The microtubule (MT) cytoskeleton is essential to eukaryotic cells: microtubules are dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. The dynamic properties of MTs are central to their function, and they derive from the biochemical properties of individual tubulin subunits and how they interact within the MT lattice. MT dynamics are modulated by a host of regulatory factors that often selectively recognize different conformations of ??-tubulin. Two distinct conformations of ?? -tubulin have been determined in atomic detail: a 'straight'one compatible with the MT lattice, and a 'curved'one that is not. Still different conformations of ?? -tubulin were revealed by lower resolution studies of ?? -tubulin assemblies that mimic the unique geometries observed at MT ends. Do any of these conformations represent the solution conformation of ?? -tubulin? How do conformation and conformational change contribute to the dynamic properties of the MT? Do regulatory proteins control MT dynamics by altering the default conformation of ?? - tubulin? Despite intense study, fundamental questions like these remain unresolved. Structural insight into these and other questions is limited: the tendency to polymerize makes it extremely difficult to obtain atomic structures of ?? -tubulin by itself or in complex with MT associated proteins (MAPs). Preliminary data demonstrate that it is now possible to prepare polymerization-blocked mutants of yeast ?? -tubulin, and to use them to determine new atomic structures of ?? -tubulin and its complexes with regulatory proteins. This unique approach will allow new experiments to understand the structural origins of microtubule dynamics and how cellular factors regulate it.
Aim 1 will determine the structure of a complex between yeast ?? -tubulin and a tubulin-binding TOG domain from an essential regulator of microtubule dynamics, the multi-TOG containing protein Stu2p. This will provide the second-ever structure of ?? -tubulin bound to a regulatory protein, and will provide a structural framework for understanding how individual TOG domains recognize ?? -tubulin.
Aim 2 will combine structural and biochemical approaches to discover how multiple TOG domains can bind to ?? -tubulin simultaneously. These experiments will lead to a better understanding of how cooperativity between TOG domains contributes to the microtubule end recognition and elongation promoting activities of Stu2p.
Aim 3 will answer questions about the conformation of un polymerized ?? -tubulin and how it depends on nucleotide state by determining structures of ?? -tubulin bound to GTP or to GDP, and by obtaining mutant ?? -tubulin with altered 'curvature'. By enabling previously impossible measurements and by closely integrating structural and functional observations, successful completion of this work will represent a major advance in the understanding of the structural determinants of microtubule behavior. )

Public Health Relevance

Microtubules are dynamic polymers of ?? -tubulin that mediate the internal organization of cells and the faithful segregation of genetic material, and that are the direct targets of some chemotherapeutic drugs. Dynamic microtubule behavior and its regulation by cellular factors are poorly understood, partly because it is hard to make altered versions of ?? -tubulin to test hypotheses and make new structural and biochemical measurements. This work takes advantage of selectively modified ?? - tubulins, using them in a series of new experiments to better understand the structural origins of microtubule dynamics and how they are regulated.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Gindhart, Joseph G
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University of Texas Sw Medical Center Dallas
Schools of Medicine
United States
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Brouhard, Gary J; Rice, Luke M (2014) The contribution of ??-tubulin curvature to microtubule dynamics. J Cell Biol 207:323-34
Ayaz, Pelin; Munyoki, Sarah; Geyer, Elisabeth A et al. (2014) A tethered delivery mechanism explains the catalytic action of a microtubule polymerase. Elife 3:e03069