bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2023‒06‒25
eighteen papers selected by
Valentina Piano
Uniklinik Köln
  1. Interdependence of a microtubule polymerase and a motor protein in establishment of kinetochore end-on attachments.
  2. Distinct effects of heat shock temperatures on mitotic progression by influencing the spindle assembly checkpoint.
  3. Conserved and divergent mechanisms of inner kinetochore assembly onto centromeric chromatin.
  4. A fine balance among key biophysical factors is required for recovery of bipolar mitotic spindle from monopolar and multipolar abnormalities.
  5. Oncogenic Ras deregulates cell-substrate interactions during mitotic rounding and respreading to alter cell division orientation.
  6. Identification of CDC20 in various forms of acute and chronic kidney injury in mice.
  7. NADPH increase in mitosis in aneuploid tumour cells protects against genomic aberrations.
  8. A mitotic NADPH upsurge promotes chromosome segregation and tumour progression in aneuploid cancer cells.
  9. Centromere Innovations Within a Mouse Species.
  10. Chromosome Conformation Capture of Mitotic Chromosomes.
  11. Biallelic variants in MAD2L1BP (p31comet) cause female infertility characterized by oocyte maturation arrest.
  12. Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes in Xenopus laevis egg extract.
  13. Budding uninhibited by benzimidazoles 1 promotes cell proliferation, invasion, and epithelial-mesenchymal transition via the Wnt/β-catenin signaling in glioblastoma.
  14. Nitrous Oxide-Induced Metaphase Arrest: A Technique for Somatic Chromosome Analysis.
  15. Multi-Omic Analysis of CIC's Functional Networks Reveals Novel Interaction Partners and a Potential Role in Mitotic Fidelity.
  16. Preparation of Mitotic Chromosomes with the Dropping Technique.
  17. Whole-Genome Doubling as a source of cancer: how, when, where, and why?
  18. Preparation of Mitotic Chromosomes with the Squash Technique.
  1. bioRxiv. 2023 Jun 08. pii: 2023.06.08.544255. [Epub ahead of print]
    Interdependence of a microtubule polymerase and a motor protein in establishment of kinetochore end-on attachments.
    Julia R Torvi, Jonathan Wong, David G Drubin, Georjana Barnes.
      Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast, Saccharomyces cerevisiae . Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.
    DOI:  https://doi.org/10.1101/2023.06.08.544255
  2. Exp Cell Res. 2023 Jun 18. pii: S0014-4827(23)00219-7. [Epub ahead of print]429(2): 113672
    Distinct effects of heat shock temperatures on mitotic progression by influencing the spindle assembly checkpoint.
    Saki Ota, Yui Tanaka, Ryuji Yasutake, Yuki Ikeda, Ryuzaburo Yuki, Yuji Nakayama, Youhei Saito.
      Heat shock is a physiological and environmental stress that leads to the denaturation and inactivation of cellular proteins and is used in hyperthermia cancer therapy. Previously, we revealed that mild heat shock (42 °C) delays the mitotic progression by activating the spindle assembly checkpoint (SAC). However, it is unclear whether SAC activation is maintained at higher temperatures than 42 °C. Here, we demonstrated that a high temperature of 44 °C just before mitotic entry led to a prolonged mitotic delay in the early phase, which was shortened by the SAC inhibitor, AZ3146, indicating SAC activation. Interestingly, mitotic slippage was observed at 44 °C after a prolonged delay but not at 42 °C heat shock. Furthermore, the multinuclear cells were generated by mitotic slippage in 44 °C-treated cells. Immunofluorescence analysis revealed that heat shock at 44 °C reduces the kinetochore localization of MAD2, which is essential for mitotic checkpoint activation, in nocodazole-arrested mitotic cells. These results indicate that 44 °C heat shock causes SAC inactivation even after full activation of SAC and suggest that decreased localization of MAD2 at the kinetochore is involved in heat shock-induced mitotic slippage, resulting in multinucleation. Since mitotic slippage causes drug resistance and chromosomal instability, we propose that there may be a risk of cancer malignancy when the cells are exposed to high temperatures.
