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Methods in Molecular Biology (Clifton,... 2018This chapter describes a microfluidic device that enables immobilization and culturing of single rod-shaped S. pombe cells in a stand-up mode. The wide-band electrical... (Review)
Review
This chapter describes a microfluidic device that enables immobilization and culturing of single rod-shaped S. pombe cells in a stand-up mode. The wide-band electrical impedance spectroscopy (EIS) has been integrated in the microfluidic device to continuously measure cell growth of single S. pombe cells. Cell growth curves showing cellular and intracellular features at high spatiotemporal resolution can be obtained from EIS signals. The features include longitudinal cell elongation in the G2 phase, mitosis, and cell division during an entire cell cycle of S. pombe cells. Microfluidics-based EIS systems provide, hence, a tool for dynamic single-cell studies.
Topics: Dielectric Spectroscopy; G2 Phase; Mitosis; Schizosaccharomyces
PubMed: 29423854
DOI: 10.1007/978-1-4939-7546-4_13 -
Journal of Cell Science Oct 2010The motions of mitotic chromosomes are complex and show considerable variety across species. A wealth of evidence supports the idea that microtubule-dependent motor... (Review)
Review
The motions of mitotic chromosomes are complex and show considerable variety across species. A wealth of evidence supports the idea that microtubule-dependent motor enzymes contribute to this variation and are important both for spindle formation and for the accurate completion of chromosome segregation. Motors that walk towards the spindle pole are, however, dispensable for at least some poleward movements of chromosomes in yeasts, suggesting that depolymerizing spindle microtubules can generate mitotic forces in vivo. Tubulin protofilaments that flare outward in association with microtubule shortening may be the origin of such forces, because they can move objects that are appropriately attached to a microtubule wall. For example, some kinetochore-associated proteins can couple experimental objects, such as microspheres, to shortening microtubules in vitro, moving them over many micrometers. Here, we review recent evidence about such phenomena, highlighting the force-generation mechanisms and different coupling strategies. We also consider bending filaments of the tubulin-like protein FtsZ, which form rings girding bacteria at their sites of cytokinesis. Mechanical similarities between these force-generation systems suggest a deep phylogenetic relationship between tubulin depolymerization in eukaryotic mitosis and FtsZ-mediated ring contraction in bacteria.
Topics: Animals; Bacteria; Humans; Microtubules; Mitosis; Protein Multimerization; Tubulin
PubMed: 20930138
DOI: 10.1242/jcs.067611 -
Current Biology : CB Dec 2009Mitosis depends on the mitotic spindle, a subcellular protein machine that uses dynamic microtubules and mitotic motors to assemble itself and to coordinate chromosome... (Review)
Review
Mitosis depends on the mitotic spindle, a subcellular protein machine that uses dynamic microtubules and mitotic motors to assemble itself and to coordinate chromosome movements. Spindle function depends critically on the interplay of microtubule polymer dynamics and the motor proteins and non-motor microtubule-associated proteins (MAPs) that crosslink adjacent microtubules. These microtubule crosslinkers can organize microtubules into bundles with specific polarity patterns and some of them can slide adjacent microtubules in relation to one another. Here, we discuss the functions and mechanisms of action of three such crosslinkers: the motors kinesin-5 and kinesin-14, and the non-motor MAPs of the Ase1p family.
Topics: Animals; Microtubules; Mitosis; Molecular Motor Proteins; Protein Transport
PubMed: 20064413
DOI: 10.1016/j.cub.2009.10.047 -
Philosophical Transactions of the Royal... Sep 1999Proteolysis controls key transitions at several points in the cell cycle. In mitosis, the activation of a large ubiquitin-protein ligase, the anaphase-promoting complex... (Review)
Review
Proteolysis controls key transitions at several points in the cell cycle. In mitosis, the activation of a large ubiquitin-protein ligase, the anaphase-promoting complex (APC), is required for anaphase initiation and for exit from mitosis. We show that APC is under complex control by a network of regulatory factors, CDC20, CDH1 and MAD2. CDC20 and CDH1 are activators of APC; they bind directly to APC and activate its cyclin ubiquitination activity. CDC20 activates APC at the onset of anaphase in a destruction box (DB)-dependent manner, while CDH1 activates APC from late anaphase through G1 with apparently a much relaxed specificity for the DB. Therefore, CDC20 and CDH1 control both the temporal order of activation and the substrate specificity of APC, and hence regulate different events during mitosis and G1. Counteracting the effect of CDC20, the checkpoint protein MAD2 acts as an inhibitor of APC. When the spindle-assembly checkpoint is activated, MAD2 forms a ternary complex with CDC20 and APC to prevent activation of APC, and thereby arrests cells at prometaphase. Thus, a combination of positive and negative regulators establishes a regulatory circuit of APC, ensuring an ordered progression of events through cell division.
Topics: Anaphase-Promoting Complex-Cyclosome; Animals; Enzyme Activation; Ligases; Mitosis; Signal Transduction; Spindle Apparatus; Ubiquitin-Protein Ligase Complexes; Ubiquitin-Protein Ligases
PubMed: 10582244
DOI: 10.1098/rstb.1999.0502 -
Cell Reports Methods Jun 2022Single-cell imaging of individual mRNAs has revealed core mechanisms of the central dogma. However, most approaches require cell fixation or have limited sensitivity for...
