scholarly journals High-resolution imaging reveals how the spindle midzone impacts chromosome movement

2019 ◽  
Vol 218 (8) ◽  
pp. 2529-2544 ◽  
Author(s):  
Melissa C. Pamula ◽  
Lina Carlini ◽  
Scott Forth ◽  
Priyanka Verma ◽  
Subbulakshmi Suresh ◽  
...  
Keyword(s):  
High Resolution ◽  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Chromosome Movement ◽  
Daughter Cells ◽  
Filament Bundles ◽  
Spindle Midzone ◽  
Resolution Imaging ◽  
Spindle Elongation ◽  
Length Reduction

In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.

Kinesinsklp5+ andklp6+ are required for normal chromosome movement in mitosis

2002 ◽  
Vol 115 (5) ◽  
pp. 931-940 ◽  
Author(s):  
Robert R. West ◽  
Terra Malmstrom ◽  
J. Richard McIntosh
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Extended Period ◽  
Normal Chromosome ◽  
Chromosome Movement ◽  
Dynamic Interactions ◽  
Spindle Elongation ◽  
Synthetically Lethal ◽  
Anaphase B ◽  
Null Mutants

Proper mitotic chromosome segregation requires dynamic interactions between spindle microtubules and kinetochores. Here we demonstrate that two related fission yeast kinesins, klp5+ and klp6+, are required for normal chromosome segregation in mitosis. Null mutants frequently lack a normal metaphase chromosome alignment. Chromosome pairs move back and forth along the spindle for an extended period prior to sister chromatid separation, a phenotype reminiscent of the loss of CENP-E in metazoans. Ultimately, sister chromatids segregate, regardless of chromosome position along the spindle, and viable daughter cells are usually produced. The initiation of anaphase B is sometimes delayed, but the rate of spindle elongation is similar to wildtype. Despite a delay, anaphase B often begins before anaphase A is completed. The klp5Δ and klp6Δ null mutants are synthetically lethal with a deletion of the spindle assembly checkpoint gene, bub1+, several mutants in components of the anaphase promoting complex, and a cold sensitive allele of the kinetochore and microtubule-binding protein, Dis1p. Klp5p-GFP and Klp6p-GFP localize to kinetochores from prophase to the onset of anaphase A, but relocalize to the spindle midzone during anaphase B. These data indicate that Klp5p and Klp6p are kinetochore kinesins required for normal chromosome movement in prometaphase.

scholarly journals Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake

2011 ◽  
Vol 193 (2) ◽  
pp. 285-294 ◽  
Author(s):  
Daniel K. Rozelle ◽  
Scott D. Hansen ◽  
Kenneth B. Kaplan
Keyword(s):  
Chromosome Segregation ◽  
Budding Yeast ◽  
Mitotic Exit ◽  
Mitotic Exit Network ◽  
Spindle Midzone ◽  
Spindle Elongation ◽  
Spindle Stability

During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)–dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.

The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A

1996 ◽  
Vol 109 (5) ◽  
pp. 961-969 ◽  
Author(s):  
K.D. Brown ◽  
K.W. Wood ◽  
D.W. Cleveland
Keyword(s):  
Chromosome Segregation ◽  
Chromosome Movement ◽  
Initial Alignment ◽  
Late Prophase ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Elongation ◽  
Anaphase B

The kinesin-like protein CENP-E transiently associates with kinetochores following nuclear envelope breakdown in late prophase, remains bound throughout metaphase, but sometime after anaphase onset it releases and by telophase becomes bound to interzonal microtubules of the mitotic spindle. Inhibition of poleward chromosome movement in vitro by CENP-E antibodies and association of CENP-E with minus-end directed microtubule motility in vitro have combined to suggest a key role for CENP-E as an anaphase chromosome motor. For this to be plausible in vivo depends on whether CENP-E remains kinetochore associated during anaphase. Using Indian muntjac cells whose seven chromosomes have large, easily tracked kinetochores, we now show that CENP-E is kinetochore-associated throughout the entirety of anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to the interzonal microtubules. These observations support roles for CENP-E not only in the initial alignment of chromosomes at metaphase and in spindle elongation in anaphase-B, but also in poleward chromosome movement in anaphase-A.

