scholarly journals TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts

2014 ◽  
Vol 206 (3) ◽  
pp. 385-393 ◽  
Author(s):  
Kara J. Helmke ◽  
Rebecca Heald
Keyword(s):  
Parallel Architecture ◽  
Cell Types ◽  
Meiotic Spindle ◽  
Spindle Poles ◽  
Frog Species ◽  
Two Factors ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Meiotic Spindles ◽  
Xenopus Egg Extracts

The spindle segregates chromosomes in dividing eukaryotic cells, and its assembly pathway and morphology vary across organisms and cell types. We investigated mechanisms underlying differences between meiotic spindles formed in egg extracts of two frog species. Small Xenopus tropicalis spindles resisted inhibition of two factors essential for assembly of the larger Xenopus laevis spindles: RanGTP, which functions in chromatin-driven spindle assembly, and the kinesin-5 motor Eg5, which drives antiparallel microtubule (MT) sliding. This suggested a role for the MT-associated protein TPX2 (targeting factor for Xenopus kinesin-like protein 2), which is regulated by Ran and binds Eg5. Indeed, TPX2 was threefold more abundant in X. tropicalis extracts, and elevated TPX2 levels in X. laevis extracts reduced spindle length and sensitivity to Ran and Eg5 inhibition. Higher TPX2 levels recruited Eg5 to the poles, where MT density increased. We propose that TPX2 levels modulate spindle architecture through Eg5, partitioning MTs between a tiled, antiparallel array that promotes spindle expansion and a cross-linked, parallel architecture that concentrates MTs at spindle poles.

scholarly journals A computational model predicts Xenopus meiotic spindle organization

2010 ◽  
Vol 191 (7) ◽  
pp. 1239-1249 ◽  
Author(s):  
Rose Loughlin ◽  
Rebecca Heald ◽  
François Nédélec
Keyword(s):  
Dynamic Instability ◽  
Length Distribution ◽  
Spindle Pole ◽  
Two Dimensions ◽  
Meiotic Spindle ◽  
Bipolar Structure ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Meiotic Spindles ◽  
Xenopus Egg Extracts

The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5–like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.

scholarly journals Probing the Force-Balancing Mechanism of the Meiotic Spindle in Xenopus Egg Extracts

2010 ◽  
Vol 98 (3) ◽  
pp. 365a
Author(s):  
Jun Takagi ◽  
Takeshi Itabashi ◽  
Yuta Shimamoto ◽  
Tarun M. Kapoor ◽  
Shin'ichi Ishiwata
Keyword(s):  
Meiotic Spindle ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

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

1998 ◽  
Vol 143 (3) ◽  
pp. 673-685 ◽  
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.

scholarly journals Augmin promotes meiotic spindle formation and bipolarity in Xenopus egg extracts

2011 ◽  
Vol 108 (35) ◽  
pp. 14473-14478 ◽  
Author(s):  
S. Petry ◽  
C. Pugieux ◽  
F. J. Nedelec ◽  
R. D. Vale
Keyword(s):  
Meiotic Spindle ◽  
Spindle Formation ◽  
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 Aurora A Phosphorylates MCAK to Control Ran-dependent Spindle Bipolarity

2008 ◽  
Vol 19 (7) ◽  
pp. 2752-2765 ◽  
Author(s):  
Xin Zhang ◽  
Stephanie C. Ems-McClung ◽  
Claire E. Walczak
Keyword(s):  
Phosphorylation Site ◽  
Aurora B ◽  
Aurora A ◽  
Spindle Formation ◽  
Chromatin Region ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Bipolar Spindles

During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition, we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity.

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 Anaphase A Chromosome Movement and Poleward Spindle Microtubule Flux Occur At Similar Rates in Xenopus Extract Spindles

1998 ◽  
Vol 141 (3) ◽  
pp. 703-713 ◽  
Author(s):  
Arshad Desai ◽  
Paul S. Maddox ◽  
Timothy J. Mitchison ◽  
E.D. Salmon
Keyword(s):  
Somatic Cells ◽  
Cell Types ◽  
Chromosome Movement ◽  
Microtubule Depolymerization ◽  
Spindle Microtubule ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
The Difference ◽  
Xenopus Egg Extracts ◽  
Different Cell Types

We have used local fluorescence photoactivation to mark the lattice of spindle microtubules during anaphase A in Xenopus extract spindles. We find that both poleward spindle microtubule flux and anaphase A chromosome movement occur at similar rates (∼2 μm/min). This result suggests that poleward microtubule flux, coupled to microtubule depolymerization near the spindle poles, is the predominant mechanism for anaphase A in Xenopus egg extracts. In contrast, in vertebrate somatic cells a “Pacman” kinetochore mechanism, coupled to microtubule depolymerization near the kinetochore, predominates during anaphase A. Consistent with the conclusion from fluorescence photoactivation analysis, both anaphase A chromosome movement and poleward spindle microtubule flux respond similarly to pharmacological perturbations in Xenopus extracts. Furthermore, the pharmacological profile of anaphase A in Xenopus extracts differs from the previously established profile for anaphase A in vertebrate somatic cells. The difference between these profiles is consistent with poleward microtubule flux playing the predominant role in anaphase chromosome movement in Xenopus extracts, but not in vertebrate somatic cells. We discuss the possible biological implications of the existence of two distinct anaphase A mechanisms and their differential contributions to poleward chromosome movement in different cell types.

scholarly journals Bipolarization and Poleward Flux Correlate duringXenopusExtract Spindle Assembly

2004 ◽  
Vol 15 (12) ◽  
pp. 5603-5615 ◽  
Author(s):  
T.J. Mitchison ◽  
P. Maddox ◽  
A. Groen ◽  
L. Cameron ◽  
Z. Perlman ◽  
...  
Keyword(s):  
Self Organization ◽  
Microtubule Organization ◽  
Importin Alpha ◽  
Egg Extracts ◽  
Show Evidence ◽  
Xenopus Egg ◽  
Meiotic Spindles ◽  
Xenopus Egg Extracts ◽  
Sliding Force ◽  
Poleward Flux

We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.

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