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Seminars in Cell & Developmental Biology Sep 2021The establishment of a metaphase plate in which all chromosomes are attached to mitotic spindle microtubules and aligned at the cell equator is required for faithful... (Review)
Review
The establishment of a metaphase plate in which all chromosomes are attached to mitotic spindle microtubules and aligned at the cell equator is required for faithful chromosome segregation in metazoans. The achievement of this configuration relies on the precise coordination between several concurrent mechanisms that start upon nuclear envelope breakdown, mediate chromosome capture at their kinetochores during mitotic spindle assembly and culminate with the congression of all chromosomes to the spindle equator. This period is called 'prometaphase'. Because the nature of chromosome capture by mitotic spindle microtubules is error prone, the cell is provided of error correction mechanisms that sense and correct most erroneous kinetochore-microtubule attachments before committing to separate sister chromatids in anaphase. In this review, aimed for newcomers in the field, more than providing an exhaustive mechanistic coverage of each and every concurrent mechanism taking place during prometaphase, we provide an integrative overview of these processes that ultimately promote the subsequent faithful segregation of chromosomes during mitosis.
Topics: Humans; Mitosis; Prometaphase; Spindle Apparatus
PubMed: 34127384
DOI: 10.1016/j.semcdb.2021.06.004 -
Molecular Biology of the Cell May 2021In prophase of meiosis I, homologous chromosomes pair and become connected by cross-overs. Chiasmata, the connections formed by cross-overs, enable the chromosome pair,...
In prophase of meiosis I, homologous chromosomes pair and become connected by cross-overs. Chiasmata, the connections formed by cross-overs, enable the chromosome pair, called a bivalent, to attach as a single unit to the spindle. When the meiotic spindle forms in prometaphase, most bivalents are associated with one spindle pole and then go through a series of oscillations on the spindle, attaching to and detaching from microtubules until the partners of the bivalent become bioriented-attached to microtubules from opposite sides of the spindle. The conserved kinase, Mps1, is essential for the bivalents to be pulled by microtubules across the spindle in prometaphase. Here we show that is needed for efficient triggering of the migration of microtubule-attached kinetochores toward the poles and promotes microtubule depolymerization. Our data support the model Mps1 acts at the kinetochore to coordinate the successful attachment of a microtubule and the triggering of microtubule depolymerization to then move the chromosome.
Topics: Cell Polarity; Chromosome Pairing; Chromosomes; Kinetochores; Microtubules; Mutation; Prometaphase; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae Proteins; Saccharomycetales
PubMed: 33788584
DOI: 10.1091/mbc.E20-08-0525-T -
Nature Communications Jul 2021Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding...
Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes.
Topics: Amino Acid Motifs; Animals; Chromosome Segregation; Meiosis; Mice; Mutation; Oocytes; Phenylalanine; Prometaphase; Protein Binding; Securin; Separase
PubMed: 34262048
DOI: 10.1038/s41467-021-24554-2 -
Journal of Cell Science Sep 2020Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer...
Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.
Topics: Humans; Kinetochores; Mitosis; Nocodazole; Prometaphase; Retinal Pigments
PubMed: 32878943
DOI: 10.1242/jcs.247940 -
Nano Letters Apr 2021Microtubules are highly strategic targets of cancer therapies. Small molecule antimitotic agents are so far the best chemotherapeutic medication in cancer treatment....
Microtubules are highly strategic targets of cancer therapies. Small molecule antimitotic agents are so far the best chemotherapeutic medication in cancer treatment. However, the high rate of neuropathy and drug resistance limit their clinical usage. Inspired by the multicomponent-targeting feature of molecular self-assembly (MSA) overcoming drug resistance, we synthesized peptide-based rotor molecules that self-assemble in response to the surrounding environment to target the microtubule array. The MSAs self-adjust morphologically in response to the pH change and viscosity variations during Golgi-endosome trafficking, escape trafficking cargos, and eventually bind to the microtubule array physically in a nonspecific manner. Such unrefined nano-bio interactions suppress regional tubulin polymerization triggering atypical prometaphase--metaphase oscillations to inhibit various cancer cells proliferating without inducing obvious neurotoxicity. The MSA also exerts potent antiproliferative effects in the subcutaneous cervix cancer xenograft tumor model equivalent to Cisplatin, better than the classic antimitotic drug Taxol.
Topics: Female; Humans; Metaphase; Microtubules; Neoplasms; Prometaphase; Tubulin
PubMed: 33756080
DOI: 10.1021/acs.nanolett.1c00233 -
Molecular Cytogenetics 2018Detailed karyotyping using metaphase chromosomes in melon ( L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase...
BACKGROUND
Detailed karyotyping using metaphase chromosomes in melon ( L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase chromosomes. However, suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated.
RESULTS
In this study, a modified Carnoy's solution II (MC II) [6:3:1 (/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase chromosomes. We demonstrated that the prometaphase chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene chromosome preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase chromosomes of melon and × generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that × has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase chromosomes.
CONCLUSION
The MC II and ADI method are simple and effective for acquiring prometaphase and pachytene chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants.
