Live cell imaging of micronucleus formation and development

Yasui, Masayoshi; Naoki, Katsuhiko; Koizumi, Tomoko; Senda-Murata, Kaori; Takashima, Yoshio; Hayashi, Makoto; Sugimoto, Kenji; Honma, Masamitsu

doi:10.1016/j.mrfmmm.2010.07.009

Cited by 33 publications

(17 citation statements)

References 30 publications

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“…Missegregation of chromosomes at anaphase can lead to formation of micronuclei 32,42 while this missegregation combined with cytoskeletal changes will contribute to cytokinesis failure. 15 The resulting micronucleated and binucleated cells are prone to cell-cycle arrest, 43,44 apoptosis, 45,46 and chromosomal instability. 32,42,[47][48][49] Therefore, the key features of XLN (slow proliferation, increased apoptosis and chromosomal instability) can all be explained by a nonspecific mechanical disruption of cell division because of excess cytoplasmic F-actin polymerization.…”

Section: Discussionmentioning

confidence: 99%

Excess F-actin mechanically impedes mitosis leading to cytokinesis failure in X-linked neutropenia by exceeding Aurora B kinase error correction capacity

Moulding

Moeendarbary

Valon

et al. 2012

Blood

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The constitutively active mutant of the Wiskott-Aldrich Syndrome protein (CA-WASp) is the cause of X-linked neutropenia and is linked with genomic instability and myelodysplasia. CA-WASp generates abnormally high levels of cytoplasmic F-actin through dysregulated activation of the Arp2/3 complex leading to defects in cell division. As WASp has no reported role in cell division, we hypothesized that alteration of cell mechanics because of increased F-actin may indirectly disrupt dynamic events during mitosis. Inhibition of the Arp2/3 complex revealed that excess cytoplasmic F-actin caused increased cellular viscosity, slowed all phases of mitosis, and perturbed mitotic mechanics. Comparison of chromosome velocity to the cytoplasmic viscosity revealed that cells compensated for increased viscosity by up-regulating force applied to chromosomes and increased the density of microtubules at kinetochores. Mitotic abnormalities were because of overload of the aurora signaling pathway as subcritical inhibition of Aurora in CA-WASp cells caused increased cytokinesis failure, while overexpression reduced defects. These findings demonstrate that changes in cell mechanics can cause significant mitotic abnormalities leading to genomic instability, and highlight the importance of mechanical sensors such as Aurora B in maintaining the fidelity of hematopoietic cell division.

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Section: Discussionmentioning

confidence: 99%

Excess F-actin mechanically impedes mitosis leading to cytokinesis failure in X-linked neutropenia by exceeding Aurora B kinase error correction capacity

Moulding

Moeendarbary

Valon

et al. 2012

Blood

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“…B, Relative fluorescence efficiencies of the Venus variants. Plasmid pmCherry 15) encoding the improved red fluorescent protein was used as internal control for protein expression. C3H10T1/2 cells were transfected with Venus and pmCherry plasmids at a molar ratio of 3:1.…”

mentioning

confidence: 99%

Improvement of a Venus-Based Bimolecular Fluorescence Complementation Assay to Visualize bFos-bJun Interaction in Living Cells

Nakagawa

Inahata

Nishimura

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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Bimolecular fluorescence complementation (BiFC) assay makes it possible to visualize protein-protein interactions in living cells. In this assay, Venus, a brightyellow variant of green fluorescent protein, is known to produce fluorescent backgrounds due to non-specific interactions. In this study we found that the V150A mutation increased by 8.6-fold the signal-to-noise ratio in the BiFC assay of bFos-bJun interaction.

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“…This was proven by supplementing the micronucleated cells with inhibitors of caspase-8 and caspase-9: the presence of the inhibitors increased the number of cells with MN, confi rming that micronucleated cells can undergo apoptosis [ 46 ]. Moreover, in HeLa cells an altered gene expression in MN was associated to the ability of micronucleated cells to die via programmed cell death [ 37 ], and MN-bearing cells were shown to suffer from apoptosis more frequently than cells without MN [ 47 ].…”

Section: Post-mitotic Fate Of Micronucleimentioning

confidence: 88%

“…To date, the MN assay has been validated for some cell lines such as the rodent-derived ones V79 [ 47 ], CHL/ IU [ 48 ], CHO and L5178Y [ 49 ], but also other cell lines like TK6 [ 50 ], HepG2 [ 51 ], A549 [ 52 ] and Syrian Hamster Embryo (SHE) cells [ 53 ] are reported in the literature.…”

Section: Post-mitotic Fate Of Micronucleimentioning

confidence: 99%

The In Vitro Micronucleus Assay and FISH Analysis

Migliore

Bucchianico

Uboldi

2014

Genotoxicity and DNA Repair

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The cytokinesis-block micronucleus cytome (CBMN-cyt) assay was originally established as an ideal system for evaluating chromosomal damage in terms of micronuclei formation. Throughout the years, the micronucleus assay evolved in a comprehensive system for assessing cytogenetic damage, cytostasis and cytotoxicity. The CBMN-cyt assay in peripheral blood lymphocytes and in other cultured mammalian cells is the most common approach to evaluate chromosomal damage induced by environmental agents, including emerging compounds as nanomaterials, and it is the most frequent test system in biomonitoring human populations.When coupled with fl uorescence in situ hybridization (FISH), CBMN-cyt assay is able to reveal the capability to induce structural chromosome aberrations (clastogenic activity) and/or numerical chromosome changes (aneuploidogenic activity).The methods for CBMN-cyt assay and FISH described here refer to the use of separate lymphocytes and whole blood cultures involving the block of cytokinesis with cytochalasin B (cyt-B) but other cell systems of different origin can be successfully used. This chapter describes in details well-established protocols for sample processing, slide preparation and scoring criteria.

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Live cell imaging of micronucleus formation and development

Cited by 33 publications

References 30 publications

Excess F-actin mechanically impedes mitosis leading to cytokinesis failure in X-linked neutropenia by exceeding Aurora B kinase error correction capacity

Excess F-actin mechanically impedes mitosis leading to cytokinesis failure in X-linked neutropenia by exceeding Aurora B kinase error correction capacity

Improvement of a Venus-Based Bimolecular Fluorescence Complementation Assay to Visualize bFos-bJun Interaction in Living Cells

The In Vitro Micronucleus Assay and FISH Analysis

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