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

2002 ◽  
Vol 13 (8) ◽  
pp. 2718-2731 ◽  
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.

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 Membrane/microtubule tip attachment complexes (TACs) allow the assembly dynamics of plus ends to push and pull membranes into tubulovesicular networks in interphase Xenopus egg extracts.

1995 ◽  
Vol 130 (5) ◽  
pp. 1161-1169 ◽  
Author(s):  
C M Waterman-Storer ◽  
J Gregory ◽  
S F Parsons ◽  
E D Salmon
Keyword(s):  
Dynamic Instability ◽  
Atp Depletion ◽  
Immunofluorescent Staining ◽  
Egg Extracts ◽  
Microtubule Motor ◽  
Xenopus Egg ◽  
Membrane Tubules ◽  
Membrane Networks ◽  
Xenopus Egg Extracts ◽  
Push And Pull

We discovered by using high resolution video microscopy, that membranes become attached selectively to the growing plus ends of microtubules by membrane/microtubule tip attachment complexes (TACs) in interphase-arrested, undiluted, Xenopus egg extracts. Persistent plus end growth of stationary microtubules pushed the membranes into thin tubules and dragged them through the cytoplasm at the approximately 20 microns/min velocity typical of free plus ends. Membrane tubules also remained attached to plus ends when they switched to the shortening phase of dynamic instability at velocities typical of free ends, 50-60 microns/min. Over time, the membrane tubules contacted and fused with one another along their lengths, forming a polygonal network much like the distribution of ER in cells. Several components of the membrane networks formed by TACs were identified as ER by immunofluorescent staining using antibodies to ER-resident proteins. TAC motility was not inhibited by known inhibitors of microtubule motor activity, including 5 mM AMP-PNP, 250 microM orthovanadate, and ATP depletion. These results show that membrane/microtubule TACs enable polymerizing ends to push and depolymerizing ends to pull membranes into thin tubular extensions and networks at fast velocities.

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

2016 ◽  
Vol 27 (9) ◽  
pp. 1451-1464 ◽  
Author(s):  
Hailing Zong ◽  
Stephanie K. Carnes ◽  
Christina Moe ◽  
Claire E. Walczak ◽  
Stephanie C. Ems-McClung
Keyword(s):  
Subcellular Localization ◽  
Point Mutation ◽  
Critical Role ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Spatial Regulation ◽  
C Terminus ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

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.

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 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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