Regulation of the γ-tubulin ring complex (γTuRC) through targeting and activation restricts nucleation of microtubules to microtubuleorganizing centers (MTOCs), aiding in the assembly of ordered microtubule arrays. However, the mechanistic basis of this important regulation remains poorly understood. Here, we show that, in human cells, γTuRC integrity, determined by the presence of γ-tubulin complex proteins (GCPs; also known as TUBGCPs) 2-6, is a prerequisite for interaction with the targeting factor NEDD1, impacting on essentially all γ-tubulin-dependent functions. Recognition of γTuRC integrity is mediated by MZT1, which binds not only to the GCP3 subunit as previously shown, but cooperatively also to other GCPs through a conserved hydrophobic motif present in the N-termini of GCP2, GCP3, GCP5 and GCP6. MZT1 knockdown causes severe cellular defects under conditions that leave γTuRC intact, suggesting that the essential function of MZT1 is not in γTuRC assembly. Instead, MZT1 specifically binds fully assembled γTuRC to enable interaction with NEDD1 for targeting, and with the CM1 domain of CDK5RAP2 for stimulating nucleation activity. Thus, MZT1 is a 'priming factor' for γTuRC that allows spatial regulation of nucleation.

The microtubule nucleator γ-tubulin ring complex (γTuRC) is essential for the function of microtubule organizing centers such as the centrosome. Since its discovery over two decades ago, γTuRC has evaded in vitro reconstitution and thus detailed structure-function studies. Here, we show that a complex of RuvB-like protein 1 (RUVBL1) and RUVBL2 “RUVBL” controls assembly and composition of γTuRC in human cells. Likewise, RUVBL assembles γTuRC from a minimal set of core subunits in a heterologous coexpression system. RUVBL interacts with γTuRC subcomplexes but is not part of fully assembled γTuRC. Purified, reconstituted γTuRC has nucleation activity and resembles native γTuRC as revealed by its cryo–electron microscopy (cryo-EM) structure at ~4.0-Å resolution. We further use cryo-EM to identify features that determine the intricate, higher-order γTuRC architecture. Our work finds RUVBL as an assembly factor that regulates γTuRC in cells and allows production of recombinant γTuRC for future in-depth mechanistic studies.

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Assaying microtubule nucleation is essential to understand the organization of microtubule networks in any cell type. In this chapter we describe methods for measuring nucleation activity at centrosomes and at mitotic chromatin in cell lines, to study interphase and mitotic microtubule organization, and for measuring non-centrosomal nucleation in cultured primary neurons, to study microtubule organization in the absence of a microtubule organizing center. While a number of different approaches and variations thereof have been reported in the literature, here we aim to keep the methodology as simple as possible and thus accessible to most research laboratories.

Background: Microtubules (MTs) are essential cytoskeletal polymers that provide structural support for the cell and play important roles in cell division, motility, and intracellular transport. The γ-tubulin ring complex (γTuRC) is the major MT nucleator in animal cells. The molecular mechanism by which the γTuRC promotes MT nucleation remains poorly understood although a template-based mechanism, remains the most widely accepted (Moritz et al., 2000, Kollman et al., 2010). According to this model γTuRC, a 2 MDa multi-subunit protein complex, forms a lock washer-like structure, in which γ-tubulin molecules are arranged in a ring-shaped structure that serves as a template for the assembly of αβ-tubulin heterodimers. Methods: We have set up an in vitro system to purify the human γTuRC using infected insect cells with recombinant baculoviruses. This complex sample was subjected to cryo-EM analysis and single-particle reconstruction. Results: We have demonstrated that RUVBL1-RUVBL2 AAA-ATPase complex (RUVBL) controls the assembly and composition of γTuRC in human cells both in vivo and in vitro. Likewise, RUVBL assembles γTuRC from a minimal set of core subunits in a heterologous co-expression system. Purified, reconstituted γTuRC has nucleation activity and resembles native γTuRC (Consolati et al., 2020, Liu et al., 2020, Wieczorek et al., 2020), as revealed by its cryo-EM structure at ~4.0 Å resolution. Conclusion: We have been able to identify novel mechanistic and structural features that determine the intricate, higher-order γTuRC architecture (Zimmermann, Serna et al., 2020).