scholarly journals CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether

2008 ◽  
Vol 181 (3) ◽  
pp. 411-419 ◽  
Author(s):  
Yumi Kim ◽  
John E. Heuser ◽  
Clare M. Waterman ◽  
Don W. Cleveland
Keyword(s):  
Motor Activity ◽  
Single Molecule ◽  
Coiled Coil ◽  
Full Length ◽  
Direct Visualization ◽  
Chromosome Missegregation ◽  
Microtubule Attachment ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Spindle Microtubules

The mitotic kinesin centromere protein E (CENP-E) is an essential kinetochore component that directly contributes to the capture and stabilization of spindle microtubules by kinetochores. Although reduction in CENP-E leads to high rates of whole chromosome missegregation, neither its properties as a microtubule-dependent motor nor how it contributes to the dynamic linkage between kinetochores and microtubules is known. Using single-molecule assays, we demonstrate that CENP-E is a very slow, highly processive motor that maintains microtubule attachment for long periods. Direct visualization of full-length Xenopus laevis CENP-E reveals a highly flexible 230-nm coiled coil separating its kinetochore-binding and motor domains. We also show that full-length CENP-E is a slow plus end–directed motor whose activity is essential for metaphase chromosome alignment. We propose that the highly processive microtubule-dependent motor activity of CENP-E serves to power chromosome congression and provides a flexible, motile tether linking kinetochores to dynamic spindle microtubules.

scholarly journals Kinetochore–microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E

2014 ◽  
Vol 25 (15) ◽  
pp. 2272-2281 ◽  
Author(s):  
Benjamin Vitre ◽  
Nikita Gudimchuk ◽  
Ranier Borda ◽  
Yumi Kim ◽  
John E. Heuser ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Biological Role ◽  
Wild Type ◽  
Microtubule Attachment ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Type Motor ◽  
Dependent Transport

Centromere protein E (CENP-E) is a highly elongated kinesin that transports pole-proximal chromosomes during congression in prometaphase. During metaphase, it facilitates kinetochore–microtubule end-on attachment required to achieve and maintain chromosome alignment. In vitro CENP-E can walk processively along microtubule tracks and follow both growing and shrinking microtubule plus ends. Neither the CENP-E–dependent transport along microtubules nor its tip-tracking activity requires the unusually long coiled-coil stalk of CENP-E. The biological role for the CENP-E stalk has now been identified through creation of “Bonsai” CENP-E with significantly shortened stalk but wild-type motor and tail domains. We demonstrate that Bonsai CENP-E fails to bind microtubules in vitro unless a cargo is contemporaneously bound via its C-terminal tail. In contrast, both full-length and truncated CENP-E that has no stalk and tail exhibit robust motility with and without cargo binding, highlighting the importance of CENP-E stalk for its activity. Correspondingly, kinetochore attachment to microtubule ends is shown to be disrupted in cells whose CENP-E has a shortened stalk, thereby producing chromosome misalignment in metaphase and lagging chromosomes during anaphase. Together these findings establish an unexpected role of CENP-E elongated stalk in ensuring stability of kinetochore–microtubule attachments during chromosome congression and segregation.

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 Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

2019 ◽  
Author(s):  
Arnaud Rondelet ◽  
Yu-Chih Lin ◽  
Divya Singh ◽  
Arthur T. Porfetye ◽  
Harish C. Thakur ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Interaction Mechanism ◽  
Adaptor Proteins ◽  
Interaction Sites ◽  
Microtubule Attachment ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Clathrin Adaptor ◽  
Spindle Stability ◽  
First Time

