scholarly journals Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase.

1995 ◽  
Vol 129 (5) ◽  
pp. 1195-1204 ◽  
Author(s):  
M S Campbell ◽  
G J Gorbsky
Keyword(s):  
Monoclonal Antibody ◽  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Metaphase Plate ◽  
Chromosome Movement ◽  
Normal Cells ◽  
Microtubule Attachment ◽  
Checkpoint Pathway ◽  
Chromosome Congression ◽  
Asymmetric Expression

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.

scholarly journals Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension

2002 ◽  
Vol 13 (10) ◽  
pp. 3706-3719 ◽  
Author(s):  
Katie B. Shannon ◽  
Julie C. Canman ◽  
E. D. Salmon
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Spindle Checkpoint ◽  
Normal Numbers ◽  
Microtubule Attachment ◽  
Checkpoint Pathway ◽  
Chromosome Congression ◽  
Full Complement ◽  
Checkpoint Genes

The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23°C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23°C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37°C. The metaphase delay at 23°C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23°C, and metaphase kinetochores at 23°C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37°C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23°C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.

scholarly journals Depletion of Centromeric MCAK Leads to Chromosome Congression and Segregation Defects Due to Improper Kinetochore Attachments

2004 ◽  
Vol 15 (3) ◽  
pp. 1146-1159 ◽  
Author(s):  
Susan L. Kline-Smith ◽  
Alexey Khodjakov ◽  
Polla Hergert ◽  
Claire E. Walczak
Keyword(s):  
Essential Role ◽  
Dominant Negative ◽  
Primary Role ◽  
Complex Behavior ◽  
Chromosome Movement ◽  
Immunofluorescence Localization ◽  
Microtubule Attachment ◽  
Chromosome Congression

The complex behavior of chromosomes during mitosis is accomplished by precise binding and highly regulated polymerization dynamics of kinetochore microtubules. Previous studies have implicated Kin Is, unique kinesins that depolymerize microtubules, in regulating chromosome positioning. We have characterized the immunofluorescence localization of centromere-bound MCAK and found that MCAK localized to inner kinetochores during prophase but was predominantly centromeric by metaphase. Interestingly, MCAK accumulated at leading kinetochores during congression but not during segregation. We tested the consequences of MCAK disruption by injecting a centromere dominant-negative protein into prophase cells. Depletion of centromeric MCAK led to reduced centromere stretch, delayed chromosome congression, alignment defects, and severe missegregation of chromosomes. Rates of chromosome movement were unchanged, suggesting that the primary role of MCAK is not to move chromosomes. Furthermore, we found that disruption of MCAK leads to multiple kinetochore–microtubule attachment defects, including merotelic, syntelic, and combined merotelic-syntelic attachments. These findings reveal an essential role for Kin Is in prevention and/or correction of improper kinetochore–microtubule attachments.

scholarly journals Xkid Is Degraded in a D-Box, KEN-Box, and A-Box-Independent Pathway

2003 ◽  
Vol 23 (12) ◽  
pp. 4126-4138 ◽  
Author(s):  
Anna Castro ◽  
Suzanne Vigneron ◽  
Cyril Bernis ◽  
Jean-Claude Labbé ◽  
Thierry Lorca
Keyword(s):  
Chromosome Segregation ◽  
Degradation Pathway ◽  
Cyclin B ◽  
Chromosome Movement ◽  
Regulatory Factor ◽  
Independent Manner ◽  
C Terminus ◽  
Chromosome Congression

ABSTRACT During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid. We demonstrate that Xkid is degraded both in vitro and in vivo by APC/Cdc20 and APC/Cdh1. We show that, despite the presence of five putative D-box motifs in its sequence, Xkid is proteolyzed in a D-box-independent manner. We identify a domain within the C terminus of this chromokinesin, with sequence GxEN, whose mutation completely stabilizes this protein by both APC/Cdc20 and APC/Cdh1. Moreover, we show that this degradation sequence acts as a transposable motif and induces the proteolysis of a GST-GXEN fusion protein. Finally, we demonstrate that both a D-box and a GXEN-containing peptides completely block APC-dependent degradation of cyclin B and Xkid, indicating that the GXEN domain might mediate the recognition and association of Xkid with the APC.

scholarly journals Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore

2016 ◽  
Vol 212 (7) ◽  
pp. 767-776 ◽  
Author(s):  
Gheorghe Cojoc ◽  
Emanuele Roscioli ◽  
Lijuan Zhang ◽  
Alfonso García-Ulloa ◽  
Jagesh V. Shah ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Mechanical Response ◽  
Viscoelastic Behavior ◽  
Length Change ◽  
Live Cells ◽  
Microtubule Severing ◽  
Microtubule Attachment ◽  
Pulling Forces

Accurate chromosome segregation depends on proper kinetochore–microtubule attachment. Upon microtubule interaction, kinetochores are subjected to forces generated by the microtubules. In this work, we used laser ablation to sever microtubules attached to a merotelic kinetochore, which is laterally stretched by opposing pulling forces exerted by microtubules, and inferred the mechanical response of the kinetochore from its length change. In both mammalian PtK1 cells and in the fission yeast Schizosaccharomyces pombe, kinetochores shortened after microtubule severing. Interestingly, the inner kinetochore–centromere relaxed faster than the outer kinetochore. Whereas in fission yeast all kinetochores relaxed to a similar length, in PtK1 cells the more stretched kinetochores remained more stretched. Simple models suggest that these differences arise because the mechanical structure of the mammalian kinetochore is more complex. Our study establishes merotelic kinetochores as an experimental model for studying the mechanical response of the kinetochore in live cells and reveals a viscoelastic behavior of the kinetochore that is conserved in yeast and mammalian cells.

