scholarly journals Characterization of a Drosophila Centrosome Protein CP309 That Shares Homology with Kendrin and CG-NAP

2004 ◽  
Vol 15 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Shin-ichi Kawaguchi ◽  
Yixian Zheng
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Biochemical Studies ◽  
Ring Complex ◽  
Centrosome Protein

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the γ-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the γ-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.

Kendrin/pericentrin-B, a centrosome protein with homology to pericentrin that complexes with PCM-1

2001 ◽  
Vol 114 (4) ◽  
pp. 797-809 ◽  
Author(s):  
Q. Li ◽  
D. Hansen ◽  
A. Killilea ◽  
H.C. Joshi ◽  
R.E. Palazzo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Evolutionary Relationship ◽  
Coiled Coil ◽  
Maturation Process ◽  
Microtubule Depolymerization ◽  
Microtubule Nucleation ◽  
Sequence Analyses ◽  
Late Event ◽  
Functional Complex ◽  
Centrosome Protein

The centrosome is responsible for nucleating microtubules and performing other cellular roles. To define the organization of the centrosome more completely, a human anti-centrosome serum was used to screen a human cDNA library, and a cDNA encoding a >350 kDa centrosome protein was identified. Sequence analyses revealed that this novel centrosome protein contains two coiled-coil domains bounded by non-coiled regions. The N-terminal region of the protein, named pericentrin-B, shares 61% identity (75% similarity) with pericentrin, suggesting an evolutionary relationship between these proteins. Antibodies against pericentrin-B stain centrosomes at all stages of the cell cycle, and pericentrin-B remains associated with centrosomes following microtubule depolymerization. Immunodepletion of neither pericentrin-B nor PCM-1 from cellular extracts inhibited the ability of salt-stripped centrosomes to recover microtubule nucleation potential, demonstrating that neither protein plays a key role in microtubule nucleation processes. Moreover, the binding of both PCM-1 and pericentrin-B with salt-stripped centrosomes required intact microtubules, demonstrating that the association of PCM-1 and pericentrin-B with centrosomes is a late event in the centrosome maturation process. Finally, pericentrin-B and PCM-1 coimmunoprecipitate, suggesting that PCM-1 and pericentrin-B form a functional complex in cells. This observation may help to explain the generation of anti-centrosome autoantibodies in certain autoimmune patients and may be important for centrosome function.

scholarly journals NME7 is a functional component of the γ-tubulin ring complex

2014 ◽  
Vol 25 (13) ◽  
pp. 2017-2025 ◽  
Author(s):  
Pengfei Liu ◽  
Yuk-Kwan Choi ◽  
Robert Z. Qi
Keyword(s):  
Cell Cycle ◽  
Crucial Role ◽  
Dependent Manner ◽  
Mitotic Spindles ◽  
Wild Type ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Functional Component ◽  
Ring Complex

As the primary microtubule nucleator in animal cells, the γ-tubulin ring complex (γTuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the γTuRC is regulated. Recently, isolated γTuRC was found to contain NME7, a poorly characterized member of the NME family. Here we report that NME7 is a γTuRC component that regulates the microtubule-nucleating activity of the γTuRC. NME7 contains two putative kinase domains, A and B, and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the γTuRC through both A and B domains, with Arg-322 in domain B being crucial to the binding. In association with the γTuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect γTuRC assembly or localization to centrosomes, but it does impair centrosome-based microtubule nucleation. Of importance, wild-type NME7 promotes γTuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the γTuRC in a kinase-dependent manner to facilitate microtubule nucleation.

scholarly journals Aurora A activates D-TACC–Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules

2005 ◽  
Vol 170 (7) ◽  
pp. 1039-1046 ◽  
Author(s):  
Teresa P. Barros ◽  
Kazuhisa Kinoshita ◽  
Anthony A. Hyman ◽  
Jordan W. Raff
Keyword(s):  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Mutant Form ◽  
Dependent Manner ◽  
Aurora A ◽  
Animal Cells

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A–dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC–Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis.

scholarly journals Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly

2018 ◽  
Author(s):  
Andrew Lyon ◽  
Alex Zelter ◽  
Shruthi Viswanath ◽  
Alison Maxwell ◽  
Richard Johnson ◽  
...  
Keyword(s):  
Structural Model ◽  
Coiled Coil ◽  
The Other ◽  
Assembly Process ◽  
X Ray ◽  
X Ray Crystallography ◽  
Small Complex ◽  
Ring Complex ◽  
Biochemical Analyses

AbstractMicrotubule (MT) nucleation in vivo is regulated by the γ-tubulin ring complex (γTuRC), an approximately 2-megadalton complex conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC assembly is a key point of regulation over the MT cytoskeleton. Budding yeast γTuRC is composed of seven γ-tubulin small complex (γTuSC) subassemblies which associate helically to form a template from which microtubules grow. This assembly process requires higher-order oligomers of the coiled-coil protein Spc110 to bind multiple γTuSCs, thereby stabilizing the otherwise low-affinity interface between γTuSCs. While Spc110 oligomerization is critical, its N-terminal domain (NTD) also plays a role that is poorly understood both functionally and structurally. In this work, we sought a mechanistic understanding of Spc110 NTD using a combination of structural and biochemical analyses. Through crosslinking-mass spectrometry (XL-MS), we determined that a segment of Spc110 coiled-coil is a major point of contact with γTuSC. We determined the structure of this coiled-coil segment by X-ray crystallography and used it in combination with our XL-MS dataset to generate an integrative structural model of the γTuSC-Spc110 complex. This structural model, in combination with biochemical analyses of Spc110 heterodimers lacking one NTD, suggests that the two NTDs within an Spc110 dimer act independently, one stabilizing association between Spc110 and γTuSC and the other stabilizing the interface between adjacent γTuSCs.

