scholarly journals Regulation of Microtubule Assembly and Organization in Mitosis by the AAA+ ATPase Pontin

2008 ◽  
Vol 19 (7) ◽  
pp. 3097-3110 ◽  
Author(s):  
Daniel Ducat ◽  
Shin-ichi Kawaguchi ◽  
Hongbin Liu ◽  
John R. Yates ◽  
Yixian Zheng
Keyword(s):  
Culture Cell ◽  
Microtubule Assembly ◽  
Tissue Culture Cell ◽  
Aaa Atpase ◽  
Novel Proteins ◽  
Egg Extracts ◽  
Ring Complex ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

To identify novel proteins important for microtubule assembly in mitosis, we have used a centrosome-based complementation assay to enrich for proteins with mitotic functions. An RNA interference (RNAi)-based screen of these proteins allowed us to uncover 13 novel mitotic regulators. We carried out in-depth analyses of one of these proteins, Pontin, which is known to have several functions in interphase, including chromatin remodeling, DNA repair, and transcription. We show that reduction of Pontin by RNAi resulted in defects in spindle assembly in Drosophila S2 cells and in several mammalian tissue culture cell lines. Further characterization of Pontin in Xenopus egg extracts demonstrates that Pontin interacts with the gamma tubulin ring complex (γ-TuRC). Because depletion of Pontin leads to defects in the assembly and organization of microtubule arrays in egg extracts, our studies suggest that Pontin has a mitosis-specific function in regulating microtubule assembly.

scholarly journals Structural Transitions at Microtubule Ends Correlate with Their Dynamic Properties in Xenopus Egg Extracts

2000 ◽  
Vol 149 (4) ◽  
pp. 767-774 ◽  
Author(s):  
Isabelle Arnal ◽  
Eric Karsenti ◽  
Anthony A. Hyman
Keyword(s):  
Dynamic Instability ◽  
Dynamic Properties ◽  
Microtubule Assembly ◽  
Two Dimensional ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
First Time ◽  
The Relationship ◽  
Dynamic Populations

Microtubules are dynamically unstable polymers that interconvert stochastically between growing and shrinking states by the addition and loss of subunits from their ends. However, there is little experimental data on the relationship between microtubule end structure and the regulation of dynamic instability. To investigate this relationship, we have modulated dynamic instability in Xenopus egg extracts by adding a catastrophe-promoting factor, Op18/stathmin. Using electron cryomicroscopy, we find that microtubules in cytoplasmic extracts grow by the extension of a two- dimensional sheet of protofilaments, which later closes into a tube. Increasing the catastrophe frequency by the addition of Op18/stathmin decreases both the length and frequency of the occurrence of sheets and increases the number of frayed ends. Interestingly, we also find that more dynamic populations contain more blunt ends, suggesting that these are a metastable intermediate between shrinking and growing microtubules. Our results demonstrate for the first time that microtubule assembly in physiological conditions is a two-dimensional process, and they suggest that the two-dimensional sheets stabilize microtubules against catastrophes. We present a model in which the frequency of catastrophes is directly correlated with the structural state of microtubule ends.

scholarly journals 2066 Functional characterization of mutant BRCA1

2018 ◽  
Vol 2 (S1) ◽  
pp. 13-13
Author(s):  
John Barrows ◽  
David Long
Keyword(s):  
Functional Characterization ◽  
Coiled Coil ◽  
Wild Type ◽  
Coiled Coil Region ◽  
Egg Extracts ◽  
Study Population ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

scholarly journals ISWI Remodeling Complexes in Xenopus Egg Extracts: Identification as Major Chromosomal Components that Are Regulated by INCENP-aurora B

2002 ◽  
Vol 13 (1) ◽  
pp. 25-39 ◽  
Author(s):  
David E. MacCallum ◽  
Ana Losada ◽  
Ryuji Kobayashi ◽  
Tatsuya Hirano
Keyword(s):  
Chromosome Condensation ◽  
Aurora B ◽  
Mitotic Chromosomes ◽  
Novel Proteins ◽  
Condensin Complex ◽  
H3 Phosphorylation ◽  
Chromatid Cohesion ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

We previously characterized major components of mitotic chromosomes assembled in Xenopus laevis egg extracts and collectively referred to them as Xenopuschromosome–associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, we have isolated XCAP-F and found it to be theXenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains theXenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, we find that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B.

scholarly journals Regulation of Op18 during Spindle Assembly in Xenopus Egg Extracts

2001 ◽  
Vol 153 (1) ◽  
pp. 149-158 ◽  
Author(s):  
Priya Prakash Budde ◽  
Akiko Kumagai ◽  
William G. Dunphy ◽  
Rebecca Heald
Keyword(s):  
Spindle Assembly ◽  
Microtubule Stabilization ◽  
Egg Extracts ◽  
Associated Proteins ◽  
Oncoprotein 18 ◽  
Xenopus Egg ◽  
Chromatin Proteins ◽  
Xenopus Egg Extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.

scholarly journals Tpx2, a Novel Xenopus Map Involved in Spindle Pole Organization

2000 ◽  
Vol 149 (7) ◽  
pp. 1405-1418 ◽  
Author(s):  
Torsten Wittmann ◽  
Matthias Wilm ◽  
Eric Karsenti ◽  
Isabelle Vernos
Keyword(s):  
Functional Characterization ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
New Family ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Dynactin Complex

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673–685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein–dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.

scholarly journals Stathmin/Op18 Phosphorylation Is Regulated by Microtubule Assembly

2001 ◽  
Vol 12 (2) ◽  
pp. 437-448 ◽  
Author(s):  
Thomas Küntziger ◽  
Olivier Gavet ◽  
Valérie Manceau ◽  
André Sobel ◽  
Michel Bornens
Keyword(s):  
Feedback Loop ◽  
Somatic Cells ◽  
Network Control ◽  
Microtubule Assembly ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromatin

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of “mitotic chromatin.” Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.

scholarly journals Characterization of Two Related Drosophila γ-tubulin Complexes that Differ in Their Ability to Nucleate Microtubules

1999 ◽  
Vol 144 (4) ◽  
pp. 721-733 ◽  
Author(s):  
Karen Oegema ◽  
Christiane Wiese ◽  
Ona C. Martin ◽  
Ronald A. Milligan ◽  
Akihiro Iwamatsu ◽  
...  
Keyword(s):  
Ring Structure ◽  
Drosophila Embryo ◽  
Cryo Electron Microscopy ◽  
Small Complex ◽  
Egg Extracts ◽  
Structural Subunit ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

γ-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1– 12). Here, we report the purification and characterization of both complexes that we name γ-tubulin small complex (γTuSC; ∼280,000 D) and Drosophila γTuRC (∼2,200,000 D). In addition to γ-tubulin, the γTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The γTuSC is a structural subunit of the γTuRC, a larger complex containing about six additional polypeptides. Like the γTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), the Drosophila γTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with ∼13 radially arranged structural repeats. The γTuSC also nucleates microtubules, but much less efficiently than the γTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the γTuSC reveals that γ-tubulin binds preferentially to GDP over GTP, rendering γ-tubulin an unusual member of the tubulin superfamily.

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