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 Nudel/NudE and Lis1 promote dynein and dynactin interaction in the context of spindle morphogenesis

2013 ◽  
Vol 24 (22) ◽  
pp. 3522-3533 ◽  
Author(s):  
Shusheng Wang ◽  
Stephanie A. Ketcham ◽  
Arne Schön ◽  
Benjamin Goodman ◽  
Yueju Wang ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Spindle Poles ◽  
Interesting Possibility ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Lis1, Nudel/NudE, and dynactin are regulators of cytoplasmic dynein, a minus end–directed, microtubule (MT)-based motor required for proper spindle assembly and orientation. In vitro studies have shown that dynactin promotes processive movement of dynein on MTs, whereas Lis1 causes dynein to enter a persistent force-generating state (referred to here as dynein stall). Yet how the activities of Lis1, Nudel/NudE, and dynactin are coordinated to regulate dynein remains poorly understood in vivo. Working in Xenopus egg extracts, we show that Nudel/NudE facilitates the binding of Lis1 to dynein, which enhances the recruitment of dynactin to dynein. We further report a novel Lis1-dependent dynein–dynactin interaction that is essential for the organization of mitotic spindle poles. Finally, using assays for MT gliding and spindle assembly, we demonstrate an antagonistic relationship between Lis1 and dynactin that allows dynactin to relieve Lis1-induced dynein stall on MTs. Our findings suggest the interesting possibility that Lis1 and dynactin could alternately engage with dynein to allow the motor to promote spindle assembly.

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

2003 ◽  
Vol 161 (6) ◽  
pp. 1041-1051 ◽  
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.

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 Mitotic spindle assembly by two different pathways in vitro.

1991 ◽  
Vol 112 (5) ◽  
pp. 925-940 ◽  
Author(s):  
K E Sawin ◽  
T J Mitchison
Keyword(s):  
Spindle Assembly ◽  
Sperm Nucleus ◽  
Free System ◽  
Microtubule Stabilization ◽  
Egg Extracts ◽  
A Cell ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
Xenopus Egg Extracts

We have used Xenopus egg extracts to study spindle morphogenesis in a cell-free system and have identified two pathways of spindle assembly in vitro using methods of fluorescent analogue cytochemistry. When demembranated sperm nuclei are added to egg extracts arrested in a mitotic state, individual nuclei direct the assembly of polarized microtubule arrays, which we term half-spindles; half-spindles then fuse pairwise to form bipolar spindles. In contrast, when sperm nuclei are added to extracts that are induced to enter interphase and arrested in the following mitosis, a single sperm nucleus can direct the assembly of a complete spindle. We find that microtubule arrays in vitro are strongly biased towards chromatin, but this does not depend on specific kinetochore-microtubule interactions. Indeed, although we have identified morphological and probably functional kinetochores in spindles assembled in vitro, kinetochores appear not to play an obligate role in the establishment of stable, bipolar microtubule arrays in either assembly pathway. Features of the two pathways suggest that spindle assembly involves a hierarchy of selective microtubule stabilization, involving both chromatin-microtubule interactions and antiparallel microtubule-microtubule interactions, and that fundamental molecular interactions are probably the same in both pathways. This in vitro reconstitution system should be useful for identifying the molecules regulating the generation of asymmetric microtubule arrays and for understanding spindle morphogenesis in general.

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

2004 ◽  
Vol 15 (12) ◽  
pp. 5318-5328 ◽  
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.

scholarly journals DNA replication of mitotic chromatin in Xenopus egg extracts

2003 ◽  
Vol 100 (23) ◽  
pp. 13241-13246 ◽  
Author(s):  
T. A. Prokhorova ◽  
K. Mowrer ◽  
C. H. Gilbert ◽  
J. C. Walter
Keyword(s):  
Dna Replication ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromatin

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.

Decondensation of Human Sperm Nuclei and Hp1 Protamine Degradation from Normospermia and Asthenospermia in Xenopus Egg Extracts

1992 ◽  
Vol 29 (2) ◽  
pp. 127-136 ◽  
Author(s):  
J. F. Griveau ◽  
M. Charbonneau ◽  
Y. Blanchard ◽  
D. Lescoat ◽  
D. Le Lannou
Keyword(s):  
Human Sperm ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
Xenopus Egg Extracts
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