-
Cytometry. Part a : the Journal of the... Apr 2021Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to... (Review)
Review
Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
Topics: Cell Cycle; Chromosomes, Plant; Flow Cytometry; Metaphase; Plants
PubMed: 33615737
DOI: 10.1002/cyto.a.24324 -
The Journal of Cell Biology Dec 2009Division plane specification in animal cells has long been presumed to involve direct contact between microtubules of the anaphase mitotic spindle and the cell cortex....
Division plane specification in animal cells has long been presumed to involve direct contact between microtubules of the anaphase mitotic spindle and the cell cortex. In this issue, von Dassow et al. (von Dassow et al. 2009. J. Cell. Biol. doi:10.1083/jcb.200907090) challenge this assumption by showing that spindle microtubules can effectively position the division plane at a distance from the cell cortex.
Topics: Animals; Centrosome; Cytokinesis; Diffusion; Embryo, Nonmammalian; Enzyme Activation; Histone Deacetylase Inhibitors; Metaphase; Microtubules; Protein Transport; Signal Transduction; Spindle Apparatus; Time Factors; Tubulin Modulators; rho GTP-Binding Proteins
PubMed: 20008561
DOI: 10.1083/jcb.200911084 -
Current Biology : CB Mar 2022Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to...
Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex. In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation, the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of +TIP association with growing microtubule ends in live cells. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics.
Topics: Animals; Mammals; Metaphase; Microtubule-Associated Proteins; Microtubules; Optogenetics; Spindle Apparatus
PubMed: 35090591
DOI: 10.1016/j.cub.2022.01.017 -
Nature Communications Nov 2022Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often...
Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often chromosomally abnormal, and many are mosaic, with the karyotype differing from one cell to another. Mosaicism presumably arises from chromosome segregation errors during the early mitotic divisions, although these events have never been visualised in living human embryos. Here, we establish live cell imaging of chromosome segregation using normally fertilised embryos from an egg-share-to-research programme, as well as embryos deselected during fertility treatment. We reveal that the first mitotic division has an extended prometaphase/metaphase and exhibits phenotypes that can cause nondisjunction. These included multipolar chromosome segregations and lagging chromosomes that lead to formation of micronuclei. Analysis of nuclear number and size provides evidence of equivalent phenotypes in 2-cell human embryos that gave rise to live births. Together this shows that errors in the first mitotic division can be tolerated in human embryos and uncovers cell biological events that contribute to preimplantation mosaicism.
Topics: Humans; Embryo, Mammalian; Chromosome Segregation; Mosaicism; Metaphase; Karyotype; Blastocyst; Aneuploidy
PubMed: 36347869
DOI: 10.1038/s41467-022-34294-6 -
Molecular & Cellular Proteomics : MCP Aug 2016The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling...
The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation.
Topics: Chromatography, Liquid; Clathrin; HeLa Cells; Humans; Metaphase; Phosphorylation; Protein Binding; Protein Interaction Maps; Proteome; Proteomics; Signal Transduction; Spindle Apparatus; Tandem Mass Spectrometry
PubMed: 27174698
DOI: 10.1074/mcp.M115.054809 -
Nucleic Acids Research Apr 2023Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation...
Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation capture (3C) sequencing. Still, direct differential visualization of a condensed chromatin fibre confirming the helical model was lacking. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy of large fluorescently labeled chromosome segments to reveal the chromonema - a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingles due to the stochastic positioning of chromatin loops inside the chromonema. Helical turn size varies along chromosome length, correlating with chromatin density. Constraints on the observable dimensions of sister chromatid exchanges further supports the helical chromonema model.
Topics: Chromatids; Chromatin; Chromosomes; Metaphase; Microscopy; Sister Chromatid Exchange; Chromosomes, Plant; Hordeum
PubMed: 36864547
DOI: 10.1093/nar/gkad028 -
Current Biology : CB Sep 2009The mitotic spindle assembles to a steady-state length at metaphase through the integrated action of molecular mechanisms that generate and respond to mechanical forces.... (Review)
Review
The mitotic spindle assembles to a steady-state length at metaphase through the integrated action of molecular mechanisms that generate and respond to mechanical forces. While molecular mechanisms that produce force have been described, our understanding of how they integrate with each other, and with the assembly/disassembly mechanisms that regulate length, is poor. We review current understanding of the basic architecture and dynamics of the metaphase spindle, and some of the elementary force-producing mechanisms. We then discuss models for force integration and spindle length determination. We also emphasize key missing data that notably include absolute values of forces and how they vary as a function of position within the spindle.
Topics: Biomechanical Phenomena; Cell Polarity; Cell Size; Elasticity; Friction; Kinetochores; Metaphase; Microtubules; Models, Biological; Spindle Apparatus
PubMed: 19906577
DOI: 10.1016/j.cub.2009.07.028 -
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 -
The Journal of Cell Biology Jun 2023Centromeres are the foundation for mitotic kinetochore assembly and thus are essential for chromosome segregation. Centromeres are epigenetically defined by nucleosomes...
Centromeres are the foundation for mitotic kinetochore assembly and thus are essential for chromosome segregation. Centromeres are epigenetically defined by nucleosomes containing the histone H3 variant CENP-A. CENP-A nucleosome assembly is uncoupled from replication and occurs in G1, but how cells control this timing is incompletely understood. The formation of CENP-A nucleosomes in vertebrates requires CENP-C and the Mis18 complex which recruit the CENP-A chaperone HJURP to centromeres. Using a cell-free system for centromere assembly in X. laevis egg extracts, we discover two activities that inhibit CENP-A assembly in metaphase. HJURP phosphorylation prevents the interaction between HJURP and CENP-C in metaphase, blocking the delivery of soluble CENP-A to centromeres. Non-phosphorylatable mutants of HJURP constitutively bind CENP-C in metaphase but are not sufficient for new CENP-A assembly. We find that the M18BP1.S subunit of the Mis18 complex also binds to CENP-C to competitively inhibit HJURP's access to centromeres. Removal of these two inhibitory activities causes CENP-A assembly in metaphase.
Topics: Animals; Autoantigens; Carrier Proteins; Centromere; Centromere Protein A; Metaphase; Nucleosomes; Phosphorylation; Xenopus laevis; Xenopus Proteins; DNA-Binding Proteins
PubMed: 37141119
DOI: 10.1083/jcb.202110124 -
Yakugaku Zasshi : Journal of the... 2022Genetic information is replicated and transmitted from a parent cell to two identical daughter cells through mitotic cell division. To accomplish this dynamic process...
Genetic information is replicated and transmitted from a parent cell to two identical daughter cells through mitotic cell division. To accomplish this dynamic process with high accuracy and precision, various motor proteins work in a concerted manner. Especially in the metaphase, mitotic chromosomes are delivered by the motor protein of centromere-associated protein E (CENP-E) to the cell equatorial plane (metaphase plate) along mitotic spindles. However, the critical functional failure of CENP-E can activate the spindle assembly checkpoint through the misalignment of chromosomes at the metaphase plate. In this symposium review, the reversibly photoswitchable CENP-E inhibitor PCEI-HU (5) is reported. Compound 5 exhibited almost quantitative trans-cis photoisomerization of the arylazopyrazole photoswitch by illuminating light at 365 nm and 510 nm. Depending on the photoisomerization, CENP-E activity was regulated not only in vitro but also in cells. We successfully established a novel technique using 5 to dynamically photocontrol the CENP-E-dependent chromosome movement and mitotic progression in a living cell.
Topics: Cell Division; Metaphase; Spindle Apparatus
PubMed: 35491157
DOI: 10.1248/yakushi.21-00203-5