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 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.

Nucleophosmin is required for chromosome congression, proper mitotic spindle formation, and kinetochore-microtubule attachment in HeLa cells

FEBS Letters ◽  
2008 ◽  
Vol 582 (27) ◽  
pp. 3839-3844 ◽  
Author(s):  
Mohammed Abdullahel Amin ◽  
Sachihiro Matsunaga ◽  
Susumu Uchiyama ◽  
Kiichi Fukui
Keyword(s):  
Hela Cells ◽  
Mitotic Spindle ◽  
Spindle Formation ◽  
Microtubule Attachment ◽  
Chromosome Congression

scholarly journals HURP controls spindle dynamics to promote proper interkinetochore tension and efficient kinetochore capture

2006 ◽  
Vol 173 (6) ◽  
pp. 879-891 ◽  
Author(s):  
Jim Wong ◽  
Guowei Fang
Keyword(s):  
Mitotic Spindle ◽  
Metaphase Plate ◽  
Mitotic Progression ◽  
Specific Mechanism ◽  
Microtubule Polymerization ◽  
Chromosome Congression ◽  
Spindle Dynamics ◽  
Spindle Stability

Through a functional genomic screen for mitotic regulators, we identified hepatoma up-regulated protein (HURP) as a protein that is required for chromosome congression and alignment. In HURP-depleted cells, the persistence of unaligned chromosomes and the reduction of tension across sister kinetochores on aligned chromosomes resulted in the activation of the spindle checkpoint. Although these defects transiently delayed mitotic progression, HeLa cells initiated anaphase without resolution of these deficiencies. This bypass of the checkpoint arrest provides a tumor-specific mechanism for chromosome missegregation and genomic instability. Mechanistically, HURP colocalized with the mitotic spindle in a concentration gradient increasing toward the chromosomes. HURP binds directly to microtubules in vitro and enhances their polymerization. In vivo, HURP stabilizes mitotic microtubules, promotes microtubule polymerization and bipolar spindle formation, and decreases the turnover rate of the mitotic spindle. Thus, HURP controls spindle stability and dynamics to achieve efficient kinetochore capture at prometaphase, timely chromosome congression to the metaphase plate, and proper interkinetochore tension for anaphase initiation.

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 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 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 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 Additive Manufacturing for Effective Smart Structures: The Idea of 6D Printing

2021 ◽  
Vol 5 (5) ◽  
pp. 119
Author(s):  
Stelios K. Georgantzinos ◽  
Georgios I. Giannopoulos ◽  
Panteleimon A. Bakalis
Keyword(s):  
Additive Manufacturing ◽  
Degrees Of Freedom ◽  
Smart Structures ◽  
Interaction Mechanism ◽  
Modeling And Simulations ◽  
Environmental Stimulus ◽  
Material Component ◽  
Design And Manufacturing ◽  
First Time ◽  
Printing Techniques

This paper aims to establish six-dimensional (6D) printing as a new branch of additive manufacturing investigating its benefits, advantages as well as possible limitations concerning the design and manufacturing of effective smart structures. The concept of 6D printing, to the authors’ best knowledge, is introduced for the first time. The new method combines the four-dimensional (4D) and five-dimensional (5D) printing techniques. This means that the printing process is going to use five degrees of freedom for creating the final object while the final produced material component will be a smart/intelligent one (i.e., will be capable of changing its shape or properties due to its interaction with an environmental stimulus). A 6D printed structure can be stronger and more effective than a corresponding 4D printed structure, can be manufactured using less material, can perform movements by being exposed to an external stimulus through an interaction mechanism, and it may learn how to reconfigure itself suitably, based on predictions via mathematical modeling and simulations.

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.

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