Schizosaccharomyces pombeBir1p, a Nuclear Protein That Localizes to Kinetochores and the Spindle Midzone, Is Essential for Chromosome Condensation and Spindle Elongation During Mitosis

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 445-456 ◽  
Author(s):  
Srividya Rajagopalan ◽  
Mohan K Balasubramanian
Keyword(s):  
Chromosome Segregation ◽  
Cell Biology ◽  
Aurora Kinase ◽  
Time Lapse ◽  
Chromosome Condensation ◽  
Multiple Processes ◽  
Spindle Midzone ◽  
Spindle Elongation ◽  
Bir Domain ◽  
Mitosis And Meiosis

AbstractThe inhibitor of apoptosis (IAP) family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat (BIR) domains. Recent work has shown that some of these BIR-domain proteins play a prominent role in the regulation of cell division, in particular at the stage of chromosome segregation and cytokinesis. We and others have shown that the Schizosaccharomyces pombe BIR-domain protein, Bir1p/Pbh1p/Cut17p, is important for the regulation of mitosis. Here we further characterize S. pombe Bir1p using methods of cell biology and genetics. We show that Bir1p is dispersed throughout the nucleus during the cell cycle. In addition, a significant part of Bir1p is also detected at the kinetochores and the spindle midzone during mitosis and meiosis. Time-lapse microscopy studies suggest that Bir1p relocates from the kinetochores to the spindle at the end of anaphase A. Bir1p colocalizes with the S. pombe Aurora kinase homolog Aim1p, a protein essential for mitosis, at the kinetochores as well as the spindle midzone during mitosis, and functional Bir1p is essential for localization of Aim1p to the kinetochores and the spindle midzone. Analyses of bir1 conditional mutants revealed that Bir1p is essential for chromosome condensation during mitosis. In addition, anaphase cells show the presence of lagging chromosomes and a defect in spindle elongation. We conclude that Bir1p is important for multiple processes that occur during mitosis in S. pombe.

scholarly journals Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation

2012 ◽  
Vol 199 (2) ◽  
pp. 251-268 ◽  
Author(s):  
Fangwei Wang ◽  
Natalia P. Ulyanova ◽  
John R. Daum ◽  
Debasis Patnaik ◽  
Anna V. Kateneva ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Metaphase Chromosome ◽  
Histone H3 ◽  
Spindle Checkpoint ◽  
Aurora B ◽  
Docking Site ◽  
Chromosome Movement ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Spindle Midzone

Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore–microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B–dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling.

scholarly journals A microfluidic array for real-time live-cell imaging of human and rodent pancreatic islets

Lab on a Chip ◽  
2016 ◽  
Vol 16 (8) ◽  
pp. 1466-1472 ◽  
Author(s):  
Mohammad Nourmohammadzadeh ◽  
Yuan Xing ◽  
Jin Wuk Lee ◽  
Matthew A. Bochenek ◽  
Joshua E. Mendoza-Elias ◽  
...  
Keyword(s):  
High Resolution ◽  
Pancreatic Islets ◽  
Real Time ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
High Resolution Imaging ◽  
Resolution Imaging

In this study, we present a microfluidic array for high-resolution imaging of individual pancreatic islets.

scholarly journals The multifunctional spindle midzone in vertebrate cells at a glance

2021 ◽  
Vol 134 (10) ◽  
Author(s):  
Patricia Wadsworth
Keyword(s):  
Chromosome Segregation ◽  
Future Research ◽  
Contractile Ring ◽  
Microtubule Associated Proteins ◽  
Ring Assembly ◽  
Open Questions ◽  
Associated Proteins ◽  
Spindle Midzone ◽  
Spindle Elongation ◽  
And Function

