factor, and a closure event, lead- ing to activated nucleation. Proposed, we seek to expand the structural studies to atomic resolution by cryoEM and discover the role of PTMs and other factors in regulation. We will place in context via structures of the SPB and centrosome determined by cryoTomography, and better understand how the pericentrioloar material forms and uncover attachment factors that connect ?TuRCs to the Golgi. Our work on MT nucleation by ?-tubulin complexes has been complemented by our progress on the structure and biochemistry of prokaryotic tubulin homologs, including a ~3.3 structure of PhuZ:GMPCPP and ~4. of PhuZ:GDP, providing new insights into filament structure and general regulation by GTP hydrolysis. Here we focus on the novel PhuZ spindle and associated cell biology.
We focus on understanding the mechanisms of prokaryotic and eukaryotic tubulin polymerization and the cellular contexts in which polymerization occurs. Errors in these processes are a common hallmark of cancer cells, and defects in the cellular machinery are linked to human ciliary diseases.
| 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: |
| Fernandez, Jose-Jesus; Li, Sam; Bharat, Tanmay A M et al. (2018) Cryo-tomography tilt-series alignment with consideration of the beam-induced sample motion. J Struct Biol 202:200-209 |
| Palovcak, Eugene; Wang, Feng; Zheng, Shawn Q et al. (2018) A simple and robust procedure for preparing graphene-oxide cryo-EM grids. J Struct Biol 204:80-84 |
| Citron, Y Rose; Fagerstrom, Carey J; Keszthelyi, Bettina et al. (2018) The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles. PLoS One 13:e0190530 |
| Chaikeeratisak, Vorrapon; Nguyen, Katrina; Khanna, Kanika et al. (2017) Assembly of a nucleus-like structure during viral replication in bacteria. Science 355:194-197 |
| Larson, Adam G; Elnatan, Daniel; Keenen, Madeline M et al. (2017) Liquid droplet formation by HP1? suggests a role for phase separation in heterochromatin. Nature 547:236-240 |
| 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 |
| 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 |
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