    Keywords:  CDC20; Heat shock; MAD2; Mitotic progression; Spindle assembly checkpoint
    DOI:  https://doi.org/10.1016/j.yexcr.2023.113672
  3. Curr Opin Struct Biol. 2023 Jun 19. pii: S0959-440X(23)00112-4. [Epub ahead of print]81 102638
    Conserved and divergent mechanisms of inner kinetochore assembly onto centromeric chromatin.
    Stanislau Yatskevich, David Barford, Kyle W Muir.
      Kinetochores are large protein complexes built on centromeric chromatin that mediate chromosome segregation. The inner kinetochore, or constitutive centromere-associated network (CCAN), assembles onto centromeres defined by centromere protein A (CENP-A) nucleosomes (CENP-ANuc), and acts as a platform for the regulated assembly of the microtubule-binding outer kinetochore. Recent cryo-EM work revealed structural conservation of CCAN, from the repeating human regional centromeres to the point centromere of budding yeast. Centromere recognition is determined mainly through engagement of duplex DNA proximal to the CENP-A nucleosome by a DNA-binding CENP-LN channel located at the core of CCAN. Additional DNA interactions formed by other CCAN modules create an enclosed DNA-binding chamber. This configuration explains how kinetochores maintain their tight grip on centromeric DNA to withstand the forces of chromosome segregation. Defining the higher-order architecture of complete kinetochore assemblies with implications for understanding the 3D organisation of regional centromeres and mechanisms of kinetochore dynamics, including how kinetochores sense and respond to tension, are important future directions.
    Keywords:  CCAN; CENP-A nucleosome; Centromere; Kinetochore; α-satellite repeats
    DOI:  https://doi.org/10.1016/j.sbi.2023.102638
  4. Mol Biol Cell. 2023 Jun 21. mbcE22100485
    A fine balance among key biophysical factors is required for recovery of bipolar mitotic spindle from monopolar and multipolar abnormalities.
    Xiaochu Li, Mathew Bloomfield, Alexandra Bridgeland, Daniela Cimini, Jing Chen.
      During mitosis, equal partitioning of chromosomes into two daughter cells requires assembly of a bipolar mitotic spindle. Since the spindle poles are each organized by a centrosome in animal cells, centrosome defects can lead to abnormal, monopolar, or multipolar spindles. However, the cell can effectively recover the bipolar spindle by separating the centrosomes in monopolar spindles and clustering them in multipolar spindles. To interrogate how a cell can separate and cluster centrosomes as needed to form a bipolar spindle, we developed a biophysical model, based on experimental data, that uses effective potential energies to describe key mechanical forces driving centrosome movements during spindle assembly. Our model identified general biophysical factors crucial for robust bipolarization of spindles that start monopolar or multipolar. These factors include appropriate force fluctuation between centrosomes, balance between repulsive and attractive forces between centrosomes, exclusion of the centrosomes from the cell center, proper cell size and geometry, and a limited centrosome number. Consistently, we found experimentally that bipolar centrosome clustering is promoted as mitotic cell aspect ratio and volume decrease in tetraploid cancer cells. Our model provides mechanistic explanations for many more experimental phenomena and a useful theoretical framework for future studies of spindle assembly. [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E22-10-0485
  5. Curr Biol. 2023 Jun 13. pii: S0960-9822(23)00723-6. [Epub ahead of print]
    Oncogenic Ras deregulates cell-substrate interactions during mitotic rounding and respreading to alter cell division orientation.
    Sushila Ganguli, Tom Wyatt, Agata Nyga, Rachel H Lawson, Tim Meyer, Buzz Baum, Helen K Matthews.