ABSTRACT During anaphase, a microtubule-containing structure called the midzone forms between the segregating chromosomes. The midzone is composed of an antiparallel array of microtubules and numerous microtubule-associated proteins that contribute to midzone formation and function. In many cells, the midzone is an important source of signals that specify the location of contractile ring assembly and constriction. The midzone also contributes to the events of anaphase by generating forces that impact chromosome segregation and spindle elongation; some midzone components contribute to both processes. The results of recent experiments have increased our understanding of the importance of the midzone, a microtubule array that has often been overlooked. This Journal of Cell Science at a Glance article will review, and illustrate on the accompanying poster, the organization, formation and dynamics of the midzone, and discuss open questions for future research.

scholarly journals The structural basis for kinetochore stabilization by Cnn1/CENP-T

2020 ◽  
Author(s):  
Stephen M. Hinshaw ◽  
Stephen C. Harrison
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Feedback Regulation ◽  
Structural Basis ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
High Resolution Structure ◽  
Spindle Microtubules ◽  
Resolution Structure

ABSTRACTChromosome segregation depends on a regulated connection between spindle microtubules and centromeric DNA. The kinetochore, a massive modular protein assembly, mediates this connection and also serves as a signaling hub that integrates and responds to changing cues during the cell cycle. Kinetochore functions evolve as the cell cycle progresses, culminating in the assurance of a persistent chromosome-microtubule connection during anaphase, when sister chromatids must transit into daughter cells uninterrupted. We previously determined the structure of the Ctf19 complex, a group of kinetochore proteins at the centromeric base of the kinetochore. We now present a high-resolution structure of a Ctf19 complex sub-assembly involved in centromere-microtubule contact: the Ctf3 complex bound to the Cnn1-Wip1 heterodimer. The resulting composite model of the Ctf19 complex and live-cell imaging experiments provide a mechanism for Cnn1-Wip1 recruitment to the kinetochore. The mechanism suggests feedback regulation of Ctf19 complex assembly and unanticipated similarities in kinetochore organization between yeast and vertebrates.

Alternative approaches to ultra-high resolution imaging

1991 ◽  
Vol 49 ◽  
pp. 650-651
Author(s):  
J.M. Cowley
Keyword(s):  
High Resolution ◽  
High Voltage ◽  
High Resolution Electron Microscopy ◽  
Crystal Defects ◽  
High Resolution Imaging ◽  
General Applicability ◽  
Resolution Electron ◽  
Radiation Resistant ◽  
Resolution Imaging ◽  
Alternative Approaches

By extrapolation of past experience, it would seem that the future of ultra-high resolution electron microscopy rests with the advances of electron optical engineering that are improving the instrumental stability of high voltage microscopes to achieve the theoretical resolutions of 1Å or better at 1MeV or higher energies. While these high voltage instruments will undoubtedly produce valuable results on chosen specimens, their general applicability has been questioned on the basis of the excessive radiation damage effects which may significantly modify the detailed structures of crystal defects within even the most radiation resistant materials in a period of a few seconds. Other considerations such as those of cost and convenience of use add to the inducement to consider seriously the possibilities for alternative approaches to the achievement of comparable resolutions.

Principle and practice of high-resolution imaging with a field-emission TEM

1992 ◽  
Vol 50 (1) ◽  
pp. 138-139
Author(s):  
Max T. Otten ◽  
Wim M.J. Coene
Keyword(s):  
High Resolution ◽  
Field Emission ◽  
Incidence Angle ◽  
Temporal Coherence ◽  
Energy Spread ◽  
High Resolution Imaging ◽  
Major Improvement ◽  
High Resolution Images ◽  
Resolution Imaging

High-resolution imaging with a LaB6 instrument is limited by the spatial and temporal coherence, with little contrast remaining beyond the point resolution. A Field Emission Gun (FEG) reduces the incidence angle by a factor 5 to 10 and the energy spread by 2 to 3. Since the incidence angle is the dominant limitation for LaB6 the FEG provides a major improvement in contrast transfer, reducing the information limit to roughly one half of the point resolution. The strong improvement, predicted from high-resolution theory, can be seen readily in diffractograms (Fig. 1) and high-resolution images (Fig. 2). Even if the information in the image is limited deliberately to the point resolution by using an objective aperture, the improved contrast transfer close to the point resolution (Fig. 1) is already worthwhile.

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