scholarly journals An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region

2015 ◽  
Vol 210 (5) ◽  
pp. 695-704 ◽  
Author(s):  
Nina Schweizer ◽  
Nisha Pawar ◽  
Matthias Weiss ◽  
Helder Maiato
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Membrane System ◽  
Correlation Spectroscopy ◽  
Molecular Crowding ◽  
Exclusion Zone ◽  
Drosophila Melanogaster S2 Cells ◽  
Spatio Temporal ◽  
And Function

The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. By combining live-cell imaging with laser microsurgery, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy in Drosophila melanogaster S2 cells, we uncovered a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, we demonstrate that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous “spindle envelope” confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.

scholarly journals Interactions between N-Terminal Modules in MPS1 Enable Spindle Checkpoint Silencing

2018 ◽  
Author(s):  
Spyridon T. Pachis ◽  
Yoshitaka Hiruma ◽  
Anastassis Perrakis ◽  
Geert J.P.L. Kops
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Intramolecular Interactions ◽  
Mitotic Progression ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Regulatory Input ◽  
Tpr Domain

ABSTRACTFaithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until all chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to unattached kinetochores to initiate SAC signaling, and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here we show that a helical fragment within the kinetochore-targeting NTE module of MPS1 is required for interactions with kinetochores, and also forms intramolecular interactions with its adjacent TPR domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, ineffecient MPS1 delocalization from kinetochores upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MSP1-NDC80-C interactions.

scholarly journals MYC Dysregulates Mitotic Spindle Function Creating a Dependency on TPX2

2018 ◽  
Author(s):  
Julia Rohrberg ◽  
Alexandra Corella ◽  
Moufida Taileb ◽  
Seda Kilinc ◽  
Marie-Lena Jokisch ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Cellular Proliferation ◽  
Spindle Assembly ◽  
Tumor Evolution ◽  
Mouse Tumor ◽  
Breast Cancers ◽  
Mitotic Progression ◽  
Myc Oncogene

AbstractThe MYC oncogene promotes tumorigenesis in part by facilitating cell cycle entry thus driving cellular proliferation. Tumors that overexpress MYC frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers, rapid tumor evolution, and poor patient outcome. While the role of MYC in overcoming the G1/S checkpoint is well established, it remains poorly understood whether MYC induces chromosomal instability (CIN). Here, we identify a direct influence of MYC on mitotic progression. MYC overexpression induces defects in microtubule nucleation and spindle assembly promoting chromosome segregation defects, micronuclei and CIN. We examined which mitotic regulators are required for the survival of MYC-overexpressing cells and found a reliance on high TPX2 expression. TPX2, a master microtubule regulator, is overexpressed together with MYC in multiple cell lines, in mouse tumor models and in aggressive human breast cancers. High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with deregulated MYC, whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Importantly, attenuation of MYC expression reverses the mitotic defects observed, even in established tumor cell lines, implicating an ongoing role for high MYC in the persistence of a CIN phenotype in tumors. Here, we implicate the MYC oncogene as a regulator of spindle assembly and dynamics and identify a new MYC-TPX2 synthetic-lethal interaction that could represent a future therapeutic strategy in MYC-overexpressing cancers. Our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

scholarly journals Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

2009 ◽  
Vol 184 (3) ◽  
pp. 373-381 ◽  
Author(s):  
Thomas J. Maresca ◽  
Edward D. Salmon
Keyword(s):  
Drosophila Melanogaster ◽  
Green Fluorescent Protein ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Fluorescent Protein ◽  
Spindle Assembly ◽  
Centromeric Chromatin ◽  
Cell Assays ◽  
Green Fluorescent

Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a “wait anaphase” signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.

scholarly journals TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

2011 ◽  
Vol 195 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Nan Ma ◽  
Janel Titus ◽  
Alyssa Gable ◽  
Jennifer L. Ross ◽  
Patricia Wadsworth
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Spindle Assembly ◽  
Artificial Chromosome ◽  
Fiber Formation ◽  
Interaction Domain ◽  
Spindle Poles ◽  
Spindle Elongation ◽  
Multiple Spindle ◽  
And Function

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2–Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.

