Centrosome-microtubule nucleation

1997 ◽  
Vol 110 (3) ◽  
pp. 295-300 ◽  
Author(s):  
G. Pereira ◽  
E. Schiebel
Keyword(s):  
Cell Types ◽  
Nucleation Site ◽  
Microtubule Nucleation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nucleation Sites ◽  
Egg Extracts ◽  
Ring Complex ◽  
Gamma Tubulin ◽  
Cycle Dependent ◽  
Xenopus Egg Extracts

In many cell types the formation of microtubules from tubulin subunits is initiated at defined nucleation sites at the centrosome. These sites contain the conserved gamma-tubulin which is in association with additional not very will characterised proteins, identified as components of a gamma-tubulin ring complex from Xenopus egg extracts or from suppressor screens in the yeast Saccharomyces cerevisiae. In this review we discuss two recently proposed models of how the gamma-tubulin complex assists in the assembly of tubulin to form microtubules. These models propose different roles for gamma-tubulin and the other proteins in the complex in tubulin assembly. While the structure and composition of a microtubule nucleation site is becoming clearer, it is still unknown how the cell-cycle dependent regulation of microtubule nucleation sites is achieved and whether they disassemble after microtubule formation in order to allow microtubule fluxes towards the centrosome which have been observed in mitotic cells.

scholarly journals Biochemical reconstitution of branching microtubule nucleation

2019 ◽  
Author(s):  
Raymundo Alfaro-Aco ◽  
Akanksha Thawani ◽  
Sabine Petry
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Chromosome Segregation ◽  
Mode Of Action ◽  
Spindle Assembly ◽  
Nucleation Site ◽  
Microtubule Nucleation ◽  
Ring Complex ◽  
Gamma Tubulin

AbstractMicrotubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways still need to be identified along with their mode of action. Here, using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, a nucleation pathway where microtubules originate from pre-existing microtubules, which is essential for spindle assembly and chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the two branching effectors augmin and TPX2 are required to efficiently nucleate branched microtubules. Specifically, TPX2 generates regularly-spaced patches that recruit augmin and γ-TuRC to microtubules, which then nucleate new microtubules at preferred branching angles of less than 90 degrees. Our work demonstrates how γ-TuRC is brought to its nucleation site for branching microtubule nucleation. It provides a blueprint for other microtubule nucleation pathways and for generating a particular microtubule architecture by regulating microtubule nucleation.

scholarly journals Characterization of the TPX2 Domains Involved in Microtubule Nucleation and Spindle Assembly in Xenopus Egg Extracts

2004 ◽  
Vol 15 (12) ◽  
pp. 5318-5328 ◽  
Author(s):  
Stéphane Brunet ◽  
Teresa Sardon ◽  
Timo Zimmerman ◽  
Torsten Wittmann ◽  
Rainer Pepperkok ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Microtubule Nucleation ◽  
Terminal Domain ◽  
Large N ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Domain Functional ◽  
Xenopus Egg Extracts

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.

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 Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (6) ◽  
pp. 3744-3755 ◽  
Author(s):  
C S Stueland ◽  
D J Lew ◽  
M J Cismowski ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Regulatory Protein ◽  
Histone H1 ◽  
Yeast Saccharomyces Cerevisiae ◽  
Kinase Activation ◽  
Egg Extracts ◽  
Xenopus Egg Extracts ◽  
Yeast Homolog

In most cells, mitosis is dependent upon completion of DNA replication. The feedback mechanisms that prevent entry into mitosis by cells with damaged or incompletely replicated DNA have been termed checkpoint controls. Studies with the fission yeast Schizosaccharomyces pombe and Xenopus egg extracts have shown that checkpoint controls prevent activation of the master regulatory protein kinase, p34cdc2, that normally triggers entry into mitosis. This is achieved through inhibitory phosphorylation of the Tyr-15 residue of p34cdc2. However, studies with the budding yeast Saccharomyces cerevisiae have shown that phosphorylation of this residue is not essential for checkpoint controls to prevent mitosis. We have investigated the basis for checkpoint controls in this organism and show that these controls can prevent entry into mitosis even in cells which have fully activated the cyclin B (Clb)-associated forms of the budding yeast homolog of p34cdc2, p34CDC28, as assayed by histone H1 kinase activity. However, the active complexes in checkpoint-arrested cells are smaller than those in cycling cells, suggesting that assembly of mitosis-inducing complexes requires additional steps following histone H1 kinase activation.

scholarly journals Spatiotemporal organization of branched microtubule networks

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Akanksha Thawani ◽  
Howard A Stone ◽  
Joshua W Shaevitz ◽  
Sabine Petry
Keyword(s):  
Single Molecule ◽  
Reaction Model ◽  
Sequential Reaction ◽  
Spatiotemporal Organization ◽  
Nucleation Sites ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Microtubule Networks ◽  
Xenopus Egg Extracts ◽  
Rate Limiting

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.

scholarly journals Branching Microtubule Nucleation in Xenopus Egg Extracts Mediated by Augmin and TPX2

Cell ◽  
2013 ◽  
Vol 152 (4) ◽  
pp. 768-777 ◽  
Author(s):  
Sabine Petry ◽  
Aaron C. Groen ◽  
Keisuke Ishihara ◽  
Timothy J. Mitchison ◽  
Ronald D. Vale
Keyword(s):  
Microtubule Nucleation ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

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.

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