bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2022–01–02
28 papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology
  1. Centromere Tension Measurement in Budding Yeast Mitosis.
  2. Identifying Cyclin A/Cdk1 Substrates in Mitosis in Human Cells.
  3. Chemical Biology of Mitotic Spindle Assembly Motors.
  4. In Vitro and In Vivo Approaches to Study Kinetochore-Microtubule Attachments During Mitosis.
  5. Using FLIM-FRET for Characterizing Spatial Interactions in the Spindle.
  6. Quantifying Changes in Chromosome Position to Assess Chromokinesin Activity.
  7. Mitotic and DNA Damage Response Proteins: Maintaining the Genome Stability and Working for the Common Good.
  8. Imaging Chambers for Arabidopsis Seedlings for Mitotic Studies.
  9. Use of Echinoderm Gametes and Early Embryos for Studying Meiosis and Mitosis.
  10. Live Fluorescence Imaging of Chromosome Segregation in Cultured Cells.
  11. Remodeling-defective GPI-anchored proteins on the plasma membrane activate the spindle assembly checkpoint.
  12. Optical Control of Mitosis with a Photoswitchable Eg5 Inhibitor.
  13. Visualizing the Dynamics of Cell Division by Live Imaging Drosophila Larval Brain Squashes.
  14. Using Microinjection of Mammalian Cultured Cells to Study Cell Division.
  15. Heliothis virescens ascovirus 3h blocks the cell cycle of Spodoptera exigua fat body cells at G2 /M phase by downregulating cyclin B1 and cyclin-dependent kinase 1.
  16. Imaging Mitosis with Lattice LightSheet.
  17. Symmetry-breaking of animal cytokinesis.
  18. Detection of Nuclear Biomarkers for Chromosomal Instability.
  19. Simulating Dynamic Chromosome Compaction: Methods for Bridging In Silico to In Vivo.
  20. Degron Tagging Using mAID and SMASh Tags in RPE-1 Cells.
  21. NuMA forms condensates through phase separation to drive spindle pole assembly.
  22. Differential Expression of Mitosis and Cell Cycle Regulatory Genes during Recovery from an Acute Respiratory Virus Infection.
  23. Staining of the Actin Cytoskeleton During Cell Division in Budding Yeast and Mammalian Cells.
  24. Synchronizing Mammalian Cells for Mitotic Analysis of the Localization of Survivin.
  25. Analysis of Autophagic Vesicles in Mitotic Cells.
  26. A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle.
  27. A Structural Ensemble of a Tau-Microtubule Complex Reveals Regulatory Tau Phosphorylation and Acetylation Mechanisms.
  28. Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy.
  1. Methods Mol Biol. 2022 ;2415 199-210
    Centromere Tension Measurement in Budding Yeast Mitosis.
    Soumya Mukherjee, Melissa K Gardner.
      During budding yeast mitosis, duplicated chromosomes are aligned at the center of the metaphase mitotic spindle, and the centromeres are stretched by forces generated within the mitotic spindle. In response to these stretching forces, mechanical tension builds up in the centromeric chromatin. The magnitude of this tension is detected by the cell to signal the attachment configuration of the sister chromosomes: a high tension signal would indicate that sister chromosomes are properly attached to opposite spindle poles, while a low tension signal could indicate the lack of a bipolar attachment. A low tension signal drives the cell to correct improper attachments in metaphase, thus preventing potential errors in anaphase chromosome segregation. In this paper, we describe a microscopy-based method to directly measure the magnitude of centromere tension in budding yeast metaphase spindles. The advantage of this method is that quantitative tension estimates are obtained without perturbing spindle and/or chromosome structure and as cells progress normally through mitosis.
    Keywords:  Centromere; Kinetochore; Metaphase; Microtubule; Mitosis; Tension
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_15
  2. Methods Mol Biol. 2022 ;2415 175-182
    Identifying Cyclin A/Cdk1 Substrates in Mitosis in Human Cells.
    Ana Maria G Dumitru, Duane A Compton.
