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Methods in Molecular Biology (Clifton,... 2017Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle...
Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle regulatory mechanisms and cell cycle progression invariably require synchronization of cell populations at specific cell cycle stages. Several methods have been established to synchronize cells, including serum deprivation, contact inhibition, centrifugal elutriation, and drug-dependent synchronization. Despite potential adverse cellular consequences of synchronizing cells by pharmacological agents, drug-dependent methods can be advantageous when studying later cell cycle events to ensure specific enrichment at selected mitotic stages. This chapter describes protocols used in our laboratory for isolating mitotic mammalian cells in a large-scale manner. In particular, we discuss the technical aspects of adherent or suspension cell isolation, the methods necessary to enrich cells at different mitotic stages and the optimized culture conditions.
Topics: Anaphase; Cell Culture Techniques; Cell Cycle; Fluorescent Antibody Technique; HeLa Cells; Humans; Metaphase; Mitosis; Prometaphase; Telophase
PubMed: 27815896
DOI: 10.1007/978-1-4939-6603-5_4 -
The EMBO Journal Mar 2024The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor...
The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor protein KIF18A therefore makes it an attractive therapeutic target. Not all cancers require KIF18A, however, and the determinants underlying this distinction remain unclear. Here, we show that KIF18A inhibition drives a modest and widespread increase in spindle assembly checkpoint (SAC) signaling from kinetochores which can result in lethal mitotic delays. Whether cells arrest in mitosis depends on the robustness of the metaphase-to-anaphase transition, and cells predisposed with weak basal anaphase-promoting complex/cyclosome (APC/C) activity and/or persistent SAC signaling through metaphase are uniquely sensitive to KIF18A inhibition. KIF18A-dependent cancer cells exhibit hallmarks of this SAC:APC/C imbalance, including a long metaphase-to-anaphase transition, and slow mitosis overall. Together, our data reveal vulnerabilities in the cell division apparatus of cancer cells that can be exploited for therapeutic benefit.
Topics: Humans; Anaphase-Promoting Complex-Cyclosome; Dyneins; Kinesins; Kinetochores; Mitosis; Neoplasms
PubMed: 38279026
DOI: 10.1038/s44318-024-00031-6 -
Methods in Molecular Biology (Clifton,... 2022The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate,...
The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.
Topics: Anaphase; HeLa Cells; Humans; Metaphase; Mitosis; Telophase
PubMed: 36045201
DOI: 10.1007/978-1-0716-2736-5_8 -
Genes Jan 2022Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of...
Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of has not been achieved using oligonucleotide sequences. Here, the chromosomes of five taxa in the mitotic metaphase and mitotic metaphase to anaphase were detected using the oligo-FISH probes (AGT), 5S rDNA, and (TTG). In total, 24 small chromosomes were clearly observed in the mitotic metaphase (0.89-3.03 μm), whereas 24-48 small chromosomes were observed in the mitotic metaphase to anaphase (0.94-3.10 μm). The signal number and intensity of (AGT), 5S rDNA, and (TTG) in the mitotic metaphase to anaphase chromosomes were nearly consistent with those in the mitotic metaphase chromosomes when the two split chromosomes were integrated as one unit. Of note, 14 chromosomes (there is a high chance that sex chromosomes are included) were exclusively identified by (AGT), 5S rDNA, and (TTG). The other 10 also showed a terminal signal with (AGT). Moreover, these oligo-probes were able to distinguish one wild taxon from four taxa. These chromosome identification and taxa differentiation data will help in elucidating visual and elaborate physical mapping and guide breeders' utilization of wild resources of .
Topics: Chromosomes, Plant; DNA, Ribosomal; Hippophae; In Situ Hybridization, Fluorescence; RNA, Ribosomal, 5S
PubMed: 35205242
DOI: 10.3390/genes13020195 -
RNA (New York, N.Y.) Dec 2020Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This...
Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This regulation involves dynamic reprogramming of gene expression at multiple transcriptional and posttranscriptional levels. In transcriptionally silent oocytes, the CPEB-family of RNAbinding proteins coordinates temporal and spatial translation regulation of stored maternal mRNAs to drive meiotic progression. CPEB1 mediates mRNA localization to the meiotic spindle, which is required to ensure proper chromosome segregation. Temporal translational regulation also takes place in mitosis, where a large repertoire of transcripts are activated or repressed in specific cell cycle phases. However, whether control of localized translation at the spindle is required for mitosis is unclear, as mitotic and acentriolar-meiotic spindles are functionally and structurally different. Furthermore, the large differences in scale-ratio between cell volume and spindle size in oocytes compared to somatic mitotic cells may generate distinct requirements for gene expression compartmentalization in meiosis and mitosis. Here we show that mitotic spindles contain CPE-localized mRNAs and translating ribosomes. Moreover, CPEB1 and CPEB4 localize in the spindles and they may function sequentially in promoting mitotic stage transitions and correct chromosome segregation. Thus, CPEB1 and CPEB4 bind to specific spindle-associated transcripts controlling the expression and/or localization of their encoded factors that, respectively, drive metaphase and anaphase/cytokinesis.
PubMed: 33323527
DOI: 10.1261/rna.077552.120 -
Current Opinion in Cell Biology Dec 2022In an active, crowded cytoplasm, eukaryotic cells construct metaphase spindles from conserved building blocks to segregate chromosomes. Yet, spindles execute their... (Review)
Review
In an active, crowded cytoplasm, eukaryotic cells construct metaphase spindles from conserved building blocks to segregate chromosomes. Yet, spindles execute their function in a stunning variety of cell shapes and sizes across orders of magnitude. Thus, the current challenge is to understand how unique mesoscale spindle characteristics emerge from the interaction of molecular collectives. Key components of these collectives are tubulin dimers, which polymerise into microtubules. Despite all conservation, tubulin is a genetically and biochemically complex protein family, and we only begin to uncover how tubulin diversity affects microtubule dynamics and thus spindle assembly. Moreover, it is increasingly appreciated that spindles are dynamically intertwined with the cytoplasm that itself exhibits cell-type specific emergent properties with yet mostly unexplored consequences for spindle construction. Therefore, on our way toward a quantitative picture of spindle function, we need to understand molecular behaviour of the building blocks and connect it to the entire cellular context.
Topics: Metaphase; Tubulin; Cell Cycle; Microtubules; Cytoplasm
PubMed: 36436307
DOI: 10.1016/j.ceb.2022.102143 -
Nature Communications Mar 2023Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms...
Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms remain unknown. Herein, we report that DYNLRB2 (Dynein light chain roadblock-type-2) is a male meiosis-upregulated dynein light chain that is indispensable for spindle formation in meiosis I. In Dynlrb2 KO mouse testes, meiosis progression is arrested in metaphase I due to the formation of multipolar spindles with fragmented pericentriolar material (PCM). DYNLRB2 inhibits PCM fragmentation through two distinct pathways; suppressing premature centriole disengagement and targeting NuMA (nuclear mitotic apparatus) to spindle poles. The ubiquitously expressed mitotic counterpart, DYNLRB1, has similar roles in mitotic cells and maintains spindle bipolarity by targeting NuMA and suppressing centriole overduplication. Our work demonstrates that two distinct dynein complexes containing DYNLRB1 or DYNLRB2 are separately used in mitotic and meiotic spindle formations, respectively, and that both have NuMA as a common target.
