Microtubule nucleation by γ-tubulin complexes and beyond

Abstract

In this short review, we give an overview of microtubule nucleation within cells. It is nearly 30 years since the discovery of γ-tubulin, a member of the tubulin superfamily essential for proper microtubule nucleation in all eukaryotes. γ-tubulin associates with other proteins to form multiprotein γ-tubulin ring complexes (γ-TuRCs) that template and catalyse the otherwise kinetically unfavourable assembly of microtubule filaments. These filaments can be dynamic or stable and they perform diverse functions, such as chromosome separation during mitosis and intracellular transport in neurons. The field has come a long way in understanding γ-TuRC biology but several important and unanswered questions remain, and we are still far from understanding the regulation of microtubule nucleation in a multicellular context. Here, we review the current literature on γ-TuRC assembly, recruitment, and activation and discuss the potential importance of γ-TuRC heterogeneity, the role of non-γ-TuRC proteins in microtubule nucleation, and whether γ-TuRCs could serve as good drug targets for cancer therapy.

Asymmetric molecular architecture of the human γ-tubulin ring complex

“…GCP4 is organized into two -helical bundles containing the conserved -tubulin r ing complex i nteracting p rotein motif 1 (GRIP1, corresponding to the N-domain) and the GRIP2 (C-domain) motifs 22 . Notably, GCP4 has also been proposed to form part of a -TuSC-like subcomplex at the -TuRC "overlap" 17,23,24 . Surprisingly, however, the crystallographic model for GCP4 could not be rigid-body fitted and refined into the -TuRC density map at the "overlap" region (i.e., positions 1 or 14; also referred to as the -TuRC "seam" 25 Further, we aligned our refined GCP4 model to the monomeric, crystallographic GCP4 model via their N-domains 22 , which revealed that within the -TuRC the GCP4 C-domain is rotated by ~10° towards the preceding subunit ( Figure S4F), and helix 21 is rotated by ~90° towards the GCP4-associated -tubulin ( Figure S4G).…”

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

“…Microtubule nucleation in cells is spatially and temporally controlled to ensure proper cytoskeleton function. The major nucleator is the γ-tubulin ring complex (γTuRC) in which several γ-tubulin complex proteins (GCPs) arrange 14 γ-tubulins into a helical arrangement so that they can serve as a template for microtubule nucleation Tovey and Conduit, 2018). The structure of γTuRC is best understood in budding yeast where 7 smaller 'Y-shaped' γTuSC complexes, each consisting of 2 γ-tubulins and one copy of GCP2 and GCP3, assemble into a conically shaped assembly upon recruitment to spindle the pole body by SPC110 (Kollman et al, 2010).…”

Architecture of the Mto1/2 microtubule nucleation complex

“…Microtubule (MT) nucleation by the g-tubulin complex (g-TuC) depends on a variety of accessory proteins that regulate g-TuC localization, activity, and assembly state (Farache et al, 2018;Kollman et al, 2011;Lin et al, 2015;Paz and Luders, 2018;Petry and Vale, 2015;Roostalu and Surrey, 2017;Tovey and Conduit, 2018). The core element of the g-TuC, the highly conserved heterotetrameric g-tubulin small complex (g-TuSC), contains two copies of g-tubulin and one copy each of the related proteins GCP2 and GCP3 (in humans).…”

“…Drosophila melanogaster, Aspergillus nidulans, and fission yeast Schizosaccharomyces pombe), these non-core proteins contribute only modestly to MT nucleation in vivo (Anders et al, 2006;Fujita et al, 2002;Venkatram et al, 2004;Verollet et al, 2006;Xiong and Oakley, 2009), and in other organisms (e.g. budding yeast Saccharomyces cerevisiae), such non-core proteins are completely absent (Lin et al, 2015;Tovey and Conduit, 2018). This diversity has suggested that in addition to the "classical" g-TuRC of metazoan cells, there may be other mechanisms by which multiple g-TuSCs can assemble to generate the functional equivalent of a g-TuRC.…”

“…The CM1 domain, a ~60 amino-acid region that interacts with the g-TuC, is conserved from yeasts to humans (Choi et al, 2010;Leong et al, 2019;Samejima et al, 2008;Sawin et al, 2004;Zhang and Megraw, 2007), and in most organisms CM1 proteins are either unique or present as a paralog pair. Characterized CM1 proteins include human CDK5RAP2/Cep215 and myomegalin/PDE4DIP, Drosophila centrosomin (Cnn), Aspergillus ApsB, budding yeast Spc110p, and fission yeast Mto1 and Pcp1 (summarized in (Lin et al, 2015;Tovey and Conduit, 2018)). Outside the CM1 domain, CM1 proteins are divergent in primary sequence; however, all CM1 proteins are ~100 kDa or greater, and all contain the CM1 domain near their N-termini and multiple predicted coiled-coils in C-terminal regions (Suppl.…”