      Cyclin A promotes Cdk activity in a cell cycle-dependent manner to facilitate specific cell cycle events and transitions with an established role for DNA replication in S phase. Recent evidence demonstrates that cyclin A also activates Cdk during early mitosis to promote faithful chromosome segregation by regulating the stability of kinetochore-microtubule (k-MT) attachments. Here we describe a methodology to identify protein substrates of cyclin A/Cdk during mitosis in human cells. The method combines selective cell cycle synchrony in mitosis with stable isotope labeling of amino acids in cell culture (SILAC) coupled to mass spectrometry. This strategy identified a catalogue of potential cyclin A/Cdk substrates in mitosis, as well as unveiled potential intersections between signaling regulated by Aurora, Polo-like, and Cdk mitotic kinases.
    Keywords:  Chromosomes; Cyclin A; Kinetochore; Mass spectrometry; Microtubule; Mitosis; Phosphorylation; Proteomics; SILAC; Spindle; Tubulin
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_13
  3. Methods Mol Biol. 2022 ;2415 151-165
    Chemical Biology of Mitotic Spindle Assembly Motors.
    April Solon, Ryoma Ohi.
      Mitotic kinesins play essential roles during mitotic spindle assembly and in ensuring proper chromosome segregation. Chemical inhibitors of mitotic kinesins are therefore valuable tools to study kinesin function in vitro and in cells. Because cancer is a disease of unregulated cell division, inhibitors also represent potential chemotherapeutic agents. Here, we present assays that can be used to evaluate the potency and specificity of mitotic kinesin inhibitors identified from high-throughput screening. By evaluating their effects in a variety of in vitro, fixed-cell, and live cell assays, screening hits can be prioritized and optimized to produce effective, on-target inhibitors.
    Keywords:  Kif15; Kinesin; Microtubule; Mitosis; Mitotic spindle
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_11
  4. Methods Mol Biol. 2022 ;2415 123-138
    In Vitro and In Vivo Approaches to Study Kinetochore-Microtubule Attachments During Mitosis.
    Sana Afreen, Amit Rahi, Adriana G Landeros, Manas Chakraborty, Richard J McKenney, Dileep Varma.
      The separation of duplicated chromosomes during mitosis is a pivotal step in the process of cellular division. Therefore, the orchestrated events that take place to ensure proper attachment and stabilization of kMTs are keen areas of interest in the mitosis field. Here we describe the methods used to study kMT attachments via in vitro biochemical methods and in vivo cell biological approaches.
    Keywords:  Cell cycle; Cell division; Cell synchronization; Chromosome; Kinetochores; Microscopy; Microtubules; Mitosis; Mitotic spindle; Photoactivation; TIR-FM; siRNA
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_9
  5. Methods Mol Biol. 2022 ;2415 221-243
    Using FLIM-FRET for Characterizing Spatial Interactions in the Spindle.
    Stephanie C Ems-McClung, Claire E Walczak.
      Proper spindle assembly and the attachment of chromosomes to the spindle are key for the accurate segregation of chromosomes to daughter cells. Errors in these processes can lead to aneuploidy, which is a hallmark of cancer. Understanding the mechanisms that drive spindle assembly will provide fundamental insights into how accurate chromosome segregation is achieved. One challenge in elucidating the complexities of spindle assembly is to visualize protein interactions in space and time. The Xenopus egg extract system has been a valuable tool to probe protein function during spindle assembly in vitro. Tagging proteins with fluorescent proteins and utilizing fluorescence-based approaches, such as Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), have provided visual clues about the mechanics of spindle assembly and its regulators. However, elucidating how spindle assembly factors are spatially regulated is still challenging. Combining the egg extract system and visual FRET approaches provides a powerful tool to probe the processes involved in spindle assembly. Here we describe how a FLIM-FRET biosensor can be used to study protein-protein interactions in spindles assembled in Xenopus egg extracts. This approach should be readily adaptable to a wide variety of proteins to allow for new insights into the regulation of spindle assembly.
    Keywords:  Biosensor; FLIM; Fluorescence; Kinesin; Protein–protein interactions; Spindle assembly
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_17
  6. Methods Mol Biol. 2022 ;2415 139-149
    Quantifying Changes in Chromosome Position to Assess Chromokinesin Activity.
