Integrated Protocol for γTuRC Expression and Purification for Microtubule Nucleation and Cryo-EM Analysis.

Microtubule nucleation is a crucial process in cell biology that describes the formation of a new microtubule from free tubulin. As one of the key steps in establishing organized microtubule networks, microtubule nucleation is fundamental to the roles of the microtubule cytoskeleton in mediating intracellular transport, mitotic spindle formation, and cellular morphology. At the core of this process is the γ-tubulin ring complex (γTuRC), a multi-protein complex that serves as a template for microtubule nucleation, thus ensuring spatial and temporal control of microtubule growth. Understanding the molecular mechanisms that govern microtubule nucleation is essential for elucidating how cells dynamically organize their cytoskeleton and how dysregulation of this process contributes to pathologies such as cancer and neurodegenerative diseases.In this method paper, we describe a straightforward and accessible approach to study microtubule nucleation without the need for advanced instrumentation. This includes a reliable and reproducible assay for assessing the activity of γTuRC and its regulatory factors that is suitable for a wide range of research settings. Detailed protocols for the recombinant production and purification of γTuRC, along with in vitro nucleation assays to assess its activity and regulation, are provided. Furthermore, we outline sample preparation for high-resolution structural analysis by cryo-electron microscopy (cryo-EM). Together, these methodologies provide a comprehensive framework for probing the mechanisms underlying γTuRC-templated microtubule nucleation and its regulation.