Topics: Mice; Animals; Male; Dyneins; Spindle Apparatus; Centrosome; Meiosis; Metaphase
PubMed: 36973253
DOI: 10.1038/s41467-023-37370-7 -
Current Cancer Drug Targets 2021The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents mitotic exit at the metaphase-to-anaphase transition until all chromosomes have... (Review)
Review
The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents mitotic exit at the metaphase-to-anaphase transition until all chromosomes have established correct bipolar attachment to spindle microtubules. Activation of SAC relies on the assembly of the mitotic checkpoint complex (MCC), which requires conformational change from inactive open Mad2 (OMad2) to the active closed Mad2 (C-Mad2) at unattached kinetochores. The Mad2-binding protein p31 plays a key role in controlling timely mitotic exit by promoting SAC silencing, through preventing Mad2 activation and promoting MCC disassembly. Besides, increasing evidences highlight the p31 potential as target for cancer therapy. Here, we provide an updated overview of the functional significance of p31 in mitotic progression, and discuss the potential of deregulated expression of p31 in cancer and in therapeutic strategies.
Topics: Adaptor Proteins, Signal Transducing; Cell Cycle Checkpoints; Cell Cycle Proteins; Drug Discovery; Gene Expression Regulation, Neoplastic; Humans; M Phase Cell Cycle Checkpoints; Molecular Targeted Therapy; Neoplasms; Nuclear Proteins
PubMed: 33511944
DOI: 10.2174/1568009621666210129095726 -
Open Biology Aug 2019The oncogenic transcription factor MYC modulates vast arrays of genes, thereby influencing numerous biological pathways including biogenesis, metabolism, proliferation,...
The oncogenic transcription factor MYC modulates vast arrays of genes, thereby influencing numerous biological pathways including biogenesis, metabolism, proliferation, apoptosis and pluripotency. When deregulated, MYC drives genomic instability via several mechanisms including aberrant proliferation, replication stress and ROS production. Deregulated MYC also promotes chromosome instability, but less is known about how MYC influences mitosis. Here, we show that deregulating MYC modulates multiple aspects of mitotic chromosome segregation. Cells overexpressing MYC have altered spindle morphology, take longer to align their chromosomes at metaphase and enter anaphase sooner. When challenged with a variety of anti-mitotic drugs, cells overexpressing MYC display more anomalies, the net effect of which is increased micronuclei, a hallmark of chromosome instability. Proteomic analysis showed that MYC modulates multiple networks predicted to influence mitosis, with the mitotic kinase PLK1 identified as a central hub. In turn, we show that MYC modulates several PLK1-dependent processes, namely mitotic entry, spindle assembly and SAC satisfaction. These observations thus underpin the pervasive nature of oncogenic MYC and provide a mechanistic rationale for MYC's ability to drive chromosome instability.
Topics: CRISPR-Cas Systems; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromosomal Instability; Chromosome Segregation; Gene Amplification; Gene Expression Regulation; Genomic Instability; Humans; Mitosis; Mutagenesis; Proto-Oncogene Proteins c-myc
PubMed: 31455158
DOI: 10.1098/rsob.190136 -
Acta Neuropathologica Communications Apr 2020Although abnormal mitosis with disarranged metaphase chromosomes or many micronuclei in astrocytes (named "Alzheimer I type astrocytes" and later "Creutzfeldt-Peters...
Although abnormal mitosis with disarranged metaphase chromosomes or many micronuclei in astrocytes (named "Alzheimer I type astrocytes" and later "Creutzfeldt-Peters cells") have been known for nearly 100 years, the origin and mechanisms of this pathology remain elusive. In experimental brain insults in rats, we show that abnormal mitoses that are not followed by cytokinesis are typical for reactive astrocytes. The pathology originates due to the inability of the cells to form normal mitotic spindles with subsequent metaphase chromosome congression, which, in turn may be due to shape constraints aggravated by cellular enlargement and to the accumulation of large amounts of cytosolic proteins. Many astrocytes escape from arrested mitosis by producing micronuclei. These polyploid astrocytes can survive for long periods of time and enter into new cell cycles.
Topics: Animals; Astrocytes; Brain Diseases; Gliosis; Mitosis; Rats; Rats, Sprague-Dawley; Rats, Wistar
PubMed: 32293551
DOI: 10.1186/s40478-020-00919-4