    Alex F Thompson, Sarah Vandal, Jason Stumpff.
      The chromokinesin KIF22 (Kid, kinesin-10 family) is the primary generator of polar ejection forces, which contribute to chromosome positioning and alignment in mitotic cells. Assessment of KIF22 function requires quantitative comparison of relative polar ejection forces between experimental conditions. This is facilitated by the generation of monopolar spindles to reduce the impact of bioriented microtubule attachment at kinetochores on chromosome positions and increase the dependence of chromosome positions on chromokinesin activity. Radial profile plots measure the intensity of chromatin signal in concentric circles around the poles of monopolar cells and represent an expedient quantitative measure of relative polar ejection forces. As such, this assay can be used to measure changes in polar ejection forces resulting from chromokinesin depletion or perturbation.
    Keywords:  Chromokinesin; Mitosis; Mitotic spindle; Monopolar spindle; Polar ejection force
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_10
  7. Front Cell Dev Biol. 2021 ;9 700162
    Mitotic and DNA Damage Response Proteins: Maintaining the Genome Stability and Working for the Common Good.
    Fernando Luna-Maldonado, Marco A Andonegui-Elguera, José Díaz-Chávez, Luis A Herrera.
      Cellular function is highly dependent on genomic stability, which is mainly ensured by two cellular mechanisms: the DNA damage response (DDR) and the Spindle Assembly Checkpoint (SAC). The former provides the repair of damaged DNA, and the latter ensures correct chromosome segregation. This review focuses on recently emerging data indicating that the SAC and the DDR proteins function together throughout the cell cycle, suggesting crosstalk between both checkpoints to maintain genome stability.
    Keywords:  DNA damage response (DDR); DNA repair; cancer; genomic stability; mitosis; spindle assembly checkpoint (SAC)
    DOI:  https://doi.org/10.3389/fcell.2021.700162
  8. Methods Mol Biol. 2022 ;2415 47-59
    Imaging Chambers for Arabidopsis Seedlings for Mitotic Studies.
    Sidney L Shaw, Mathew Siebe, Timothy Cioffi.
      Flowering plants evolved away from creating centrosomes or conventional microtubule organizing centers. Therein, plants have posed a long-standing challenge to many of the conventional ideas for mitotic spindle construction and the process of chromosome segregation. The Arabidopsis seedling has emerged as a leading model for plant cell biological studies of the cytoskeleton and vesicle trafficking. Here we describe methods for creating a reusable chamber for mitotic studies in both seedling root and shoot cells with instruction for best practices with conventional microscopic techniques.
    Keywords:  Arabidopsis mitosis; Kevin; Seedling imaging chamber
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_4
  9. Methods Mol Biol. 2022 ;2415 1-17
    Use of Echinoderm Gametes and Early Embryos for Studying Meiosis and Mitosis.
    Debadrita Pal, Florencia Visconti, Silvia P Sepúlveda-Ramírez, S Zachary Swartz, Charles B Shuster.
      The early embryos of sea urchins and other echinoderms have served as experimental models for the study of cell division since the nineteenth century. Their rapid development, optical clarity, and ease of manipulation continue to offer advantages for studying spindle assembly and cytokinesis. In the absence of transgenic lines, alternative strategies must be employed to visualize microtubules and actin. Here, we describe methods to visualize actin and microtubule using either purified, recombinant proteins, or probes in in vitro-transcribed mRNAs.
    Keywords:  Confocal microscopy; Cytokinesis actin; Meiosis; Microtubule; Mitosis; Sea star; Sea urchin
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_1
  10. Methods Mol Biol. 2022 ;2415 61-86
    Live Fluorescence Imaging of Chromosome Segregation in Cultured Cells.
    John R Daum, Casey O DuBose, Sushama Sivakumar, Gary J Gorbsky.
