Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1

Abstract

Summary Microtubule (MT) nucleation depends on the γ-tubulin complex (γ-TuC), in which multiple copies of the heterotetrameric γ-tubulin small complex (γ-TuSC) associate to form a ring-like structure (in metazoans, γ-tubulin ring complex; γ-TuRC) [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ]. Additional conserved regulators of the γ-TuC include the small protein Mzt1 (MOZART1 in human; GIP1/1B and GIP2/1A in plants) [ 8 , 9 , 10 , 11 , 12 , 13 ] and proteins containing a Centrosomin Motif 1 (CM1) domain [ 10 , 14 , 15 , 16 , 17 , 18 , 19 ]. Many insights into γ-TuC regulators have come from in vivo analysis in fission yeast Schizosaccharomyces pombe . The S. pombe CM1 protein Mto1 recruits the γ-TuC to microtubule-organizing centers (MTOCs) [ 14 , 20 , 21 , 22 ], and analysis of Mto1[bonsai], a truncated version of Mto1 that cannot localize to MTOCs, has shown that Mto1 also has a role in γ-TuC activation [ 23 ]. S. pombe Mzt1 interacts with γ-TuSC and is essential for γ-TuC function and localization to MTOCs [ 11 , 12 ]. However, the mechanisms by which Mzt1 functions remain unclear. Here we describe reconstitution of MT nucleation using purified recombinant Mto1[bonsai], the Mto1 partner protein Mto2, γ-TuSC, and Mzt1. Multiple copies of the six proteins involved coassemble to form a 34-40S ring-like “MGM” holocomplex that is a potent MT nucleator in vitro . Using purified MGM and subcomplexes, we investigate the role of Mzt1 in MT nucleation. Our results suggest that Mzt1 is critical to stabilize Alp6, the S. pombe homolog of human γ-TuSC protein GCP3, in an “interaction-competent” form within the γ-TuSC. This is essential for MGM to become a functional nucleator.

Architecture of the Mto1/2 microtubule nucleation complex

“…Figure 1C). In a recent density-gradient centrifugation analysis, Mto1/2[bonsai] sedimented with a peak at 9-10S and a very long "tail", extending to 20S and beyond, suggesting that the complex forms higher-order multimers (Leong et al, 2019). Consistent with this, using size-exclusion chromatography with multi-angle light scattering (SEC-MALS) we found that the Mto1/2[bonsai] SEC peak comprises a range of molecular masses (Suppl.…”

“…In this work we provide insights into assembly and architecture of the Mto1/2 microtubule nucleation complex, through biophysical and structural analysis of the Mto1/2[bonsai] complex, a freely-diffusible, minimal form of the Mto1/2 complex that retains Mto1/2's ability to promote g-tubulin complex-dependent microtubule nucleation (Leong et al, 2019;Lynch et al, 2014). Previous imaging of Mto1/2[bonsai] puncta in vivo suggested that higher-order structure and stability of Mto1/2[bonsai] complex may depend on underlying multimerization of Mto2, as well as additional cooperative interactions (Lynch et al, 2014).…”

“…together with a fragment of the CM1 protein Spc110p, can associate in vitro to form a lowpitch helix with near-correct orientations for MT nucleation (Kollman et al, 2015;Kollman et al, 2010). Recently we demonstrated that Mto1/2[bonsai], fission yeast g-TuSC, and the small protein Mzt1 together form active MT-nucleation complexes in vitro (Leong et al, 2019). Based on the budding yeast work, we favor the idea that disordered regions of Mto2 could allow the multimeric Mto1/2 complex to serve as a flexible "landing strip" that brings g-TuSCs into proximity and allows them to interact without constraining their intrinsic geometry of interaction.…”

“…Proteins that contain a Centrosomin Motif 1 (CM1) domain (here referred to as "CM1 proteins") regulate multiple aspects of g-TuC function, including assembly into g-TuRC-like structures (see below). 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)).…”

“…More recently, Drosophila Cnn has been shown to dynamically assemble into large-scale scaffolds that, via their ability to recruit the g-TuC, can modulate the MT-nucleation capacity of the centrosome during the cell cycle (Citron et al, 2018;Conduit et al, 2014;Feng et al, 2017). Importantly, independently of regulating localization of the g-TuC, CM1 proteins from multiple organisms have also been shown to promote MT nucleation activity of the g-TuC, in a range of in vitro and in vivo assays (Choi et al, 2010;Kollman et al, 2010;Leong et al, 2019;Lynch et al, 2014;Samejima et al, 2008). Thus, rather than serving as simple "adapters" that localize the g-TuC to MTOCs, CM1 proteins may coordinate g-TuC localization with g-TuC activation, not only promoting g-TuC activation at MTOC sites but also preventing undesired g-TuC activation at non-MTOC sites.…”