      Oncogenic Ras has been shown to change the way cancer cells divide by increasing the forces generated during mitotic rounding. In this way, RasV12 enables cancer cells to divide across a wider range of mechanical environments than normal cells. Here, we identify a further role for oncogenic Ras-ERK signaling in division by showing that RasV12 expression alters the shape, division orientation, and respreading dynamics of cells as they exit mitosis. Many of these effects appear to result from the impact of RasV12 signaling on actomyosin contractility, because RasV12 induces the severing of retraction fibers that normally guide spindle positioning and provide a memory of the interphase cell shape. In support of this idea, the RasV12 phenotype is reversed by inhibition of actomyosin contractility and can be mimicked by the loss of cell-substrate adhesion during mitosis. Finally, we show that RasV12 activation also perturbs division orientation in cells cultured in 2D epithelial monolayers and 3D spheroids. Thus, the induction of oncogenic Ras-ERK signaling leads to rapid changes in division orientation that, along with the effects of RasV12 on cell growth and cell-cycle progression, are likely to disrupt epithelial tissue organization and contribute to cancer dissemination.
    Keywords:  MAPK signaling; actin cytoskeleton; cell division; division orientation; focal adhesions; mitosis; mitotic rounding; mitotic spindle
    DOI:  https://doi.org/10.1016/j.cub.2023.05.061
  6. Am J Physiol Renal Physiol. 2023 Jun 22.
    Identification of CDC20 in various forms of acute and chronic kidney injury in mice.
    Mallory Swanson, Jiyoung Yun, Daniel M Collier, Connor Seif, Chao-Yie Yang, Kevin R Regner, Frank Park.
      Tubular epithelial cell fate following exposure to various types of injurious stimuli can be decided at distinct cell cycle checkpoints. One such checkpoint occurs during mitosis, known as the spindle assembly checkpoint (SAC), and is tightly regulated through the actions of cell division cycle protein 20 (CDC20). Due to our paucity of knowledge about the role of CDC20 in the kidney, the present study was designed to investigate the expression levels and distribution of CDC20 within the kidney, and how pharmacological inhibition on CDC20 function affects kidney recovery using various rodent models of kidney injury. CDC20 is normally detected in the distal tubules, but upon injury by either cisplatin administration or ureter obstruction, CDC20 accumulation is considerably elevated. Blockade of CDC20 activity using a selective pharmacological inhibitor, Apcin, lowered serum creatinine, tubular damage and DNA injury following AKI, compared to vehicle-treated mice. In UUO, Apcin reduced tissue KIM-1 levels, Sirius red staining, and tubulointerstitial α-SMA staining in the tissue. The findings in the present study demonstrated that elevations in CDC20 levels in the kidney is associated with kidney injury, and that inhibition of CDC20 can alleviate and reverse some of the pathological effects on kidney architecture and function.
    Keywords:  acute kidney injury; cisplatin; fibrosis; mitotic checkpoint; tubular epithelial cell
    DOI:  https://doi.org/10.1152/ajprenal.00302.2022
  7. Nat Metab. 2023 Jun 22.
    NADPH increase in mitosis in aneuploid tumour cells protects against genomic aberrations.
      
    DOI:  https://doi.org/10.1038/s42255-023-00848-1
  8. Nat Metab. 2023 Jun 22.
    A mitotic NADPH upsurge promotes chromosome segregation and tumour progression in aneuploid cancer cells.
    Aoxing Cheng, Tian Xu, Weiyi You, Ting Wang, Dongming Zhang, Huimin Guo, Haiyan Zhang, Xin Pan, Yucai Wang, Liu Liu, Kaiguang Zhang, Jue Shi, Xuebiao Yao, Jing Guo, Zhenye Yang.
      Redox metabolites have been observed to fluctuate through the cell cycle in cancer cells, but the functional impacts of such metabolic oscillations remain unknown. Here, we uncover a mitosis-specific nicotinamide adenine dinucleotide phosphate (NADPH) upsurge that is essential for tumour progression. Specifically, NADPH is produced by glucose 6-phosphate dehydrogenase (G6PD) upon mitotic entry, which neutralizes elevated reactive oxygen species (ROS) and prevents ROS-mediated inactivation of mitotic kinases and chromosome missegregation. Mitotic activation of G6PD depends on the phosphorylation of its co-chaperone protein BAG3 at threonine 285, which results in dissociation of inhibitory BAG3. Blocking BAG3T285 phosphorylation induces tumour suppression. A mitotic NADPH upsurge is present in aneuploid cancer cells with high levels of ROS, while nearly unobservable in near-diploid cancer cells. High BAG3T285 phosphorylation is associated with worse prognosis in a cohort of patients with microsatellite-stable colorectal cancer. Our study reveals that aneuploid cancer cells with high levels of ROS depend on a G6PD-mediated NADPH upsurge in mitosis to protect them from ROS-induced chromosome missegregation.