      Live-cell fluorescence microscopy is an effective tool for characterizing aberrant mitotic phenotypes resulting from exposure to chemical inhibitors and after RNA interference-mediated or CRISPR knockout-mediated depletion of protein targets. Live imaging of cultured cells during mitotic progression presents challenges in maintaining optimal health of cells while achieving the temporal and spatial resolution to accomplish the goals of the study. Herein are strategies to monitor and analyze mammalian cell mitosis utilizing either a wide field or a light sheet, inverted, fluorescence microscope.
    Keywords:  Cell division; Fluorescent protein; Light sheet fluorescence microscopy; Live-cell imaging; Microscope; Mitosis; Wide field fluorescence microscopy
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_5
  11. Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01614-4. [Epub ahead of print]37(13): 110120
    Remodeling-defective GPI-anchored proteins on the plasma membrane activate the spindle assembly checkpoint.
    Li Chen, Linna Tu, Gege Yang, David K Banfield.
      Newly synthesized glycosylphosphatidylinositol-anchored proteins (GPI-APs) undergo extensive remodeling prior to transport to the plasma membrane. GPI-AP remodeling events serve as quality assurance signatures, and complete remodeling of the anchor functions as a transport warrant. Using a genetic approach in yeast cells, we establish that one remodeling event, the removal of ethanolamine-phosphate from mannose 2 via Ted1p (yPGAP5), is essential for cell viability in the absence of the Golgi-localized putative phosphodiesterase Dcr2p. While GPI-APs in which mannose 2 has not been remodeled in dcr2 ted1-deficient cells can still be delivered to the plasma membrane, their presence elicits a unique stress response. Stress is sensed by Mid2p, a constituent of the cell wall integrity pathway, whereupon signal promulgation culminates in activation of the spindle assembly checkpoint. Our results are consistent with a model in which cellular stress response and chromosome segregation checkpoint pathways are functionally interconnected.
    Keywords:  CWI; ER; GPI; GPI-AP remodeling enzymes; Golgi; SAC; cell wall integrity pathway; glycosylphosphatidylinositol-anchored proteins; spindle assembly checkpoint; yeast
    DOI:  https://doi.org/10.1016/j.celrep.2021.110120
  12. Angew Chem Int Ed Engl. 2021 Dec 27.
    Optical Control of Mitosis with a Photoswitchable Eg5 Inhibitor.
    Dirk Trauner, Anna Impastato, Andrej Shemet, Nynke Veprek, Gadiel Saper, Henry Hess, Lu Rao, Arne Gennerich.
      Eg5 is a kinesin motor protein that is responsible for bipolar spindle formation and plays a crucial role during mitosis. Loss of Eg5 function leads to the formation of monopolar spindles, followed by mitotic arrest, and subsequent cell death. Several cell-permeable small molecules have been reported to inhibit Eg5 and some have been evaluated as anticancer agents. We now describe the design, synthesis, and biological evaluation of photoswitchable variants with five different pharmacophores. Our lead compound is a cell permeable azobenzene that inhibits Eg5 more potently in its light-induced cis form. This activity decreased movement in microtubule gliding assays, promoted formation of monopolar spindles, and led to mitotic arrest in a light dependent way.
    Keywords:  Photopharmacology; Povarov reaction; cytoskeleton; motor proteins
    DOI:  https://doi.org/10.1002/anie.202115846
  13. Methods Mol Biol. 2022 ;2415 37-46
    Visualizing the Dynamics of Cell Division by Live Imaging Drosophila Larval Brain Squashes.
    Brandt Warecki, Ian Bast, William Sullivan.
      The dramatic changes of subcellular structures during mitosis are best visualized by live imaging. In general, live imaging requires the expression of proteins of interest fused to fluorophores and a model system amenable to live microscopy. Drosophila melanogaster is an attractive model in which to perform live imaging because of the numerous transgenic stocks bearing fluorescently tagged transgenes as well as the ability to precisely manipulate gene expression. Traditionally, the early Drosophila embryo has been used for live fluorescent analysis of mitotic events such as spindle formation and chromosome segregation. More recent studies demonstrate that the Drosophila third instar neuroblasts have a number of properties that make them well suited for live analysis: (1) neuroblasts are distinct cells surrounded by plasma membranes; (2) neuroblasts undergo a complete cell cycle, consisting of G1, S, G2, and M phases; and (3) neuroblasts gene expression is not influenced by maternal load, and so the genetics are therefore relatively more simple. Finally, the Drosophila neuroblast is arguably the best system for live imaging asymmetric stem cell divisions. Here, we detail a method for live imaging Drosophila larval neuroblasts.