PubMed: 29760782
DOI: 10.1186/s13039-018-0380-6 -
Cells May 2022The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to... (Review)
Review
The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to align during prometaphase, which are characterized by distinct biomolecular requirements. Peripheral polar chromosomes are an intriguing case as their alignment depends on the activity of kinetochore motors, polar ejection forces, and a transition from lateral to end-on attachments to microtubules, all of which can result in the delayed alignment of these chromosomes. Due to their undesirable position close to and often behind the spindle pole, these chromosomes may be particularly prone to the formation of erroneous kinetochore-microtubule interactions, such as merotelic attachments. To prevent such errors, the cell employs intricate mechanisms to preposition the spindle poles with respect to chromosomes, ensure the formation of end-on attachments in restricted spindle regions, repair faulty attachments by error correction mechanisms, and delay segregation by the spindle assembly checkpoint. Despite this protective machinery, there are several ways in which polar chromosomes can fail in alignment, mis-segregate, and lead to aneuploidy. In agreement with this, polar chromosomes are present in certain tumors and may even be involved in the process of tumorigenesis.
Topics: Chromosome Segregation; Kinetochores; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35563837
DOI: 10.3390/cells11091531 -
Nature Cell Biology Oct 2007The first female meiotic division (meiosis I, MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy...
The first female meiotic division (meiosis I, MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss. Here, we show a fundamental difference in the control of mammalian meiosis that may underlie such susceptibility. It involves a reversal in the well-established timing of activation of the anaphase-promoting complex (APC) by its co-activators cdc20 and cdh1. APC(cdh1) was active first, during prometaphase I, and was needed in order to allow homologue congression, as loss of cdh1 speeded up MI, leading to premature chromosome segregation and a non-disjunction phenotype. APC(cdh1) targeted cdc20 for degradation, but did not target securin or cyclin B1. These were degraded later in MI through APC(cdc20), making cdc20 re-synthesis essential for successful meiotic progression. The switch from APC(cdh1) to APC(cdc20) activity was controlled by increasing CDK1 and cdh1 loss. These findings demonstrate a fundamentally different mechanism of control for the first meiotic division in mammalian oocytes that is not observed in meioses of other species.
Topics: Anaphase-Promoting Complex-Cyclosome; Animals; Blotting, Western; Carrier Proteins; Cdc20 Proteins; Cell Cycle Proteins; Cyclin B; Cyclin B1; Female; Meiosis; Mice; Microscopy, Fluorescence; Oocytes; Prometaphase; Securin; Time Factors; Ubiquitin-Protein Ligase Complexes
PubMed: 17891138
DOI: 10.1038/ncb1640 -
The Journal of Cell Biology Jan 2010We tested the classical hypothesis that astral, prometaphase bipolar mitotic spindles are maintained by balanced outward and inward forces exerted on spindle poles by...
We tested the classical hypothesis that astral, prometaphase bipolar mitotic spindles are maintained by balanced outward and inward forces exerted on spindle poles by kinesin-5 and -14 using modeling of in vitro and in vivo data from Drosophila melanogaster embryos. Throughout prometaphase, puncta of both motors aligned on interpolar microtubules (MTs [ipMTs]), and motor perturbation changed spindle length, as predicted. Competitive motility of purified kinesin-5 and -14 was well described by a stochastic, opposing power stroke model incorporating motor kinetics and load-dependent detachment. Motor parameters from this model were applied to a new stochastic force-balance model for prometaphase spindles, providing a good fit to data from embryos. Maintenance of virtual spindles required dynamic ipMTs and a narrow range of kinesin-5 to kinesin-14 ratios matching that found in embryos. Functional perturbation and modeling suggest that this range can be extended significantly by a disassembling lamin-B envelope that surrounds the prometaphase spindle and augments the finely tuned, antagonistic kinesin force balance to maintain robust prometaphase spindles as MTs assemble and chromosomes are pushed to the equator.
Topics: Animals; Biophysical Phenomena; Drosophila Proteins; Drosophila melanogaster; Kinesins; Lamin Type B; Microtubule-Associated Proteins; Models, Biological; Prometaphase; Spindle Apparatus
PubMed: 20065089
DOI: 10.1083/jcb.200908150 -
EMBO Reports Jun 2020The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific...
The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of the APC/C in mitosis is restrained by the spindle assembly checkpoint (SAC), which coordinates chromosome segregation with the assembly of the mitotic spindle. The SAC effector is the mitotic checkpoint complex (MCC), which binds and inhibits the APC/C. It is incompletely understood how the APC/C switches substrate specificity in a cell cycle-specific manner. For instance, it is unclear how in prometaphase, when APC/C activity towards cyclin B and securin is repressed by the MCC, the kinase Nek2A is ubiquitinated. Here, we combine biochemical and structural analysis with functional studies in cells to show that Nek2A is a conformational-specific binder of the APC/C-MCC complex (APC/C ) and that, in contrast to cyclin A, Nek2A can be ubiquitinated efficiently by the APC/C in conjunction with both the E2 enzymes UbcH10 and UbcH5. We propose that these special features of Nek2A allow its prometaphase-specific ubiquitination.
Topics: Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; Cell Cycle Proteins; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mitosis; Prometaphase; Spindle Apparatus; Ubiquitination
PubMed: 32307883
DOI: 10.15252/embr.201949831