Single-cell imaging of individual mRNAs has revealed core mechanisms of the central dogma. However, most approaches require cell fixation or have limited sensitivity for live-cell applications. Here, we describe SunRISER (SunTag-based reporter for imaging signal-enriched mRNA), a computationally and experimentally optimized approach for unambiguous detection of single mRNA molecules in living cells. When viewed by epifluorescence microscopy, SunRISER-labeled mRNAs show strong signal to background and resistance to photobleaching, which together enable long-term mRNA imaging studies. SunRISER variants, using 8× and 10× stem-loop arrays, demonstrate effective mRNA detection while significantly reducing alterations to target mRNA sequences. We characterize SunRISER to observe mRNA inheritance during mitosis and find that stressors enhance diversity among post-mitotic sister cells. Taken together, SunRISER enables a glimpse into living cells to observe aspects of the central dogma and the role of mRNAs in rare and dynamical trafficking events.
Topics: RNA, Messenger; Mitosis; Diagnostic Imaging
PubMed: 35784652
DOI: 10.1016/j.crmeth.2022.100226 -
Fungal Genetics and Biology : FG & B Jun 1999This review traces the principal advances in the study of mitosis in filamentous fungi from its beginnings near the end of the 19(th) century to the present day. Meiosis... (Review)
Review
This review traces the principal advances in the study of mitosis in filamentous fungi from its beginnings near the end of the 19(th) century to the present day. Meiosis and mitosis had been accurately described and illustrated by the second decade of the present century and were known to closely resemble nuclear divisions in higher eukaryotes. This information was effectively lost in the mid-1950s, and the essential features of mitosis were then rediscovered from about the mid-1960s to the mid-1970s. Interest in the forces that separate chromatids and spindle poles during fungal mitosis followed closely on the heels of detailed descriptions of the mitotic apparatus in vivo and ultrastructurally during this and the following decade. About the same time, fundamental studies of the structure of fungal chromatin and biochemical characterization of fungal tubulin were being carried out. These cytological and biochemical studies set the stage for a surge of renewed interest in fungal mitosis that was issued in by the age of molecular biology. Filamentous fungi have provided model studies of the cytology and genetics of mitosis, including important advances in the study of mitotic forces, microtubule-associated motor proteins, and mitotic regulatory mechanisms.
Topics: Fungi; Mitosis
PubMed: 10413611
DOI: 10.1006/fgbi.1999.1146 -
Cell Structure and Function Jul 1984
Review
Topics: Anaphase; Animals; Cell Cycle; Cell Division; Cell Survival; Meiosis; Mitosis; Spindle Apparatus
PubMed: 6383636
DOI: 10.1247/csf.9.supplement_s73 -
Molecular Biology of the Cell Apr 2021Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase...
Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore-microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Tools to sensitively, accurately, and quantitatively measure chromosome alignment at metaphase will facilitate understanding of the contribution of chromosome segregation errors to the development of aneuploidy. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semiautomated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.
Topics: Chromatids; Chromosome Segregation; HeLa Cells; Humans; Image Processing, Computer-Assisted; Kinetochores; Metaphase; Microscopy, Fluorescence; Microtubules; Mitosis; Models, Theoretical; Spindle Apparatus
PubMed: 33085580
DOI: 10.1091/mbc.E20-09-0585 -
Nature Genetics May 2024How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to...
How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to disentangle and quantify distinct mechanisms underlying genome evolution in single cells, during single mitoses and at single-strand resolution. To distinguish between chronic (reactive oxygen species (ROS)) and acute (ultraviolet light (UV)) mutagenesis, we microfluidically separate pairs of sister cells from the first mitosis following burst UV damage. Strikingly, UV mutations manifest as sister-specific events, revealing mirror-image mutation phasing genome-wide. In contrast, ROS mutagenesis in transcribed regions is reduced strand agnostically. Successive rounds of genome replication over persisting UV damage drives multiallelic variation at CC dinucleotides. Finally, we show that mutation phasing can be resolved to single strands across the entire genome of liver tumors from F1 mice. This strategy can be broadly used to distinguish the contributions of overlapping cancer relevant mutational processes.
Topics: Mutagenesis; Animals; Mice; DNA Repair; Ultraviolet Rays; DNA Damage; Mitosis; Reactive Oxygen Species; Mutation; Humans
PubMed: 38627597
DOI: 10.1038/s41588-024-01712-y -
Molecules (Basel, Switzerland) Mar 2019Obesity is recognized as a worldwide health crisis. Obesity and its associated health complications such as diabetes, dyslipidemia, hypertension, and cardiovascular... (Review)
Review
Obesity is recognized as a worldwide health crisis. Obesity and its associated health complications such as diabetes, dyslipidemia, hypertension, and cardiovascular diseases impose a big social and economic burden. In an effort to identify safe, efficient, and long-term effective methods to treat obesity, various natural products with potential for inhibiting adipogenesis were revealed. This review aimed to discuss the molecular mechanisms underlying adipogenesis and the inhibitory effects of various phytochemicals, including those from natural sources, on the early stage of adipogenesis. We discuss key steps (proliferation and cell cycle) and their regulators (cell-cycle regulator, transcription factors, and intracellular signaling pathways) at the early stage of adipocyte differentiation as the mechanisms responsible for obesity.
Topics: Adipocytes; Adipogenesis; Biological Products; Cell Cycle; Cell Differentiation; Humans; Mitosis; Obesity; Signal Transduction
PubMed: 30909556
DOI: 10.3390/molecules24061157