scholarly journals Microtubule nucleation without a ring?

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Andreas Merdes
Keyword(s):  
Structure Function ◽  
Regulatory Mechanisms ◽  
Microtubule Nucleation ◽  
Ring Complex

The native γ-tubulin ring complex is an asymmetric, imperfect template for microtubule nucleation. Wieczorek et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202009146) and Zimmermann et al. (2020. Sci. Adv.https://doi.org/10.1126/sciadv.abe0894) have reconstituted a recombinant complex that allows study of structure–function relationships and regulatory mechanisms.

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

2019 ◽  
Author(s):  
Tanja Consolati ◽  
Julia Locke ◽  
Johanna Roostalu ◽  
Jayant Asthana ◽  
Wei Ming Lim ◽  
...  
Keyword(s):  
Real Time ◽  
Regulatory Mechanisms ◽  
Microtubule Nucleation ◽  
Tirf Microscopy ◽  
Structural Asymmetry ◽  
Closed Conformation ◽  
The Core ◽  
Ring Complex ◽  
Kinetic Inhibition ◽  
Outer Perimeter

SUMMARYThe γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. However, the mechanism of its regulation is not understood. Here, we purified human γTuRC and quantitatively characterized its nucleation properties in a TIRF microscopy-based real-time nucleation assay. We find that microtubule nucleation by γTuRC is kinetically inhibited compared to microtubule elongation. Determining the cryo-EM structure of γTuRC at 4 Å resolution reveals an asymmetric conformation with only part of the complex in a ‘closed’ conformation matching the microtubule geometry. Several factors stabilise the closed conformation. One is actin in the core of the complex and others, likely MZT1 or MZT2, line the outer perimeter of the closed part of γTuRC. The opposed side of γTuRC is in an ‘open’, nucleation-incompetent conformation, leading to a structural asymmetry, explaining the kinetic inhibition of nucleation by human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.

Identification of an Spc110p-related protein in vertebrates

1997 ◽  
Vol 110 (20) ◽  
pp. 2533-2545 ◽  
Author(s):  
A.M. Tassin ◽  
C. Celati ◽  
M. Paintrand ◽  
M. Bornens
Keyword(s):  
Mammalian Cells ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Partial Purification ◽  
Related Protein ◽  
Microtubule Nucleation ◽  
Pole Body ◽  
Egg Extract ◽  
Ring Complex ◽  
Gamma Tubulin

Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the gamma-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.

scholarly journals Characterization and Reconstitution of Drosophila γ-Tubulin Ring Complex Subunits

2000 ◽  
Vol 151 (7) ◽  
pp. 1513-1524 ◽  
Author(s):  
Ruwanthi N. Gunawardane ◽  
Ona C. Martin ◽  
Kan Cao ◽  
Lijun Zhang ◽  
Kimberly Dej ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Sequence Motifs ◽  
Microtubule Nucleation ◽  
Sequence Comparisons ◽  
Baculovirus Expression ◽  
Ring Complex ◽  
And Function ◽  
First Time ◽  
Centrosomal Proteins

The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin, the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC, we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.

scholarly journals NEDD1-dependent recruitment of the γ-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly

2006 ◽  
Vol 172 (4) ◽  
pp. 505-515 ◽  
Author(s):  
Laurence Haren ◽  
Marie-Hélène Remy ◽  
Ingrid Bazin ◽  
Isabelle Callebaut ◽  
Michel Wright ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Somatic Cells ◽  
Spindle Assembly ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Centrosomal Protein ◽  
Centriole Duplication ◽  
Large Complex ◽  
Ring Complex ◽  
Carboxy Terminal

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein γ-tubulin. In mammals, γ-tubulin associates with additional proteins into a large complex, the γ-tubulin ring complex (γTuRC). We characterize NEDD1, a centrosomal protein that associates with γTuRCs. We show that the majority of γTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of γ-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to γTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of γ-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of γ-tubulin to the centrosome.

Structure of the γ-tubulin ring complex: a template for microtubule nucleation

2000 ◽  
Vol 2 (6) ◽  
pp. 365-370 ◽  
Author(s):  
Michelle Moritz ◽  
Michael B. Braunfeld ◽  
Vincent Guénebaut ◽  
John Heuser ◽  
David A. Agard
Keyword(s):  
Microtubule Nucleation ◽  
Ring Complex

scholarly journals CDK5RAP2 Is a Pericentriolar Protein That Functions in Centrosomal Attachment of the γ-Tubulin Ring Complex

2008 ◽  
Vol 19 (1) ◽  
pp. 115-125 ◽  
Author(s):  
Ka-Wing Fong ◽  
Yuk-Kwan Choi ◽  
Jerome B. Rattner ◽  
Robert Z. Qi
Keyword(s):  
Autosomal Recessive ◽  
Macromolecular Complex ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Primary Microcephaly ◽  
Conserved Sequence ◽  
Ring Complex ◽  
Pericentriolar Material ◽  
Related Proteins ◽  
Autosomal Recessive Primary Microcephaly

Microtubule nucleation and organization by the centrosome require γ-tubulin, a protein that exists in a macromolecular complex called the γ-tubulin ring complex (γTuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including γ-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the γTuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for γTuRC attachment to the centrosome but not for γTuRC assembly. Perturbing CDK5RAP2 function delocalized γ-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in γTuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly.

scholarly journals Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

1998 ◽  
Vol 142 (3) ◽  
pp. 775-786 ◽  
Author(s):  
Michelle Moritz ◽  
Yixian Zheng ◽  
Bruce M. Alberts ◽  
Karen Oegema
Keyword(s):  
Soluble Fraction ◽  
Protein Complexes ◽  
Drosophila Embryo ◽  
Microtubule Nucleation ◽  
Complementation Assay ◽  
Embryo Extract ◽  
Ring Complex ◽  
Centrosomal Proteins

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.

scholarly journals XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex

2018 ◽  
Vol 20 (5) ◽  
pp. 575-585 ◽  
Author(s):  
Akanksha Thawani ◽  
Rachel S. Kadzik ◽  
Sabine Petry
Keyword(s):  
Microtubule Nucleation ◽  
Ring Complex
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