scholarly journals Assembly of the asymmetric human γ-tubulin ring complex by RUVBL1-RUVBL2 AAA ATPase

2020 ◽  
Vol 6 (51) ◽  
pp. eabe0894
Author(s):  
Fabian Zimmermann ◽  
Marina Serna ◽  
Artur Ezquerra ◽  
Rafael Fernandez-Leiro ◽  
Oscar Llorca ◽  
...  
Keyword(s):  
Mechanistic Studies ◽  
Detailed Structure ◽  
Minimal Set ◽  
Aaa Atpase ◽  
Cryo Electron Microscopy ◽  
In Vitro Reconstitution ◽  
Ring Complex ◽  
Assembly Factor ◽  
Nucleation Activity

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.

scholarly journals Structural insight into TPX2-stimulated microtubule assembly

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Rui Zhang ◽  
Johanna Roostalu ◽  
Thomas Surrey ◽  
Eva Nogales
Keyword(s):  
Molecular Mechanism ◽  
Structural Information ◽  
Microtubule Assembly ◽  
Cryo Electron Microscopy ◽  
Interaction Mode ◽  
In Vitro Reconstitution ◽  
Assembly Factor ◽  
Ran Gtp ◽  
Mitosis And Meiosis

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements (’ridge’ and ‘wedge’) in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.

scholarly journals Genetically inspired in vitro reconstitution of Saccharomyces cerevisiae actin cables from seven purified proteins

2020 ◽  
Vol 31 (5) ◽  
pp. 335-347 ◽  
Author(s):  
Luther W. Pollard ◽  
Mikael V. Garabedian ◽  
Salvatore L. Alioto ◽  
Shashank Shekhar ◽  
Bruce L. Goode
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Scale Structure ◽  
Minimal Set ◽  
In Vitro Reconstitution ◽  
Actin Cables ◽  
Powerful Demonstration

Yeast actin cables are reconstituted from seven purified proteins, providing a powerful demonstration of how a minimal set of components can self-organize into a micron-scale structure that has many of the same features of actin cables found in vivo.

scholarly journals Molecular insight into how γ-TuRC makes microtubules

2021 ◽  
Vol 134 (14) ◽  
Author(s):  
Akanksha Thawani ◽  
Sabine Petry
Keyword(s):  
Single Molecule ◽  
Cell Function ◽  
Building Blocks ◽  
Ring Complex ◽  
Single Molecule Studies ◽  
Nucleation Activity ◽  
Open Question ◽  
Insight Into ◽  
Made In

ABSTRACT As one of four filament types, microtubules are a core component of the cytoskeleton and are essential for cell function. Yet how microtubules are nucleated from their building blocks, the αβ-tubulin heterodimer, has remained a fundamental open question since the discovery of tubulin 50 years ago. Recent structural studies have shed light on how γ-tubulin and the γ-tubulin complex proteins (GCPs) GCP2 to GCP6 form the γ-tubulin ring complex (γ-TuRC). In parallel, functional and single-molecule studies have informed on how the γ-TuRC nucleates microtubules in real time, how this process is regulated in the cell and how it compares to other modes of nucleation. Another recent surprise has been the identification of a second essential nucleation factor, which turns out to be the well-characterized microtubule polymerase XMAP215 (also known as CKAP5, a homolog of chTOG, Stu2 and Alp14). This discovery helps to explain why the observed nucleation activity of the γ-TuRC in vitro is relatively low. Taken together, research in recent years has afforded important insight into how microtubules are made in the cell and provides a basis for an exciting era in the cytoskeleton field.

scholarly journals Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules

2018 ◽  
Vol 217 (7) ◽  
pp. 2417-2428 ◽  
Author(s):  
Jae-Geun Song ◽  
Matthew R. King ◽  
Rui Zhang ◽  
Rachel S. Kadzik ◽  
Akanksha Thawani ◽  
...  
Keyword(s):  
Cell Shape ◽  
Insect Cells ◽  
The Other ◽  
Negative Stain ◽  
In Vitro Reconstitution ◽  
Ring Complex ◽  
Negative Stain Electron Microscopy ◽  
The Right ◽  
And Function

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin’s architecture and function, we purified Xenopus laevis augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs.

scholarly journals The cryo-EM structure of a γ-TuSC elucidates architecture and regulation of minimal microtubule nucleation systems

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Erik Zupa ◽  
Anjun Zheng ◽  
Annett Neuner ◽  
Martin Würtz ◽  
Peng Liu ◽  
...  
Keyword(s):  
De Novo ◽  
Relative Positioning ◽  
Microtubule Nucleation ◽  
Cryo Electron Microscopy ◽  
Small Complex ◽  
Ring Complex ◽  
Regulation Mechanisms ◽  
Microtubule Formation ◽  
Nucleation Activity ◽  
Higher Eukaryotes

