A guide to classifying mitotic stages and mitotic defects in fixed cells

Baudoin, Nicolaas C; Cimini, Daniela

doi:10.1007/s00412-018-0660-2

Cited by 36 publications

(35 citation statements)

References 105 publications

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“…Then we measured spindle length and calculated spindle orientation along the z-axis ( Figure 3A). We found that average distances between the two spindle poles did not 9 differ significantly between WT-and S62A-expressing cells (Figures 3B and 3D), indicating that the overall structure of the spindle and the dynamics of spindle microtubule fibers were not affected (Baudoin and Cimini, 2018). Notably, in WT-expressing cells, both spindle poles were detected within the same z-section and were aligned parallel to the substratum.…”

Section: Mitotic Phosphorylation Of Importin α Controls Spindle Orienmentioning

confidence: 85%

TPX2 activation by GM130 controls astral microtubule formation and spindle orientation

Guo

Wei

Seemann

2020

Preprint

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Spindle orientation is important in multiple developmental processes as it determines cell fate and function. The correct orientation of the mitotic spindle depends on the assembly of a proper astral microtubule network. Here, we report that the spindle assembly factor TPX2 regulates the astral microtubule network. TPX2 in the spindle pole area is activated by GM130 on mitotic Golgi membranes to promote astral microtubule growth. GM130 relieves TPX2 inhibition by competing for importin α binding. During mitosis, phosphorylation of importin α at Serine 62 by Cdk1 switches its substrate preference from TPX2 to GM130, thereby enabling competitionbased activation. Importin α S62A impedes local TPX2 activation and compromises astral microtubule formation, ultimately resulting in misoriented spindles. Blocking the GM130importin α-TPX2 pathway impairs astral microtubule growth. Our results reveal a novel role for TPX2 in the organization of astral microtubules. Furthermore, we show that the substrate preference of the important mitotic modulator importin α is regulated by Cdk1 phosphorylation.

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Section: Mitotic Phosphorylation Of Importin α Controls Spindle Orienmentioning

confidence: 85%

TPX2 activation by GM130 controls astral microtubule formation and spindle orientation

Guo

Wei

Seemann

2020

Preprint

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“…One hundred mitoses per sample were counted and spindle anomalies were classified according to Baudoin and Cimini (). In order to discriminate between true monopolar spindles and bipolar spindles with poles on different focal planes (Supporting Information Fig.…”

Section: Methodsmentioning

confidence: 99%

In vitro effects of 1‐MHz ultrasound on the mitotic spindle

Udroiu

Coluzzi

Bedini

et al. 2019

Environ and Mol Mutagen

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The effects of ultrasound on the cytoskeleton, comprising microtubules, had been studied decades ago. Nonetheless, very little attention has been paid to the effects of ultrasound on the mitotic spindle, which is also formed by microtubules. In this study, we treated human fibroblasts and human cancer cells (HeLa and MCF‐7) with 1‐MHz ultrasound at low intensities (70, 140, and 300 mW/cm2). In all cell lines, 5 min after the end of sonication, we found an intensity‐dependent increase of mitotic abnormalities (including multipolar spindles). Two hours after sonication, these abnormalities were present, but at much lower frequencies. Twenty‐four hours after sonication, mitotic abnormalities were at the same level of untreated samples, suggesting a transient effect due to ultrasound. Beside abnormalities of the mitotic spindle, we also observed an increase of metaphases with nonaligned chromosomes. The mitotic index of fibroblasts and HeLa cells, two hours after sonication, showed an intensity‐dependent decrease; this was not observed in MCF‐7 cells. In agreement with this last result, ultrasound‐induced growth inhibition (which was also intensity‐dependent) was more marked in fibroblasts and HeLa cells compared to MCF‐7 cells. This work indicates that therapeutic ultrasound, even at intensities below the cavitation threshold, can affect genome integrity, showing the need to increase the knowledge of the potential risks of ultrasound to human health. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

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“…In other organisms, depletion of kinetochore components results in mitotic defects [32]. Previous studies in B. mori have shown that, consistently, depletion of outer kinetochore components also results in mitotic defects and increased numbers of aneuploid cells [33].…”

Section: Cenp-t and Other Kinetochore Components Are Required For Accmentioning

confidence: 97%

CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

Cortes-Silva

Ulmer

Kiuchi

et al. 2019

Preprint

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Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromereassociated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPRmediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a 2 conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore. IntroductionThe centromere is an essential chromosomal region which ensures equal partitioning of chromosomal DNA during cell division [1]. In all eukaryotes, faithful chromosome segregation requires each chromosome to interact accurately with microtubule fibers from the mitotic or meiotic spindle. This interaction is mediated by the kinetochore, a macromolecular protein complex that assembles on centromeric DNA [2]. The centromere-proximal inner kinetochore hosts components of the Constitutive Centromere Associated Network (CCAN), a group of up to 16 different proteins present throughout the cell cycle that create the centromere-kinetochore interface. Upon cell division, the CCAN recruits the centromere distal outer kinetochore complex, which is composed of the KMN network (Knl1, the Mis12 and the Ndc80 complex) [3]. This recruitment is enabled by two CCAN subunits, CENP-C and CENP-T, that physically interact with subunits of the Mis12 and Ndc80 complexes [4][5][6][7][8][9][10][11]. The KMN network then in turn mediates the interaction with spindle microtubules to drive chromosome segregation during cell division [12].Previous work identified the histone H3 variant CenH3 (also called CENP-A [13,14]) as a core constituent for defining the site of a functional kinetochore in most eukaryotes. This is because CenH3 forms specialized nucleosomes preferentially found at centromeres that are the target sites for kinetochore assembly [15][16][17][18]. To do so, CenH3 physically in...

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A guide to classifying mitotic stages and mitotic defects in fixed cells

Cited by 36 publications

References 105 publications

TPX2 activation by GM130 controls astral microtubule formation and spindle orientation

TPX2 activation by GM130 controls astral microtubule formation and spindle orientation

In vitro effects of 1‐MHz ultrasound on the mitotic spindle

CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

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