Decision letter: Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting.

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Centromere and Kinetochore Assembly in Xenopus laevis Egg Extract

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot102509 ◽

2018 ◽

Vol 2018 (9) ◽

pp. pdb.prot102509 ◽

Cited By ~ 1

Author(s):

Julio C. Flores Servin ◽

Aaron F. Straight

Keyword(s):

Xenopus Laevis ◽

Kinetochore Assembly ◽

Egg Extract

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Identification of an Spc110p-related protein in vertebrates

Journal of Cell Science ◽

10.1242/jcs.110.20.2533 ◽

1997 ◽

Vol 110 (20) ◽

pp. 2533-2545 ◽

Cited By ~ 1

Author(s):

A.M. Tassin ◽

C. Celati ◽

M. Paintrand ◽

M. Bornens

Keyword(s):

Mammalian Cells ◽

Spindle Pole Body ◽

Spindle Pole ◽

Partial Purification ◽

Related Protein ◽

Microtubule Nucleation ◽

Pole Body ◽

Egg Extract ◽

Ring Complex ◽

Gamma Tubulin

Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the gamma-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.

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Extracts from eggs and oocytes of Xenopus laevis differ in their capacities for nuclear assembly and DNA replication

Journal of Cell Science ◽

10.1242/jcs.97.1.177 ◽

1990 ◽

Vol 97 (1) ◽

pp. 177-184

Author(s):

L.S. Cox ◽

G.H. Leno

Keyword(s):

Dna Replication ◽

Xenopus Laevis ◽

High Speed ◽

De Novo ◽

Soluble Fraction ◽

Sperm Chromatin ◽

Nuclear Assembly ◽

Egg Extract ◽

Egg Extracts ◽

Sperm Nuclei

We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.

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Poleward transport of Eg5 by dynein–dynactin in Xenopus laevis egg extract spindles

The Journal of Cell Biology ◽

10.1083/jcb.200801125 ◽

2008 ◽

Vol 182 (4) ◽

pp. 715-726 ◽

Cited By ~ 63

Author(s):

Marianne Uteng ◽

Christian Hentrich ◽

Kota Miura ◽

Peter Bieling ◽

Thomas Surrey

Keyword(s):

Cell Division ◽

Xenopus Laevis ◽

Molecular Motors ◽

Motor Proteins ◽

Motor Protein ◽

Spindle Assembly ◽

Cross Linking ◽

Motor Movement ◽

Spindle Poles ◽

Egg Extract

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein–dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.

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Nucleus Assembly and Import in Xenopus laevis Egg Extract

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot097196 ◽

2018 ◽

Vol 2018 (6) ◽

pp. pdb.prot097196 ◽

Cited By ~ 6

Author(s):

Pan Chen ◽

Daniel L. Levy

Keyword(s):

Xenopus Laevis ◽

Egg Extract

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Atomic Force Microscope Imaging of Chromatin Assembled in Xenopus Laevis Egg Extract

Biophysical Journal ◽

10.1016/j.bpj.2009.12.2580 ◽

2010 ◽

Vol 98 (3) ◽

pp. 474a

Author(s):

Hongxia Fu ◽

Benjamin Freedman ◽

Chwee Teck Lim ◽

Rebecca Heald ◽

Jie Yan

Keyword(s):

Xenopus Laevis ◽

Atomic Force Microscope ◽

Atomic Force ◽

Egg Extract ◽

Microscope Imaging

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Use of dna end joining activity of a xenopus laevis egg extract for construction of deletions and expression vectors for hiv-1 tat and rev proteins

Gene ◽

10.1016/0378-1119(93)90405-r ◽

1993 ◽

Vol 124 (2) ◽

pp. 275-280 ◽

Cited By ~ 13

Author(s):

Heiner Schaal ◽

Petra Pfeiffer ◽

Michael Klein ◽

Peter Gehrmann ◽

Andreas Scheid

Keyword(s):

Xenopus Laevis ◽

Expression Vectors ◽

End Joining ◽

Egg Extract ◽

Hiv 1

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The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles

The Journal of Cell Biology ◽

10.1083/jcb.200407126 ◽

2004 ◽

Vol 167 (5) ◽

pp. 813-818 ◽

Cited By ~ 118

Author(s):

David T. Miyamoto ◽

Zachary E. Perlman ◽

Kendra S. Burbank ◽

Aaron C. Groen ◽

Timothy J. Mitchison

Keyword(s):

Xenopus Laevis ◽

Chromosome Segregation ◽

Spindle Poles ◽

Egg Extract ◽

Constant Rate ◽

Quantitative Image ◽

Meiotic Spindles ◽

Kinesin Eg5 ◽

Poleward Flux

Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This “poleward flux” of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose–responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.

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