Abstract The nucleation of microtubules from αβ-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms. Aiming to understand how de novo microtubule formation is achieved and regulated by a minimal microtubule nucleation system, we here determined the cryo-electron microscopy structure of the heterotetrameric γ-tubulin small complex (γ-TuSC) from C. albicans at near-atomic resolution. Compared to the vertebrate γ-tubulin ring complex (γ-TuRC), we observed a vastly remodeled interface between the SPC/GCP-γ-tubulin spokes, which stabilizes the complex and defines the γ-tubulin arrangement. The relative positioning of γ-tubulin subunits indicates that a conformational rearrangement of the complex is required for microtubule nucleation activity, which follows opposing directionality as predicted for the vertebrate γ-TuRC. Collectively, our data suggest that the assembly and regulation mechanisms of γ-tubulin complexes fundamentally differ between the microtubule nucleation systems in lower and higher eukaryotes.

scholarly journals The AAA+ ATPase TorsinA polymerizes into hollow tubes with a helical periodicity of 8.5 subunits per turn

2019 ◽  
Author(s):  
F. Esra Demircioglu ◽  
Weili Zheng ◽  
Alexander J. McQuown ◽  
Nolan Maier ◽  
Nicki Watson ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Neuromuscular Disease ◽  
Central Channel ◽  
Peptide Substrate ◽  
Aaa Atpase ◽  
Helical Reconstruction ◽  
Cryo Electron Microscopy ◽  
Primary Dystonia ◽  
Aaa Atpases

AbstractTorsinA is an ER-resident AAA+ ATPase, whose single residue deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is a highly unusual AAA+ ATPase in that it needs an external activator. Also, it appears not to thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of the still enigmatic function of it. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 Å structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner cavity of ∼4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA+ ATPases. We discuss the implications of these observations for TorsinA function.

scholarly journals Abstract OR-9: Cryo-EM Structure of the Reconstituted Human γ-Tubulin Ring Complex

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S10-S10
Author(s):  
Marina Serna ◽  
Fabian Zimmermann ◽  
Artur Ezquerra ◽  
Rafael Fernandez-Leiro ◽  
Jens Luders ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Expression System ◽  
Structural Features ◽  
Animal Cells ◽  
Single Particle Reconstruction ◽  
Aaa Atpase ◽  
Structural Support ◽  
Ring Complex

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).

scholarly journals Mutual Dependence of Candida albicans Est1p and Est3p in Telomerase Assembly and Activation

2007 ◽  
Vol 6 (8) ◽  
pp. 1330-1338 ◽  
Author(s):  
Min Hsu ◽  
Eun Young Yu ◽  
Sunitha M. Singh ◽  
Neal F. Lue
Keyword(s):  
Candida Albicans ◽  
Protein Complex ◽  
Null Mutant ◽  
Mechanistic Studies ◽  
Protein Subunit ◽  
Telomere Repeat ◽  
Mutual Dependence ◽  
Affinity Isolation ◽  
In Vitro Reconstitution

ABSTRACT Telomerase is an RNA-protein complex responsible for extending one strand of the telomere terminal repeats. Analysis of the telomerase complex in budding yeasts has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The TERT subunit is essential for telomerase catalysis, while the functions of Est1p and Est3p have not been precisely elucidated. In an earlier study, we showed that telomerase derived from a Candida est1-null mutant is defective in primer utilization in vitro; it exhibits reduced initiation and processivity on primers that terminate in two regions of the telomere repeat. Here we show that telomerase derived from a Candida est3-null mutant has nearly identical defects in primer utilization and processivity. Further analysis revealed an unexpected mutual dependence of Est1p and Est3p in their assembly into the full telomerase complex, which accounts for the similarity between the mutant enzymes. We also developed an affinity isolation and an in vitro reconstitution protocol for the telomerase complex that will facilitate future mechanistic studies.

scholarly journals TRiC’s tricks inhibit huntingtin aggregation

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Sarah H Shahmoradian ◽  
Jesus G Galaz-Montoya ◽  
Michael F Schmid ◽  
Yao Cong ◽  
Boxue Ma ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Structural Description ◽  
Mutant Huntingtin ◽  
Cryo Electron Microscopy ◽  
Amyloid Aggregates ◽  
Cryo Electron Tomography ◽  
Ring Complex ◽  
Exon 1

In Huntington’s disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC’s inhibition of mhttQ51 aggregation in vitro.

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