The far C-terminus of MCAK regulates its conformation and spindle pole focusing

To ensure proper spindle assembly, microtubule (MT) dynamics needs to be spatially regulated within the cell. The kinesin-13 MCAK is a potent MT depolymerase with a complex subcellular localization, yet how MCAK spatial regulation contributes to spindle assembly is not understood. Here we show that the far C-terminus of MCAK plays a critical role in regulating MCAK conformation, subspindle localization, and spindle assembly in Xenopus egg extracts. Alteration of MCAK conformation by the point mutation E715A/E716A in the far C-terminus increased MCAK targeting to the poles and reduced MT lifetimes, which induced spindles with unfocused poles. These effects were phenocopied by the Aurora A phosphomimetic mutation, S719E. Furthermore, addition of the kinesin-14 XCTK2 to spindle assembly reactions rescued the unfocused-pole phenotype. Collectively our work shows how the regional targeting of MCAK regulates MT dynamics, highlighting the idea that multiple phosphorylation pathways of MCAK cooperate to spatially control MT dynamics to maintain spindle architecture.

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Tpx2, a Novel Xenopus Map Involved in Spindle Pole Organization

The Journal of Cell Biology ◽

10.1083/jcb.149.7.1405 ◽

2000 ◽

Vol 149 (7) ◽

pp. 1405-1418 ◽

Cited By ~ 256

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-05-0385 ◽

2004 ◽

Vol 15 (12) ◽

pp. 5318-5328 ◽

Cited By ~ 68

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.138.3.615 ◽

1997 ◽

Vol 138 (3) ◽

pp. 615-628 ◽

Cited By ~ 248

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.

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Localization of the Kinesin-like Protein Xklp2 to Spindle Poles Requires a Leucine Zipper, a Microtubule-associated Protein, and Dynein

The Journal of Cell Biology ◽

10.1083/jcb.143.3.673 ◽

1998 ◽

Vol 143 (3) ◽

pp. 673-685 ◽

Cited By ~ 131

Author(s):

Torsten Wittmann ◽

Haralabia Boleti ◽

Claude Antony ◽

Eric Karsenti ◽

Isabelle Vernos

Keyword(s):

Leucine Zipper ◽

Molecular Mechanisms ◽

Single Point ◽

Spindle Pole ◽

Single Point Mutation ◽

Spindle Poles ◽

Centrosome Separation ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Xklp2 is a plus end–directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein–dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.

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The Microtubule-destabilizing Kinesin XKCM1 Regulates Microtubule Dynamic Instability in Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e01-12-0143 ◽

2002 ◽

Vol 13 (8) ◽

pp. 2718-2731 ◽

Cited By ~ 112

Author(s):

Susan L. Kline-Smith ◽

Claire E. Walczak

Keyword(s):

Mammalian Cells ◽

Dynamic Instability ◽

Critical Role ◽

Dependent Manner ◽

Related Protein ◽

Microtubule Dynamic ◽

Mitotic Delay ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.

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

The Journal of Cell Biology ◽

10.1083/jcb.136.4.859 ◽

1997 ◽

Vol 136 (4) ◽

pp. 859-870 ◽

Cited By ~ 101

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.

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

Current Biology ◽

10.1016/j.cub.2019.02.061 ◽

2019 ◽

Vol 29 (8) ◽

pp. 1273-1285.e5 ◽

Cited By ~ 2

Author(s):

Jitender S. Bisht ◽

Miroslav Tomschik ◽

Jesse C. Gatlin

Keyword(s):

Spindle Assembly ◽

M Phase ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

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

Molecular Biology of the Cell ◽

10.1091/mbc.e05-11-1061 ◽

2006 ◽

Vol 17 (9) ◽

pp. 3806-3818 ◽

Cited By ~ 109

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.200303185 ◽

2003 ◽

Vol 161 (6) ◽

pp. 1041-1051 ◽

Cited By ~ 38

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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