SUMMARYClathrin plays an important role to ensure mitotic spindle stability and efficient chromosome alignment, independently of its well-characterized functions in vesicle trafficking. While clathrin clearly localizes to the mitotic spindle and kinetochore-fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules have remained elusive. Here we show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the mitotic spindle. Structural analyses reveal that multiple clathrin-box motifs on GTSE1 interact directly with different clathrin adaptor interaction sites on CHC, in a manner structurally analogous to that which occurs between adaptor proteins and CHC near membranes. Specific disruption of this interaction in cells releases GTSE1 from spindles and causes defects in chromosome alignment. Surprisingly, this disruption causes destabilization of astral microtubules, but not kinetochore-microtubule attachments, and the resulting chromosome alignment defect is due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. Finally, we show that GTSE1 recruited to the spindle by clathrin stabilizes microtubules and promotes chromosome congression by inhibiting the activity of the microtubule depolymerase MCAK. This work thus uncovers a novel role of clathrin to stabilize non-kinetochore-fiber microtubules to support chromosome congression. This role is carried out via clathrin adaptor-type interactions of CHC with GTSE1, defining for the first time an important repurposing of this endocytic interaction mechanism during mitosis.

scholarly journals The internal loop of fission yeast Ndc80 binds Alp7/TACC-Alp14/TOG and ensures proper chromosome attachment

2013 ◽  
Vol 24 (8) ◽  
pp. 1122-1133 ◽  
Author(s):  
Ngang Heok Tang ◽  
Hirofumi Takada ◽  
Kuo-Shun Hsu ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Critical Role ◽  
Substantial Reduction ◽  
Coiled Coil ◽  
Internal Loop ◽  
Microtubule Attachment ◽  
Chromosome Alignment ◽  
Ndc80 Complex ◽  
Chromosome Attachment ◽  
Mutant Phenotypes

The Ndc80 outer kinetochore complex plays a critical role in kinetochore–microtubule attachment, yet our understanding of the mechanism by which this complex interacts with spindle microtubules for timely and accurate chromosome segregation remains limited. Here we address this issue using an ndc80 mutant (ndc80-NH12) from fission yeast that contains a point mutation within a ubiquitous internal loop. This mutant is normal for assembly of the Ndc80 complex and bipolar spindle formation yet defective in proper end-on attachment to the spindle microtubule, with chromosome alignment defects and missegregation happening later during mitosis. We find that ndc80-NH12 exhibits impaired localization of the microtubule-associated protein complex Alp7/transforming acidic coiled coil (TACC)-Alp14/tumor-overexpressed gene (TOG) to the mitotic kinetochore. Consistently, wild-type Ndc80 binds these two proteins, whereas the Ndc80-NH12 mutant protein displays a substantial reduction of interaction. Crucially, forced targeting of Alp7–Alp14 to the outer kinetochore rescues ndc80-NH12-mutant phenotypes. The loop was previously shown to bind Dis1/TOG, by which it ensures initial chromosome capture during early mitosis. Strikingly, ndc80-NH12 is normal in Dis1 localization. Genetic results indicate that the loop recruits Dis1/TOG and Alp7/TACC-Alp14/TOG independently. Our work therefore establishes that the Ndc80 loop plays sequential roles in spindle–kinetochore attachment by connecting the Ndc80 complex to Dis1/TOG and Alp7/TACC-Alp14/TOG.

scholarly journals Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

2020 ◽  
Vol 219 (2) ◽  
Author(s):  
Arnaud Rondelet ◽  
Yu-Chih Lin ◽  
Divya Singh ◽  
Arthur T. Porfetye ◽  
Harish C. Thakur ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Interaction Mechanism ◽  
Interaction Sites ◽  
Microtubule Attachment ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Clathrin Adaptor ◽  
Spindle Stability ◽  
First Time

Clathrin ensures mitotic spindle stability and efficient chromosome alignment, independently of its vesicle trafficking function. Although clathrin localizes to the mitotic spindle and kinetochore fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules are unclear. We show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the spindle. Structural analyses reveal that these sites interact directly with clathrin-box motifs on GTSE1. Disruption of this interaction releases GTSE1 from spindles, causing defects in chromosome alignment. Surprisingly, this disruption destabilizes astral microtubules, but not kinetochore-microtubule attachments, and chromosome alignment defects are due to a failure of chromosome congression independent of kinetochore–microtubule attachment stability. GTSE1 recruited to the spindle by clathrin stabilizes microtubules by inhibiting the microtubule depolymerase MCAK. This work uncovers a novel role of clathrin adaptor-type interactions to stabilize nonkinetochore fiber microtubules to support chromosome congression, defining for the first time a repurposing of this endocytic interaction mechanism during mitosis.

