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 A requirement for MAP kinase in the assembly and maintenance of the mitotic spindle

2003 ◽  
Vol 161 (6) ◽  
pp. 1021-1028 ◽  
Author(s):  
Melinda M. Horne ◽  
Thomas M. Guadagno
Keyword(s):  
Map Kinase ◽  
Mitotic Spindle ◽  
Kinase Inhibitor ◽  
Map Kinase Phosphatase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Multiple Spindle ◽  
Xenopus Egg Extracts ◽  
Nih 3T3 ◽  
Bipolar Spindles

Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.

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 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 Chromokinesin Xklp1 Contributes to the Regulation of Microtubule Density and Organization during Spindle Assembly

2006 ◽  
Vol 17 (3) ◽  
pp. 1451-1460 ◽  
Author(s):  
Mirco Castoldi ◽  
Isabelle Vernos
Keyword(s):  
Cell Division ◽  
Spindle Assembly ◽  
Embryonic Cell ◽  
Microtubule Polymerization ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Directed Motility ◽  
Xenopus Egg Extracts ◽  
Bipolar Spindles

Xklp1 is a chromosome-associated kinesin required for Xenopus early embryonic cell division. Function blocking experiments in Xenopus egg extracts suggested that it is required for spindle assembly. We have reinvestigated Xklp1 function(s) by monitoring spindle assembly and microtubule behavior under a range of Xklp1 concentrations in egg extracts. We found that in the absence of Xklp1, bipolar spindles form with a reduced efficiency and display abnormalities associated with an increased microtubule mass. Likewise, centrosomal asters assembled in Xklp1-depleted extract show an increased microtubule mass. Conversely, addition of recombinant Xklp1 to the extract reduces the microtubule mass associated with spindles and asters. Our data suggest that Xklp1 affects microtubule polymerization during M-phase. We propose that these attributes, combined with Xklp1 plus-end directed motility, contribute to the assembly of a functional bipolar spindle.

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 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.

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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