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.

scholarly journals Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
N. Tavernier ◽  
Y. Thomas ◽  
S. Vigneron ◽  
P. Maisonneuve ◽  
S. Orlicky ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Activation Mechanism ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Entry ◽  
Amino Acid Region ◽  
Egg Extracts ◽  
Activation Segment ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

AbstractPolo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

scholarly journals Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization

1997 ◽  
Vol 138 (3) ◽  
pp. 615-628 ◽  
Author(s):  
Rebecca Heald ◽  
Régis Tournebize ◽  
Anja Habermann ◽  
Eric Karsenti ◽  
Anthony Hyman
Keyword(s):  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
Xenopus Egg Extracts ◽  
Microtubule Stabilizing Agent ◽  
Mitotic Chromatin

In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end–directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

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 XCTK2: A Kinesin-related Protein That Promotes Mitotic Spindle Assembly in Xenopus laevis Egg Extracts

1997 ◽  
Vol 136 (4) ◽  
pp. 859-870 ◽  
Author(s):  
Claire E. Walczak ◽  
Suzie Verma ◽  
Timothy J. Mitchison
Keyword(s):  
Spindle Assembly ◽  
Motor Domain ◽  
Cdna Expression Library ◽  
Globular Domain ◽  
Mitotic Spindles ◽  
Vitro Assay ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Bipolar Spindles

We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central α-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor- arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.

scholarly journals Induction of a Spindle-Assembly-Competent M Phase in Xenopus Egg Extracts

2019 ◽  
Vol 29 (8) ◽  
pp. 1273-1285.e5 ◽  
Author(s):  
Jitender S. Bisht ◽  
Miroslav Tomschik ◽  
Jesse C. Gatlin
Keyword(s):  
Spindle Assembly ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals The Nup107-160 Nucleoporin Complex Is Required for Correct Bipolar Spindle Assembly

2006 ◽  
Vol 17 (9) ◽  
pp. 3806-3818 ◽  
Author(s):  
Arturo V. Orjalo ◽  
Alexei Arnaoutov ◽  
Zhouxin Shen ◽  
Yekaterina Boyarchuk ◽  
Samantha G. Zeitlin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Sperm Chromatin ◽  
Nuclear Pore ◽  
Checkpoint Proteins ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

scholarly journals The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts

2003 ◽  
Vol 161 (6) ◽  
pp. 1041-1051 ◽  
Author(s):  
Sarah M. Wignall ◽  
Renée Deehan ◽  
Thomas J. Maresca ◽  
Rebecca Heald
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly ◽  
Condensin Complex ◽  
Egg Extracts ◽  
Chromosomal Architecture ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
And Function ◽  
Xenopus Egg Extracts

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.

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