scholarly journals Coordination of NDC80 and Ska complexes at the kinetochore-microtubule interface in human cells

2019 ◽  
Author(s):  
Robert Wimbish ◽  
Keith F. DeLuca ◽  
Jeanne E. Mick ◽  
Jack Himes ◽  
Ignacio J. Sánchez ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Human Cells ◽  
Tail Domain ◽  
Mitotic Chromosomes ◽  
Microtubule Attachment ◽  
Coiled Coil Domain ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Lines Of Evidence

AbstractThe conserved kinetochore-associated NDC80 complex (comprised of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including (1) connecting mitotic chromosomes to spindle microtubules to establish force-transducing kinetochore-microtubule attachments, and (2) regulating the binding strength between kinetochores and microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to microtubules, additional factors support these processes as well, including the spindle and kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind microtubules, especially to depolymerizing microtubule plus-ends, but how this is accomplished remains unclear. Using cell-based and in vitro assays, we demonstrate that the Hec1 tail domain is dispensable for Ska complex recruitment to kinetochores and for generation of kinetochore-microtubule attachments in human cells. We further demonstrate that Hec1 tail phosphorylation regulates kinetochore-microtubule attachment stability independently of the Ska complex. Finally, we map the location of the Ska complex in cells to a region near the coiled-coil domain of the NDC80 complex, and demonstrate that this region is required for Ska complex recruitment to the NDC80 complex-microtubule interface.

scholarly journals Silencing Mitosin Induces Misaligned Chromosomes, Premature Chromosome Decondensation before Anaphase Onset, and Mitotic Cell Death

2005 ◽  
Vol 25 (10) ◽  
pp. 4062-4074 ◽  
Author(s):  
Zhenye Yang ◽  
Jing Guo ◽  
Qi Chen ◽  
Chong Ding ◽  
Juan Du ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
Cytoplasmic Dynein ◽  
Dependent Manner ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
M Phase ◽  
Dying Cells ◽  
Chromosome Decondensation

ABSTRACT Mitosin (also named CENP-F) is a large human nuclear protein transiently associated with the outer kinetochore plate in M phase. Using RNA interference and fluorescence microscopy, we showed that mitosin depletion attenuated chromosome congression and led to metaphase arrest with misaligned polar chromosomes whose kinetochores showed few cold-stable microtubules. Kinetochores of fully aligned chromosomes often failed to show orientation in the direction of the spindle long axis. Moreover, tension across their sister kinetochores was decreased by 53% on average. These phenotypes collectively imply defects in motor functions in mitosin-depleted cells and are similar to those of CENP-E depletion. Consistently, the intensities of CENP-E and cytoplasmic dynein and dynactin, which are motors controlling microtubule attachment and chromosome movement, were reduced at the kinetochore in a microtubule-dependent manner. In addition, after being arrested in pseudometaphase for approximately 2 h, mitosin-depleted cells died before anaphase initiation through apoptosis. The dying cells exhibited progressive chromosome arm decondensation, while the centromeres were still associated with spindles. Mitosin is therefore essential for full chromosome alignment, possibly by promoting proper kinetochore attachments through modulating CENP-E and dynein functions. Its depletion also prematurely triggers chromosome decondensation, a process that normally occurs from telophase for the nucleus reassembly, thus resulting in apoptosis.

scholarly journals Leaving no-one behind: how CENP-E facilitates chromosome alignment

2020 ◽  
Vol 64 (2) ◽  
pp. 313-324 ◽  
Author(s):  
Benjamin Craske ◽  
Julie P.I. Welburn
Keyword(s):  
Motor Activity ◽  
Recent Work ◽  
Transport Mechanism ◽  
Genomic Stability ◽  
Mitotic Progression ◽  
Kinesin Motor ◽  
Independent Manner ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Microtubule Motor