scholarly journals CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes

2020 ◽  
Vol 219 (5) ◽  
Author(s):  
Philip Auckland ◽  
Emanuele Roscioli ◽  
Helena Louise Elvidge Coker ◽  
Andrew D. McAinsh
Keyword(s):  
Chromosome Segregation ◽  
Direct Interaction ◽  
Antagonistic Activity ◽  
Early Event ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Microtubule Attachment ◽  
Dynein Motor ◽  
Chromosome Congression ◽  
Kinetochore Function

Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation.

scholarly journals Direct kinetochore–spindle pole connections are not required for chromosome segregation

2014 ◽  
Vol 206 (2) ◽  
pp. 231-243 ◽  
Author(s):  
Vitali Sikirzhytski ◽  
Valentin Magidson ◽  
Jonathan B. Steinman ◽  
Jie He ◽  
Maël Le Berre ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Genetic Material ◽  
Spindle Pole ◽  
Chromosome Movement ◽  
Parallel Mechanisms ◽  
Laser Microbeam ◽  
Kinetochore Assembly ◽  
Spindle Poles

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes′ kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.

Kinetochore–microtubule interactions in chromosome segregation: lessons from yeast and mammalian cells

2017 ◽  
Vol 474 (21) ◽  
pp. 3559-3577 ◽  
Author(s):  
Geethu Emily Thomas ◽  
Marira R. Renjith ◽  
Tapas K. Manna
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Structural Organization ◽  
Vital Role ◽  
Present Review ◽  
The Past ◽  
Chromosome Congression ◽  
Spindle Microtubules ◽  
Higher Eukaryotes ◽  
Structural Aspects

Chromosome congression and segregation require robust yet dynamic attachment of the kinetochore with the spindle microtubules. Force generated at the kinetochore–microtubule interface plays a vital role to drive the attachment, as it is required to move chromosomes and to provide signal to sense correct attachments. To understand the mechanisms underlying these processes, it is critical to describe how the force is generated and how the molecules at the kinetochore–microtubule interface are organized and assembled to withstand the force and respond to it. Research in the past few years or so has revealed interesting insights into the structural organization and architecture of kinetochore proteins that couple kinetochore attachment to the spindle microtubules. Interestingly, despite diversities in the molecular players and their modes of action, there appears to be architectural similarity of the kinetochore-coupling machines in lower to higher eukaryotes. The present review focuses on the most recent advances in understanding of the molecular and structural aspects of kinetochore–microtubule interaction based on the studies in yeast and vertebrate cells.

scholarly journals An unbiased, quantitative and versatile method for determining misaligned and lagging chromosome during mitosis

2020 ◽  
Author(s):  
Luciano Gama Braga ◽  
Diogjena Katerina Prifti ◽  
Chantal Garand ◽  
Pawan Kumar Saini ◽  
Sabine Elowe
Keyword(s):  
Chromosome Segregation ◽  
Hallmarks Of Cancer ◽  
Analytical Geometry ◽  
Daughter Cells ◽  
Chromosome Missegregation ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Lagging Chromosome

ABSTRACTAccurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore-microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Therefore, tools to sensitively and quantitatively measure chromosome alignment at metaphase will facilitate understanding of how changes in the composition and regulation of the microtubule attachment machinery impinge on this process. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semi-automated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive, unbiased, and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.

scholarly journals Tetraploidy causes chromosomal instability in acentriolar mouse embryos

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lia Mara Gomes Paim ◽  
Greg FitzHarris
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Chromosomal Instability ◽  
Current Model ◽  
Cell Stage ◽  
Multipolar Spindle ◽  
Genome Doubling ◽  
Microtubule Attachment ◽  
Multipolar Spindles ◽  
Bipolar Spindles

Abstract Tetraploidisation is considered a common event in the evolution of chromosomal instability (CIN) in cancer cells. The current model for how tetraploidy drives CIN in mammalian cells is that a doubling of the number of centrioles that accompany the genome doubling event leads to multipolar spindle formation and chromosome segregation errors. By exploiting the unusual scenario of mouse blastomeres, which lack centrioles until the ~64-cell stage, we show that tetraploidy can drive CIN by an entirely distinct mechanism. Tetraploid blastomeres assemble bipolar spindles dictated by microtubule organising centres, and multipolar spindles are rare. Rather, kinetochore-microtubule turnover is altered, leading to microtubule attachment defects and anaphase chromosome segregation errors. The resulting blastomeres become chromosomally unstable and exhibit a dramatic increase in whole chromosome aneuploidies. Our results thus reveal an unexpected mechanism by which tetraploidy drives CIN, in which the acquisition of chromosomally-unstable microtubule dynamics contributes to chromosome segregation errors following tetraploidisation.

scholarly journals DUSP7 Regulates the Activity of ERK2 to Promote Proper Chromosome Alignment During Cell Division

2020 ◽  
Author(s):  
Xiao Guo ◽  
Yenni A. Garcia ◽  
Ivan Ramirez ◽  
Erick F. Velasquez ◽  
Lucy W. Gao ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Human Cell ◽  
Mitotic Chromosome ◽  
Marked Decrease ◽  
Metaphase Plate ◽  
Chromosome Congression ◽  
Dual Specificity ◽  
Chromosome Alignment ◽  
Chemical Inhibition

SUMMARYHuman cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. Here we show that the dual specificity phosphatase DUSP7 is important for regulating chromosome alignment. DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytic dead mutants, led to a marked decrease in phopho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, chemical inhibition of the MEK kinase that phosphorylates ERK2 or ERK2 itself led to chromosome alignment defects. Our results support a model where MEK phosphorylation and DUSP7 dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.

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