    Keywords:  Division; Drosophila; Live imaging; Mitosis; Neuroblast; Stem cell
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_3
  14. Methods Mol Biol. 2022 ;2415 105-122
    Using Microinjection of Mammalian Cultured Cells to Study Cell Division.
    Charles Day, Alyssa Langfald, Edward H Hinchcliffe.
      The introduction of macromolecules directly into individual cells by microinjection is an important technique for manipulating mitotic cells. mRNA, purified proteins, or concentrated antibodies can all be injected directly into a single cell, and their effects monitored by live-cell imaging. The equipment necessary is relatively simple, and the technique can be easily mastered. Here we describe our microinjection setup, how to microinject cultured mammalian cells in mitosis, and how to analyze those cells by same-cell live and fixed imaging.
    Keywords:  Antibody; Cell cycle; Fluorescence; Injector; Micromanipulator; Microneedle; Microscopy; Mitosis; Phosphorylation
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_8
  15. J Cell Physiol. 2021 Dec 26.
    Heliothis virescens ascovirus 3h blocks the cell cycle of Spodoptera exigua fat body cells at G2 /M phase by downregulating cyclin B1 and cyclin-dependent kinase 1.
    Xiao-Hui Song, Ni Li, Chang-Jin Yang, Yanyang Wu, Guo-Hua Huang.
      Ascoviruses are double-stranded DNA viruses that are pathogenic to noctuid larvae. In vitro infection causes the cells to fail to replicate and proliferate normally. However, the molecular mechanisms are unclear. In this study, the transmission electron microscopy data of infected-Spodoptera exigua (Hübner) fat body cells (SeFB, IOZCAS-SpexII-A cells) showed that virions were internalized in phagocytic vesicles, but not in the nucleus. FACS of cell-cycle progression was performed in SeFB cells infected with Heliothis virescens ascovirus 3h (HvAV-3h). The cell cycle phase distributions of the SeFB cells were G1  = 29.52 ± 1.10%, S = 30.33 ± 1.19%, and G2 /M = 40.06 ± 0.75%. The cell culture doubling time was approximately 24 h. The G1 , S, and G2 /M phases were each approximately 8 h. The unsynchronized or synchronized cells were arrested at G2 /M phase after infection with HvAV-3h. Our data also showed that cells with more than 4N DNA content appeared in the HvAV-3h-treated group. While the mRNA levels of cyclin B1 , cyclin H, and cyclin-dependent kinase 1 (CDK1) were downregulated after HvAV-3h infection, the mRNA expression levels of cyclin A, cyclin D, and cyclin B2 were not significantly changed. Western blotting results showed that the expression of cyclin B1 and CDK1 in infected SeFB cells within 24 h postinfection (hpi), and HvAV-3h infection inhibited the expression of cyclin B1 and CDK1 at 12-24 hpi. Overall, these data implied that HvAV-3h infection leads to an accumulation of cells in the G2 /M phases by downregulating the expression of cyclin B1 and CDK1.
    Keywords:  CDK1; Heliothis virescens ascovirus 3h; SeFB cell line; cell cycle; cyclin B
    DOI:  https://doi.org/10.1002/jcp.30665
  16. Methods Mol Biol. 2022 ;2415 245-252
    Imaging Mitosis with Lattice LightSheet.
    Andrew Barazia, Colin Monks.
      Mitosis is one of the most fundamental processes of life, allowing organisms to grow, develop, and evolve. Acquiring microscopic images and understanding the detailed mechanism of this process is critical in the fields of cell and developmental biology. Modern fluorescence microscopy is the standard for imaging specific molecules and proteins as they interact during this complicated process. However, researchers must take care to ensure that they are maintaining the basal cell processes during mitosis without disruption by placing the sample on a microscope. In addition, mitosis in itself is an incredibly dynamic process that requires both high-speed and high-resolution imaging (McIntosh and Hays. Biology. 5:55, 2016). The Lattice LightSheet is an advanced system, developed in the lab of Eric Betzig (Chen et al. Science. 346:1257998), that offers imaging speeds in the volumes/second while still resolving fine, intracellular structures. Here we describe how to prepare cell culture samples for ideal mitotic imaging on this cutting-edge light sheet fluorescence microscope.