Abstract Chromosome alignment and biorientation is essential for mitotic progression and genomic stability. Most chromosomes align at the spindle equator in a motor-independent manner. However, a subset of polar kinetochores fail to bi-orient and require a microtubule motor-based transport mechanism to move to the cell equator. Centromere Protein E (CENP-E/KIF10) is a kinesin motor from the Kinesin-7 family, which localizes to unattached kinetochores during mitosis and utilizes plus-end directed microtubule motility to slide mono-oriented chromosomes to the spindle equator. Recent work has revealed how CENP-E cooperates with chromokinesins and dynein to mediate chromosome congression and highlighted its role at aligned chromosomes. Additionally, we have gained new mechanistic insights into the targeting and regulation of CENP-E motor activity at the kinetochore. Here, we will review the function of CENP-E in chromosome congression, the pathways that contribute to CENP-E loading at the kinetochore, and how CENP-E activity is regulated during mitosis.

scholarly journals Mps1 regulates spindle morphology through MCRS1 to promote chromosome alignment

2019 ◽  
Vol 30 (9) ◽  
pp. 1060-1068 ◽  
Author(s):  
Hongdan Yang ◽  
Fengxia Zhang ◽  
Ching-Jung Huang ◽  
Jun Liao ◽  
Ying Han ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Underlying Mechanism ◽  
Genome Maintenance ◽  
Microtubule Attachment ◽  
Downstream Effectors ◽  
Chromosome Alignment ◽  
Assembly Factor ◽  
Spindle Microtubules

Accurate partitioning of chromosomes during mitosis is essential for genetic stability and requires the assembly of the dynamic mitotic spindle and proper kinetochore–microtubule attachment. The spindle assembly checkpoint (SAC) monitors the incompleteness and errors in kinetochore–microtubule attachment and delays anaphase. The SAC kinase Mps1 regulates the recruitment of downstream effectors to unattached kinetochores. Mps1 also actively promotes chromosome alignment during metaphase, but the underlying mechanism is not completely understood. Here, we show that Mps1 regulates chromosome alignment through MCRS1, a spindle assembly factor that controls the dynamics of the minus end of kinetochore microtubules. Mps1 binds and phosphorylates MCRS1. This mechanism enables KIF2A localization to the minus end of spindle microtubules. Thus, our study reveals a novel role of Mps1 in regulating the dynamics of the minus end of microtubules and expands the functions of Mps1 in genome maintenance.

scholarly journals Delayed Chromosome Alignment to the Spindle Equator Increases the Rate of Chromosome Missegregation in Cancer Cell Lines

Biomolecules ◽  
2018 ◽  
Vol 9 (1) ◽  
pp. 10 ◽  
Author(s):  
Kinue Kuniyasu ◽  
Kenji Iemura ◽  
Kozo Tanaka
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Chromosome Missegregation ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
In Cells

For appropriate chromosome segregation, kinetochores on sister chromatids have to attach to microtubules from opposite spindle poles (bi-orientation). Chromosome alignment at the spindle equator, referred to as congression, can occur through the attachment of kinetochores to the lateral surface of spindle microtubules, facilitating bi-orientation establishment. However, the contribution of this phenomenon to mitotic fidelity has not been clarified yet. Here, we addressed whether delayed chromosome alignment to the spindle equator increases the rate of chromosome missegregation. Cancer cell lines depleted of Kid, a chromokinesin involved in chromosome congression, showed chromosome alignment with a slight delay, and increased frequency of lagging chromosomes. Delayed chromosome alignment concomitant with an increased rate of lagging chromosomes was also seen in cells depleted of kinesin family member 4A (KIF4A), another chromokinesin. Cells that underwent chromosome missegregation took relatively longer time to align chromosomes in both control and Kid/KIF4A-depleted cells. Tracking of late-aligning chromosomes showed that they exhibit a higher rate of lagging chromosomes. Intriguingly, the metaphase of cells that underwent chromosome missegregation was shortened, and delaying anaphase onset ameliorated the increased chromosome missegregation. These data suggest that late-aligning chromosomes do not have sufficient time to establish bi-orientation, leading to chromosome missegregation. Our data imply that delayed chromosome alignment is not only a consequence, but also a cause of defective bi-orientation establishment, which can lead to chromosomal instability in cells without severe mitotic defects.

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