    DOI:  https://doi.org/10.1038/s42255-023-00832-9
  9. bioRxiv. 2023 May 13. pii: 2023.05.11.540353. [Epub ahead of print]
    Centromere Innovations Within a Mouse Species.
    Craig W Gambogi, Nootan Pandey, Jennine M Dawicki-McKenna, Uma P Arora, Mikhail A Liskovykh, Jun Ma, Piero Lamelza, Vladimir Larionov, Michael A Lampson, Glennis A Logsdon, Beth L Dumont, Ben E Black.
      Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying CENP-A nucleosomes at the nexus of a satellite repeat that we identified and term π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ∼6 Mbp of a homogenized π-sat-related repeat, π-sat B , that contains >20,000 functional CENP-B boxes. There, CENP-B abundance drives accumulation of microtubule-binding components of the kinetochore, as well as a microtubule-destabilizing kinesin of the inner centromere. The balance of pro and anti-microtubule-binding by the new centromere permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.Teaser: Chromatin and kinetochore alterations arise in response to evolutionarily rapid changes to underlying repetitive centromere DNA.
    DOI:  https://doi.org/10.1101/2023.05.11.540353
  10. Methods Mol Biol. 2023 ;2672 485-500
    Chromosome Conformation Capture of Mitotic Chromosomes.
    Petr Cápal.
      Despite more than a century of intensive study of mitotic chromosomes, their three-dimensional organization remains enigmatic. The last decade established Hi-C as a method of choice for study of spatial genome-wide interactions. Although its utilization has been focused mainly on studying genomic interactions in interphase nuclei, the method can be also successfully applied to study 3D architecture and genome folding in mitotic chromosomes. However, obtaining sufficient number of mitotic chromosomes as an input material and effective coupling with Hi-C method is challenging in plant species. An elegant way to overcome hindrances with obtaining a pure mitotic chromosome fraction is their isolation via flow cytometric sorting. This chapter presents a protocol describing plant sample preparation for chromosome conformation studies, for flow-sorting of plant mitotic metaphase chromosomes and for the Hi-C procedure.
    Keywords:  3D architecture; Chromatin interaction; Flow sorting; Hi-C; Plant chromosomes
    DOI:  https://doi.org/10.1007/978-1-0716-3226-0_29
  11. Elife. 2023 Jun 19. pii: e85649. [Epub ahead of print]12
    Biallelic variants in MAD2L1BP (p31comet) cause female infertility characterized by oocyte maturation arrest.
    Lingli Huang, Wenqing Li, Xingxing Dai, Shuai Zhao, Bo Xu, Fengsong Wang, Ren-Tao Jin, Lihua Luo, Liming Wu, Xue Jiang, Yu Cheng, Jiaqi Zou, Caoling Xu, Xianhong Tong, Heng-Yu Fan, Han Zhao, Jianqiang Bao.
      Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human disease remain largely unknown. The spindle assembly checkpoint (SAC) is an intricate surveillance mechanism that ensures accurate segregation of chromosomes throughout cell cycles. Once the kinetochores of chromosomes are correctly attached to bipolar spindles and the SAC is satisfied, the MAD2L1BP, best known as p31comet, binds MAD2 and recruits the AAA+-ATPase TRIP13 to disassemble the mitotic checkpoint complex (MCC), leading to the cell cycle progression. In this study, by whole-exome sequencing (WES), we identified homozygous and compound heterozygous MAD2L1BP variants in three families with female patients diagnosed with primary infertility owing to oocyte metaphase I (MI) arrest. Functional studies revealed that the protein variants resulting from the C-terminal truncation of MAD2L1BP lost their binding ability to MAD2. cRNA microinjection of full-length or truncated MAD2L1BP uncovered their discordant roles in driving the extrusion of polar body 1 (PB1) in mouse oocytes. Furthermore, the patient's oocytes carrying the mutated MAD2L1BP variants resumed polar body extrusion (PBE) when rescued by microinjection of full-length MAD2L1BP cRNAs. Together, our studies identified and characterized novel biallelic variants in MAD2L1BP responsible for human oocyte maturation arrest at MI, and thus prompted new therapeutic avenues for curing female primary infertility.
