Regulatory mechanisms for the biogenesis and polymerization of alpha/beta tubulin and their impact on Microtubule Function

University Of California At Davis, Davis CA

Investigators

Abstract

Project Summary The dynamic microtubule cytoskeleton mediates intracellular organization, generates forces for dividing or migrating eukaryotic cells, and forms tracks for intracellular trafficking. The fundamental properties of microtubules, including polarized growth and “dynamic instability” stem directly from the activities of their building blocks, the αβ-tubulin heterodimers. Tubulins are among the most highly expressed proteins in eukaryotic cells. Despite advances in understanding special mechanisms regulating tubulin translation and folding, we lack understanding of how -tubulins are topologically assembled by three conserved tubulin cofactors (C, D, and E) and the dedicated Arf-like 2 G-protein. These molecules form multi-subunit platforms for the GTP-hydrolysis-dependent biogenesis and degradation of αβ-tubulins and maintain their high concentration within the cytoplasm. However, the mechanisms of these assemblies remain mostly mysterious, due in part to a lack of biochemical and structural information. We do not understand how the unique microtubule polymerization regulators, chTOG and CLASP, with arrays of tumor overexpressed gene (TOG) domains, recruit -tubulins while persistently tracking dynamic microtubule ends. Understanding these cellular regulation pathways is critical since genetic defects that impair either soluble -tubulin biogenesis or microtubule regulators are linked to inherited neurological and developmental disorders and are observed in human cancers, respectively. This proposal explores the biochemical and structural mechanisms of -tubulin biogenesis and microtubule polymerization regulators and their impact on microtubule function. Our strategy combines methods across multiple resolution scales, including in vitro reconstitution of purified assemblies, structural studies by cryo-electron microscopy (cryo-EM), reconstitution of assemblies with microtubule dynamics using in vitro fluorescence microscopy-based assays, and in vivo live imaging within living cells in collaboration with two expert cell biology groups. In the first research theme, we will build on our recently determined cryo-EM structures for yeast tubulin cofactor Arf-like 2 assemblies with -tubulin in the pre- and post-catalytic states to explore the biological roles of assembly interactions using an in vivo model system for -tubulin biogenesis, and we will determine novel cryo-EM structures for human tubulin cofactor assemblies purified from eukaryotic cells bound to novel monomeric tubulin biogenesis intermediates. These studies will fill in missing structural states and establish the functional relevance of the “catalytic chaperone” -tubulin biogenesis model. These studies will also deepen our understanding of how -tubulin biogenesis is regulated and its functional impact on microtubule function. In the second research theme, we will build on our understanding of the role of the self-folding of yeast TOG arrays in the recruitment and polymerization of -tubulin by using a new strategy to reconstitute human chTOG and CLASP, bound to -tubulins in different states and determine their cryo-EM structures, and explore the functional impact of these self-folded states using structure-based mutants using in vitro reconstitution and in vivo novel CRISPER-based replacement and imaging in human cells. We will expand on the “polarized unfurling” model we developed based on structural and biochemical studies for the self-folding TOG domain arrays in mediating -tubulin recruitment and polymerization. These studies will help determine the biochemical states of the two classes of microtubule regulators with TOG domain arrays in regulating tubulin recruitment and promoting polymerization. Our focused biochemical strategies will allow cryo-EM structure determination of assemblies in unique states that will help refine and deepen our new models for soluble -tubulin biogenesis, TOG array-mediated recruitment, and incorporation of -tubulin during microtubule polymerization. Our studies will pave the way to a deeper understanding of tubulin regulation, which will in turn point toward new strategies for addressing defects in tubulin biogenesis and regulation in patients with a range of developmental and neurological disorders.