scholarly journals γ-Tubulin ring complexes regulate microtubule plus end dynamics

2009 ◽  
Vol 187 (3) ◽  
pp. 327-334 ◽  
Author(s):  
Anaïs Bouissou ◽  
Christel Vérollet ◽  
Aureliana Sousa ◽  
Paula Sampaio ◽  
Michel Wright ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Live Imaging ◽  
S2 Cells ◽  
Imaging Analysis ◽  
Mitotic Progression ◽  
Small Complex ◽  
Drosophila S2 Cells ◽  
Ring Complex ◽  
Ring Complexes

γ-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the γ-tubulin small complex (γ-TuSC) and the γ-tubulin ring complex (γ-TuRC). Proteins specific of the γ-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during interphase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the γ-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the γ-TuRC localizes along interphase MTs and that distal γ-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the γ-TuRC associated to MTs may regulate their dynamics by limiting catastrophes.

scholarly journals Biochemical reconstitution of branching microtubule nucleation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Raymundo Alfaro-Aco ◽  
Akanksha Thawani ◽  
Sabine Petry
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Chromosome Segregation ◽  
Mode Of Action ◽  
Microtubule Nucleation ◽  
Ring Complex ◽  
Gamma Tubulin

Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways and their mode of action still need to be identified. Using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, which is critical for chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the branching effectors augmin and TPX2 are required to efficiently nucleate microtubules from pre-existing microtubules. TPX2 has the unexpected capacity to directly recruit γ-TuRC as well as augmin, which in turn targets more γ-TuRC along the microtubule lattice. TPX2 and augmin enable γ-TuRC-dependent microtubule nucleation at preferred branching angles of less than 90 degrees from regularly-spaced patches along microtubules. This work provides a blueprint for other microtubule nucleation pathways and helps explain how microtubules are generated in the spindle.

scholarly journals Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Tien-chen Lin ◽  
Annett Neuner ◽  
Yvonne T Schlosser ◽  
Annette ND Scharf ◽  
Lisa Weber ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Basis ◽  
Dependent Manner ◽  
Microtubule Nucleation ◽  
Biochemical Studies ◽  
N Terminus ◽  
Complex Receptor ◽  
A Cell ◽  
Cycle Dependent ◽  
Receptor Family

Budding yeast Spc110, a member of γ-tubulin complex receptor family (γ-TuCR), recruits γ-tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies suggest that Spc110 facilitates higher-order γ-tubulin complex assembly (<xref ref-type="bibr" rid="bib45">Kollman et al., 2010</xref>). Nevertheless the molecular basis for this activity and the regulation are unclear. Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates γ-TuSC oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between the N-terminus of the γ-TuSC subunit Spc98 and Spc110 is important for this activity. Besides the conserved CM1 motif in γ-TuCRs (<xref ref-type="bibr" rid="bib65">Sawin et al., 2004</xref>), a second motif that we named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/myomegalin) types of γ-TuCRs. The two types of γ-TuCRs contain distinct but conserved C-terminal MTOC targeting domains.

scholarly journals MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template

2016 ◽  
Vol 215 (6) ◽  
pp. 823-840 ◽  
Author(s):  
Tien-chen Lin ◽  
Annett Neuner ◽  
Dirk Flemming ◽  
Peng Liu ◽  
Takumi Chinen ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Human Cells ◽  
Molecular Function ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Ring Complex

MOZART1/Mzt1 is required for the localization of γ-tubulin complexes to microtubule (MT)–organizing centers from yeast to human cells. Nevertheless, the molecular function of MOZART1/Mzt1 is largely unknown. Taking advantage of the minimal MT nucleation system of Candida albicans, we reconstituted the interactions of Mzt1, γ-tubulin small complex (γ-TuSC), and γ-tubulin complex receptors (γ-TuCRs) Spc72 and Spc110 in vitro. With affinity measurements, domain deletion, and swapping, we show that Spc110 and Mzt1 bind to distinct regions of the γ-TuSC. In contrast, both Mzt1 and γ-TuSC interact with the conserved CM1 motif of Spc110/Spc72. Spc110/Spc72 and Mzt1 constitute “oligomerization chaperones,” cooperatively promoting and directing γ-TuSC oligomerization into MT nucleation-competent rings. Consistent with the functions of Mzt1, human MOZART1 directly interacts with the CM1-containing region of the γ-TuCR CEP215. MOZART1 depletion in human cells destabilizes the large γ-tubulin ring complex and abolishes CEP215CM1-induced ectopic MT nucleation. Together, we reveal conserved functions of MOZART1/Mzt1 through interactions with γ-tubulin complex subunits and γ-TuCRs.

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 γ-Tubulin–γ-Tubulin Interactions as the Basis for the Formation of a Meshwork

2018 ◽  
Vol 19 (10) ◽  
pp. 3245 ◽  
Author(s):  
Catalina Rosselló ◽  
Lisa Lindström ◽  
Greta Eklund ◽  
Matthieu Corvaisier ◽  
Maria Kristensson
Keyword(s):  
Present Review ◽  
Cellular Functions ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Ring Complex ◽  
Ring Shaped Structure

In cytoplasm, protein γ-tubulin joins with various γ-tubulin complex proteins (GCPs) to form a heterotetramer γ-tubulin small complex (γ-TuSC) that can grow into a ring-shaped structure called the γ-tubulin ring complex (γ-TuRC). Both γ-TuSC and γ-TuRC are required for microtubule nucleation. Recent knowledge on γ-tubulin with regard to its cellular functions beyond participation in its creation of microtubules suggests that this protein forms a cellular meshwork. The present review summarizes the recognized functions of γ-tubulin and aims to unite the current views on this protein.

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