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

Consolati, Tanja; Locke, Julia; Roostalu, Johanna; Asthana, Jayant; Wm, Lim; Gannon, Julian; Martino, Fabrizio; Costa, Alessandro; Surrey, Thomas

doi:10.1101/853218

Cited by 8 publications

(17 citation statements)

References 61 publications

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“…We demonstrate that GCPs 4, 5, and 6 form a stable sub-complex that permits the association with γTuSCs into a functional γTuRC. Within the sub-complex, the stoichiometric ratio of these GCPs matches the values found in recently reported structures of native γTuRCs (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). We find that GCPs 2 and 3 are also present in our preparations of the sub-complex, but at a molar ratio equivalent to half-a-γTuSC.…”

Section: Discussionsupporting

confidence: 87%

“…GCPs 4, 5, and 6 are essential for the assembly and/or for the stabilization of γTuRCs, since depletion of either component causes a reduction of γTuRCs both at the centrosome and in the cytoplasm (Izumi et al, 2008; Bahtz et al, 2012; Scheidecker et al, 2015; Farache et al, 2016; Cota et al, 2017). Besides GCPs 4, 5, 6, additional factors may still be needed, such as Mozart1, actin, or other proteins corresponding to unassigned densities in cryo-electron microscopy structures of native γTuRCs (Lin et al, 2016; Cota et al, 2017; Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). It is now clear that GCPs 4, 5, and 6 integrate into the helical wall of the γTuRC, and that they are laterally bound to γTuSCs, but their specific role within the γTuRC still remains to be determined (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

“…Besides GCPs 4, 5, 6, additional factors may still be needed, such as Mozart1, actin, or other proteins corresponding to unassigned densities in cryo-electron microscopy structures of native γTuRCs (Lin et al, 2016; Cota et al, 2017; Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). It is now clear that GCPs 4, 5, and 6 integrate into the helical wall of the γTuRC, and that they are laterally bound to γTuSCs, but their specific role within the γTuRC still remains to be determined (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). Our results show that they form a nucleus promoting the stable assembly of the 2MDa complex.…”

Section: Discussionmentioning

confidence: 99%

“…The assembly of γTuRCs via GCPs 4, 5, 6 may be important for the regulation of its microtubule-nucleation activity: cryo-electron microscopy structures show an asymmetric architecture, incompatible with the geometry of microtubules (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). The presence of GCPs 4, 5, and 6 may therefore prevent soluble γTuRCs from acquiring an active conformation, unless bound to additional activating factors, such as Cdk5rap2/Cep215, at designated microtubule-organizing centres.…”

Section: Discussionmentioning

confidence: 99%

“…Because these proteins are present only in one or two copies per complex (Murphy et al, 2001; Choi et al, 2010), and because the rescue experiments with chimeras suggest that GCPs 4, 5, 6 occupy non-random positions, their localisation within the complex is of particular interest. During the course of this work, three studies have described the structure of native γTuRCs by cry-electron microscopy, and have found a lateral association of four γTuSCs, bound to a lateral array of GCP4/GCP5/GCP4/GCP6, to which an additional γTuSC was associated (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). Altogether, this has raised the question whether GCPs 4, 5, 6 form assembly intermediates equivalent to γTuSCs.…”

Section: Introductionmentioning

confidence: 99%

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A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

Merdes

Haren

Farache

et al. 2020

Preprint

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Abstractγ-tubulin is a major protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (gamma-tubulin complex proteins): γ-tubulin small complexes (γTuSCs), containing γ-tubulin and GCPs 2 and 3, and γ-tubulin ring complexes (γTuRCs), containing multiple γTuSCs, in addition to GCPs 4, 5, and 6. Whereas the structure and assembly properties of γTuSCs have been intensively studied, little is known about the assembly of γTuRCs, and about the specific roles of GCPs 4, 5, and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt-resistant sub-complex within the γTuRC that assembles independently of the presence of γTuSCs. Incubation of this sub-complex with cytoplasmic extracts containing γTuSCs leads to the reconstitution of full-sized γTuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the amino-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs, and we demonstrate that these are involved in the assembly or stabilization of the γTuRC.Summary statementγ-tubulin ring complexes are templates for microtubule nucleation, composed of γ-tubulin and GCP proteins. GCPs 4, 5, 6 form a stable sub-complex, driving the assembly of the full complex.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

Merdes

Haren

Farache

et al. 2020

Preprint

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Einblicke in die Entstehung von Mikrotubuli

Pfeffer

Schiebel

2020

Biospektrum

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Microtubules are part of the cytoskeleton and promote various essential cellular functions. Microtubules are dynamic polymers composed of heterodimeric α/ß-tubulin subunits and can assemble de novo in a ‘structural templating’ mechanism assisted by ring-like complexes containing the protein γ-tubulin. Recent cryo-electron microscopy structures of such γ-tubulin ring complexes from vertebrates propelled our understanding of their architecture, assembly and activation mechanism.

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The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

Wagner

Raunser

2020

Commun Biol

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Particle selection is a crucial step when processing electron cryo microscopy data. Several automated particle picking procedures were developed in the past but most struggle with non-ideal data sets. In our recent Communications Biology article, we presented crYOLO, a deep learning based particle picking program. It enables fast, automated particle picking at human levels of accuracy with low effort. A general model allows the use of crYOLO for selecting particles in previously unseen data sets without further training. Here we describe how crYOLO has evolved since its initial release. We have introduced filament picking, a new denoising technique, and a new graphical user interface. Moreover, we outline its usage in automated processing pipelines, which is an important advancement on the horizon of the field.The crYOLO particle picking procedure A major goal of electron cryo microscopy (cryo-EM) is to obtain high-resolution threedimensional (3D) reconstructions of proteins and protein complexes to gain novel biological insights. This process involves the selection of thousands to millions of noisy two-dimensional (2D) particle projections, a number that only keeps increasing with recent advances in hardware and software development.In our recent work in Communications Biology 1 we introduced the "crYOLO" particle picking procedure. It is based on a deep neural network and the You Only Look Once (YOLO) object detection framework 2 . This approach enables the automated picking of particles within cryo-EM micrographs with a low signal-to-noise ratio requiring minimal human supervision or intervention. CrYOLO is easy to configure and train on a specific data set. It is fast and can process up to six micrographs per second. As crYOLO sees the complete micrograph, it is able to learn the

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

Cited by 8 publications

References 61 publications

A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

Einblicke in die Entstehung von Mikrotubuli

The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

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