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.

Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts

Nature ◽  
1996 ◽  
Vol 382 (6590) ◽  
pp. 420-425 ◽  
Author(s):  
Rebecca Heald ◽  
Régis Tournebize ◽  
Thiemo Blank ◽  
Raphael Sandaltzopoulos ◽  
Peter Becker ◽  
...  
Keyword(s):  
Self Organization ◽  
Artificial Chromosomes ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Bipolar Spindles

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 Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

1994 ◽  
Vol 5 (2) ◽  
pp. 217-226 ◽  
Author(s):  
K E Sawin ◽  
T J Mitchison
Keyword(s):  
Basic Element ◽  
Low Light ◽  
Self Organized ◽  
Egg Extracts ◽  
Microtubule Motor ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Microtubule Motor Protein ◽  
Poleward Flux

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.

scholarly journals The Cytoskeleton and Its Roles in Self-Organization Phenomena: Insights from Xenopus Egg Extracts

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2197
Author(s):  
Zachary M. Geisterfer ◽  
Gabriel Guilloux ◽  
Jesse C. Gatlin ◽  
Romain Gibeaux
Keyword(s):  
Experimental System ◽  
Structure And Function ◽  
Self Organization ◽  
African Clawed Frog ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
The Many ◽  
And Function ◽  
Xenopus Egg Extracts ◽  
Clawed Frog

Self-organization of and by the cytoskeleton is central to the biology of the cell. Since their introduction in the early 1980s, cytoplasmic extracts derived from the eggs of the African clawed-frog, Xenopus laevis, have flourished as a major experimental system to study the various facets of cytoskeleton-dependent self-organization. Over the years, the many investigations that have used these extracts uniquely benefited from their simplified cell cycle, large experimental volumes, biochemical tractability and cell-free nature. Here, we review the contributions of egg extracts to our understanding of the cytoplasmic aspects of self-organization by the microtubule and the actomyosin cytoskeletons as well as the importance of cytoskeletal filaments in organizing nuclear structure and function.

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 The N-terminal coiled-coil of Ndel1 is a regulated scaffold that recruits LIS1 to dynein

2011 ◽  
Vol 192 (3) ◽  
pp. 433-445 ◽  
Author(s):  
Eliza Żyłkiewicz ◽  
Monika Kijańska ◽  
Won-Chan Choi ◽  
Urszula Derewenda ◽  
Zygmunt S. Derewenda ◽  
...  
Keyword(s):  
Experimental Approach ◽  
Coiled Coil ◽  
Self Organization ◽  
Binding Partners ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.

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