    Keywords:  developmental biology; genetics; genomics; human; mouse
    DOI:  https://doi.org/10.7554/eLife.85649
  12. Nat Commun. 2023 Jun 21. 14(1): 3696
    Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes in Xenopus laevis egg extract.
    Bernardo Gouveia, Sagar U Setru, Matthew R King, Aaron Hamlin, Howard A Stone, Joshua W Shaevitz, Sabine Petry.
      Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstitute microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and find that chromosomes alone can form spindles. We visualize microtubule nucleation near chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. By inhibiting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient that spatially biases branching microtbule nucleation. In the presence of motors, these branched networks are ultimately organized into functional spindles, where the number of emergent spindle poles scales with the number of chromosomes and total chromatin area.
    DOI:  https://doi.org/10.1038/s41467-023-39041-z
  13. Heliyon. 2023 Jun;9(6): e16996
    Budding uninhibited by benzimidazoles 1 promotes cell proliferation, invasion, and epithelial-mesenchymal transition via the Wnt/β-catenin signaling in glioblastoma.
    Jinshi Zhang, Wenjin Wei, Qinglin Zhong, Kaiming Feng, Ruijin Yang, Qiuhua Jiang.
      The pathogenesis and progression of GBM (glioblastoma), as one of the most frequently occurring malignancies of the central nervous system, are regulated by several genes. BUB1 (budding uninhibited by benzimidazoles 1) is a mitotic checkpoint that plays an important role in chromosome segregation as well as in various tumors. However, its role in glioma is unknown. The current study discovered prominently elevated BUB1 in glioma and a significant relationship between BUB1 expression, a high World Health Organization grade, and a poor prognosis in glioma patients. Moreover, BUB1 triggered EMT (epithelial-mesenchymal transition) apart from promoting glioma cell proliferation, migration, and infiltration. Besides, BUB1 promoted EMT by activating the Wnt/β-catenin axis. As implied by our study, BUB1 probably has the potential as a target for GBM management.
    Keywords:  BUB1; Cell proliferation; EMT; Invasion; Wnt/β-catenin signaling
    DOI:  https://doi.org/10.1016/j.heliyon.2023.e16996
  14. Methods Mol Biol. 2023 ;2672 129-139
    Nitrous Oxide-Induced Metaphase Arrest: A Technique for Somatic Chromosome Analysis.
    Patrice S Albert, James A Birchler.
      Procedures to arrest metaphase chromosomes are used for determining chromosome numbers, chromosomal aberrations, and natural chromosome variation, as well as chromosome sorting. Here is described a technique of nitrous oxide gas treatment of freshly harvested root tips that is highly effective at producing an excellent mitotic index together with well-spread chromosomes. The details of the treatment and equipment used are provided. The metaphase spreads can be used directly for determining chromosome numbers or for in situ hybridization to reveal chromosomal features.
    Keywords:  Antimitotic agent; Metaphase accumulation; Metaphase arrest; Mitotic plant chromosomes; Nitrous oxide; Plant cytogenetics
    DOI:  https://doi.org/10.1007/978-1-0716-3226-0_6
  15. Cancers (Basel). 2023 May 17. pii: 2805. [Epub ahead of print]15(10):
    Multi-Omic Analysis of CIC's Functional Networks Reveals Novel Interaction Partners and a Potential Role in Mitotic Fidelity.
    Yuka Takemon, Véronique G LeBlanc, Jungeun Song, Susanna Y Chan, Stephen Dongsoo Lee, Diane L Trinh, Shiekh Tanveer Ahmad, William R Brothers, Richard D Corbett, Alessia Gagliardi, Annie Moradian, J Gregory Cairncross, Stephen Yip, Samuel A J R Aparicio, Jennifer A Chan, Christopher S Hughes, Gregg B Morin, Sharon M Gorski, Suganthi Chittaranjan, Marco A Marra.
      CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CIC's interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, as well as novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines, predominantly at 3' and 5' untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CIC's functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CIC's potential roles in cancer. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts.
    Keywords:  CIC; cell cycle; genetic networks; mitosis; proteomic interactions; single-cell sequencing; splicing
    DOI:  https://doi.org/10.3390/cancers15102805
  16. Methods Mol Biol. 2023 ;2672 151-162
    Preparation of Mitotic Chromosomes with the Dropping Technique.
    Nicola Schmidt, Beatrice Weber, Jessica Klekar, Susan Liedtke, Sarah Breitenbach, Tony Heitkam.
      The quality of chromosome preparation influences all downstream analyses and is therefore crucial. Hence, numerous protocols exist to produce microscopic slides with mitotic chromosomes. Nevertheless, due to the high content of fibers in and around a plant cell, preparation of plant chromosomes is still far from trivial and needs to be fine-tuned for each species and tissue type. Here, we outline the "dropping method," a straightforward and efficient protocol to prepare multiple slides with uniform quality from a single chromosome preparation. In this method, nuclei are extracted and cleaned to produce a nuclei suspension. In a drop-by-drop manner, this suspension is then applied from a certain height onto the slides, causing the nuclei to rupture and the chromosomes to spread. Due to the physical forces that accompany the dropping and spreading process, this method is best suited for species with small- to medium-sized chromosomes.
    Keywords:  Chromosome preparation; Dropping technique; Karyotyping; Metaphase preparation; Microscopy; Molecular cytogenetics; Plant chromosomes
    DOI:  https://doi.org/10.1007/978-1-0716-3226-0_8
  17. Front Cell Dev Biol. 2023 ;11 1209136
    Whole-Genome Doubling as a source of cancer: how, when, where, and why?
    Natalia Sanz-Gómez, María González-Álvarez, Javier De Las Rivas, Guillermo de Cárcer.
      Chromosome instability is a well-known hallmark of cancer, leading to increased genetic plasticity of tumoral cells, which favors cancer aggressiveness, and poor prognosis. One of the main sources of chromosomal instability are events that lead to a Whole-Genome Duplication (WGD) and the subsequently generated cell polyploidy. In recent years, several studies showed that WGD occurs at the early stages of cell transformation, which allows cells to later become aneuploid, thus leading to cancer progression. On the other hand, other studies convey that polyploidy plays a tumor suppressor role, by inducing cell cycle arrest, cell senescence, apoptosis, and even prompting cell differentiation, depending on the tissue cell type. There is still a gap in understanding how cells that underwent WGD can overcome the deleterious effect on cell fitness and evolve to become tumoral. Some laboratories in the chromosomal instability field recently explored this paradox, finding biomarkers that modulate polyploid cells to become oncogenic. This review brings a historical view of how WGD and polyploidy impact cell fitness and cancer progression, and bring together the last studies that describe the genes helping cells to adapt to polyploidy.
    Keywords:  aneuploidy; chromosomal instability; endoreduplication; mitotic slippage; oncogene; polyploidy; whole-genome doubling
    DOI:  https://doi.org/10.3389/fcell.2023.1209136
  18. Methods Mol Biol. 2023 ;2672 141-149
    Preparation of Mitotic Chromosomes with the Squash Technique.
    Alexis J Maravilla, Marcela Rosato, Josep A Rosselló.
      Plant chromosomes are usually obtained from meristematic tissue of active root tips through the conventional squash method. Nevertheless, cytogenetic work usually implies a great effort and some modifications of standard procedures need to be evaluated. In this chapter, we describe our outline for handling chromosomes using the squash method. By using these protocols, high-quality chromosome spreads are obtained, which allow chromosome counting, building karyotypes, and assessing chromosomal landmarks, and enable genome mapping by fluorochrome banding and in situ hybridization techniques.
    Keywords:  Feulgen; Plant chromosomes; Somatic chromosomes; Squash method
    DOI:  https://doi.org/10.1007/978-1-0716-3226-0_7
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