Driving chromosome segregation: lessons from the human and Drosophila centromere–kinetochore machinery

2010 ◽  
Vol 38 (6) ◽  
pp. 1667-1675 ◽  
Author(s):  
Bernardo Orr ◽  
Olga Afonso ◽  
Tália Feijão ◽  
Claudio E. Sunkel
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Genomic Stability ◽  
Mitotic Progression ◽  
Chromosomal Locus ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
And Function ◽  
Drosophila Cells

The kinetochore is a complex molecular machine that serves as the interface between sister chromatids and the mitotic spindle. The kinetochore assembles at a particular chromosomal locus, the centromere, which is essential to maintain genomic stability during cell division. The kinetochore is a macromolecular puzzle of subcomplexes assembled in a hierarchical manner and fulfils three main functions: microtubule attachment, chromosome and sister chromatid movement, and regulation of mitotic progression though the spindle assembly checkpoint. In the present paper we compare recent results on the assembly, organization and function of the kinetochore in human and Drosophila cells and conclude that, although essential functions are highly conserved, there are important differences that might help define what is a minimal chromosome segregation machinery.

scholarly journals Concentrating on the mitotic spindle

2015 ◽  
Vol 210 (5) ◽  
pp. 691-693 ◽  
Author(s):  
Paul S. Maddox ◽  
Anne-Marie Ladouceur
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Molecular Crowding ◽  
Cell Biol ◽  
New Feature

In eukaryotes, the microtubule-based spindle drives chromosome segregation. In this issue, Schweizer et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506107) find that the spindle area is demarcated by a semipermeable organelle barrier. Molecular crowding, which is microtubule independent, causes the enrichment and/or retention of crucial factors in the spindle region. Their results add an important new feature to the models of how this structure assembles and is regulated.

scholarly journals Conditional Mutations in γ-Tubulin Reveal Its Involvement in Chromosome Segregation and Cytokinesis

2001 ◽  
Vol 12 (8) ◽  
pp. 2469-2481 ◽  
Author(s):  
Triscia W. Hendrickson ◽  
Joyce Yao ◽  
Saswata Bhadury ◽  
Anita H. Corbett ◽  
Harish C. Joshi
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Homology Model ◽  
Deleterious Mutations ◽  
Alanine Scanning Mutagenesis ◽  
Structural Mapping ◽  
Lethal Mutations ◽  
M Phase ◽  
Cold Sensitive ◽  
And Function

γ-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human γ-tubulin can replace the γ-tubulin gene inSchizosaccharomyces pombe. To understand the structural/functional domains of γ-tubulin, we performed a systematic alanine-scanning mutagenesis of human γ-tubulin (TUBG1) and studied phenotypes of each mutant allele inS. pombe. Our screen, both in the presence and absence of the endogenous S. pombe γ-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human γ-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with α- and/or β-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, γ-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.

Spatial gradients controlling spindle assembly

2015 ◽  
Vol 43 (1) ◽  
pp. 7-12 ◽  
Author(s):  
Lesley N. Weaver ◽  
Claire E. Walczak
Keyword(s):  
Self Assembly ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Present Review ◽  
Aurora B ◽  
Spatial Gradients ◽  
Self Organized ◽  
Ran Gtp ◽  
And Function ◽  
In Cells

The mitotic spindle is the macromolecular machine utilized to accurately segregate chromosomes in cells. How this self-organized structure assembles is a key aspect of understanding spindle morphogenesis. In the present review, we focus on understanding mechanisms of spindle self-assembly and address how subcellular signalling gradients, such as Ran-GTP and Aurora B, contribute to spindle organization and function.

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