Chapter 20 The Use of Xenopus Egg Extracts to Study Mitotic Spindle Assembly and Function in Vitro

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a ∼200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to “rescue” experiments. Defects in the “early” step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the “late” step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e13-05-0283 ◽

2013 ◽

Vol 24 (22) ◽

pp. 3522-3533 ◽

Cited By ~ 42

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.200303185 ◽

2003 ◽

Vol 161 (6) ◽

pp. 1041-1051 ◽

Cited By ~ 38

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.

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The Use of Dominant Negative Mutants to Study the Function of Mitotic Motors in the In Vitro Spindle Assembly Assay in Xenopus Egg Extracts

Kinesin Protocols ◽

10.1385/1-59259-069-1:173 ◽

2001 ◽

pp. 173-189 ◽

Cited By ~ 1

Author(s):

Haralabia Boleti ◽

Eric Karsenti ◽

Isabelle Vernos

Keyword(s):

Spindle Assembly ◽

Dominant Negative ◽

Egg Extracts ◽

Mitotic Motors ◽

Xenopus Egg ◽

Xenopus Egg Extracts

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XMAP215–EB1 Interaction Is Required for Proper Spindle Assembly and Chromosome Segregation in Xenopus Egg Extract

Molecular Biology of the Cell ◽

10.1091/mbc.e08-10-1051 ◽

2009 ◽

Vol 20 (11) ◽

pp. 2684-2696 ◽

Cited By ~ 31

Author(s):

Iva Kronja ◽

Anamarija Kruljac-Letunic ◽

Maïwen Caudron-Herger ◽

Peter Bieling ◽

Eric Karsenti

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly ◽

Xenopus Egg Extract ◽

Egg Extract ◽

Egg Extracts ◽

Xenopus Egg ◽

Microtubule Growth ◽

And Function ◽

Xenopus Egg Extracts ◽

Reduced Density

In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of ΔEB1 or ΔXMAP215 spindles leads to faulty kinetochore–microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215–EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215–EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts.

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In vitro Assays for Mitotic Spindle Assembly and Function

Cell Biology ◽

10.1016/b978-012164730-8/50120-9 ◽

2006 ◽

pp. 379-386

Author(s):

C ANTONIO ◽

R HEALD ◽

I VERNOS

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

In Vitro Assays ◽

Mitotic Spindle Assembly ◽

And Function

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Mitotic spindle assembly by two different pathways in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.112.5.925 ◽

1991 ◽

Vol 112 (5) ◽

pp. 925-940 ◽

Cited By ~ 159

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.

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Spindle Checkpoint Protein Bub1 Is Required for Kinetochore Localization of Mad1, Mad2, Bub3, and Cenp-E, Independently of Its Kinase Activity

The Journal of Cell Biology ◽

10.1083/jcb.153.6.1239 ◽

2001 ◽

Vol 153 (6) ◽

pp. 1239-1250 ◽

Cited By ~ 141

Author(s):

Hilary Sharp-Baker ◽

Rey-Huei Chen

Keyword(s):

Mitotic Spindle ◽

Kinase Activity ◽

Spindle Checkpoint ◽

Wild Type ◽

Checkpoint Proteins ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Checkpoint Signal

The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1–Mad2. However, Bub1 differs from Mad1–Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

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