A quantitative and semiautomated method for determining misaligned and lagging chromosomes during mitosis

Braga, Luciano Gama; Prifti, Diogjena Katerina; Garand, Chantal; Saini, Pawan; Elowe, Sabine

doi:10.1091/mbc.e20-09-0585

Cited by 7 publications

(5 citation statements)

References 37 publications

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“…This is particularly imprecise when using chromosome labels, since the position of bulky chromosome arms do not always represent the position of kinetochores where microtubules attach to segregate them. Recent efforts to quantify lagging chromosomes from fixed cell analyses are an important step forward 60 . However, these approaches do not use a time-series, and therefore cannot capture the temporal and spatial evolution of short-lived events.…”

Section: Discussionmentioning

confidence: 99%

Kinetochore life histories reveal the origins of chromosome mis-segregation and correction mechanisms

Sen

Harrison

Burroughs

et al. 2021

Preprint

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Chromosome mis-segregation during mitosis leads to daughter cells with deviant karyotypes (aneuploidy) and an increased mutational burden through chromothripsis of mis-segregated chromosomes. The rate of mis-segregation and the aneuploidy state are hallmarks of cancer and linked to cancer genome evolution. Errors can manifest as lagging chromosomes in anaphase, although the mechanistic origins and likelihood of correction are incompletely understood. Here we combine lattice light sheet microscopy, endogenous protein labelling and computational analysis to define the life history of >10^4 kinetochores throughout metaphase and anaphase from over 200 cells. By defining the laziness of kinetochores in anaphase, we reveal that chromosomes are at a considerable and continual risk of mis-segregation. We show that the majority of kinetochores are corrected rapidly in early anaphase through an Aurora B dependent process. Moreover, quantitative analyses of the kinetochore life histories reveal a unique dynamic signature of metaphase kinetochore oscillations that forecasts their fate in the subsequent anaphase. We propose that in diploid human cells chromosome segregation is fundamentally error prone, with a new layer of early anaphase error correction required for stable karyotype propagation.

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Section: Discussionmentioning

confidence: 99%

Kinetochore life histories reveal the origins of chromosome mis-segregation and correction mechanisms

Sen

Harrison

Burroughs

et al. 2021

Preprint

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“…To test for alignment, we treated control and TEM4 depleted cells with the Eg5 inhibitor S-Trityl-L-Cysteine (STLC) to disrupt the formation of bipolar spindles. Cells were then released into media containing the proteasome inhibitor MG132 to allow for bipolar spindle formation and alignment, and kinetochore positions relative to the metaphase plate were monitored as previously reported (Gama Braga et al, 2021). We found that cells depleted of TEM4 exhibited a significant increase in misaligned kinetochores compared to control cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Angular histograms were plotted using a custom R script (Scarpa et al, 2018). Misaligned and lagging chromosomes were quantified in fixed cells as previously described (Gama Braga et al, 2021). Briefly, spindle pole position was defined, followed by the establishment of an alignment region after equally dividing the area in between the poles of the cell into 5 zones.…”

Section: Image Quantification and Statistical Analysismentioning

confidence: 99%

ARHGEF17/TEM4 regulates the cell cycle through control of G1 progression

Prifti,

Calvo,

Lauzier

et al. 2024

Preprint

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The Ras homolog (Rho) small GTPases, via their role in regulating the actin cytoskeleton, coordinate diverse cellular functions including cell morphology, adhesion and motility, as well as cell cycle progression, survival and apoptosis. The upstream Rho regulators for many of these functions are unknown. ARHGEF17 (also known as TEM4) is a Rho family guanine nucleotide exchange factor (GEF) that been implicated in cell migration, cell-cell junction formation and the mitotic checkpoint. In this study we characterize the regulation of the cell cycle by TEM4. We demonstrate that TEM4 depleted cells exhibit multiple defects in mitotic entry and duration, spindle morphology, and spindle orientation. In addition, we find that TEM4 insufficiency leads to excessive cortical actin polymerization and cell rounding defects. Mechanistically, we demonstrate that TEM4 depleted cells delay in G1 as a consequence of elevated levels of the G1/S inhibitor p21waf1/cip1and that TEM4 depleted cells that progress through to mitosis, do so with decreased transcription ofCCNB1and thus attenuated levels of cyclin B. Importantly, cyclin B overexpression in TEM4-depleted cells largely rescues mitotic progression and chromosome segregation defects in anaphase. Our study thus illustrates the consequences of Rho signalling imbalance on cell cycle progression and identifies TEM4 as the first GEF governing Rho GTPase-mediated regulation of G1/S.

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“…To classify cells with misaligned chromosome, an automated analysis was employed as described previously ( Gama Braga et al., 2021 ). The standard/control used were the cells arrested at metaphase, and the distance between the two spindle poles in the control cells was accurately measured.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of CENP-R by Aurora B regulates kinetochore–microtubule attachment for accurate chromosome segregation

Sedzro

Yuan

Mullen

et al. 2022

Journal of Molecular Cell Biology

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Error-free mitosis depends on accurate chromosome attachment to spindle microtubules via a fine structure called the centromere that is epigenetically specified by the enrichment of CENP-A nucleosomes. Centromere maintenance during mitosis requires CENP-A-mediated deposition of constitutive centromere-associated network (CCAN) that establishes the inner kinetochore and connects centromeric chromatin to spindle microtubules during mitosis. Although previously proposed to be an adaptor of retinoic acid receptor, here, we show that CENP-R synergizes with CENP-OPQU to regulate kinetochore–microtubule attachment stability and ensure accurate chromosome segregation in mitosis. We found that a phospho-mimicking mutation of CENP-R weakened its localization to the kinetochore, suggesting that phosphorylation may regulate its localization. Perturbation of CENP-R phosphorylation is shown to prevent proper kinetochore–microtubule attachment at metaphase. Mechanistically, CENP-R phosphorylation disrupts its binding with CENP-U. Thus, we speculate that Aurora B-mediated CENP-R phosphorylation promotes the correction of improper kinetochore–microtubule attachment in mitosis. As CENP-R is absent from yeast, we reasoned that metazoan evolved an elaborate chromosome stability control machinery to ensure faithful chromosome segregation in mitosis.

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A quantitative and semiautomated method for determining misaligned and lagging chromosomes during mitosis

Cited by 7 publications

References 37 publications

Kinetochore life histories reveal the origins of chromosome mis-segregation and correction mechanisms

Kinetochore life histories reveal the origins of chromosome mis-segregation and correction mechanisms

ARHGEF17/TEM4 regulates the cell cycle through control of G1 progression

Phosphorylation of CENP-R by Aurora B regulates kinetochore–microtubule attachment for accurate chromosome segregation

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