    Keywords:  Deconvolution; Fluorescence Microscopy; Laser; Light sheet; Live cell imaging; Mitosis
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_18
  17. Semin Cell Dev Biol. 2021 Dec 23. pii: S1084-9521(21)00316-5. [Epub ahead of print]
    Symmetry-breaking of animal cytokinesis.
    Kenji Sugioka.
      Cytokinesis is a mechanism that separates dividing cells via constriction of a supramolecular structure, the contractile ring. In animal cells, three modes of symmetry-breaking of cytokinesis result in unilateral cytokinesis, asymmetric cell division, and oriented cell division. Each mode of cytokinesis plays a significant role in tissue patterning and morphogenesis by the mechanisms that control the orientation and position of the contractile ring relative to the body axis. Despite its significance, the mechanisms involved in the symmetry-breaking of cytokinesis remain unclear in many cell types. Classical embryologists have identified that the geometric relationship between the mitotic spindle and cell cortex induces cytokinesis asymmetry; however, emerging evidence suggests that a concerted flow of compressional cell-cortex materials (cortical flow) is a spindle-independent driving force in spatial cytokinesis control. This review provides an overview of both classical and emerging mechanisms of cytokinesis asymmetry and their roles in animal development.
    Keywords:  Asymmetric cell division; Cell cortex; Cell polarity; Contractile ring; Cytokinesis; Cytoskeleton
    DOI:  https://doi.org/10.1016/j.semcdb.2021.12.008
  18. Methods Mol Biol. 2022 ;2445 117-125
    Detection of Nuclear Biomarkers for Chromosomal Instability.
    Carles Pons, Eugenia Almacellas, Albert Tauler, Caroline Mauvezin.
      Chromosomal instability (CIN) is a hallmark of cancer, which is characterized by the gain or loss of chromosomes as well as the rearrangement of the genetic material during cell division. Detection of mitotic errors such as misaligned chromosomes or chromosomal bridges (also known as lagging chromosomes) is challenging as it requires the analysis and manual discrimination of chromosomal aberrations in mitotic cells by molecular techniques. In interphase cells, more frequent in the cell population than mitotic cells, two distinct nuclear phenotypes are associated with CIN: the micronucleus and the toroidal nucleus. Several methods are available for the detection of micronuclei, but none for toroidal nuclei. Here, we provide a method to quantify the presence of both nuclear biomarkers for the evaluation of CIN status in non-mitotic cells particularly suited for genotoxicity screens.
    Keywords:  Biological image processing; Chromosomal instability; Genotoxicity biomarkers; Micronucleus; Nuclear phenotype; Toroidal nucleus
    DOI:  https://doi.org/10.1007/978-1-0716-2071-7_8
  19. Methods Mol Biol. 2022 ;2415 211-220
    Simulating Dynamic Chromosome Compaction: Methods for Bridging In Silico to In Vivo.
    Yunyan He, David Adalsteinsson, Benjamin Walker, Josh Lawrimore, M Gregory Forest, Kerry Bloom.
      The application of polymer models to chromosome structure and dynamics is a powerful approach for dissecting functional properties of the chromosome. The models are based on well-established bead-spring models of polymers and are distinct from molecular dynamics studies used in structural biology. In this work, we outline a polymer dynamics model that simulates budding yeast chromatin fibers in a viscous environment inside the nucleus using DataTank as a user interface for the C++ simulation. We highlight features for creating the nucleolus, a dynamic region of chromatin with protein-mediated, transient chromosomal cross-links, providing a predictive, stochastic polymer-physics model for versatile analyses of chromosome spatiotemporal organization. DataTank provides real-time visualization and data analytics methods during simulation. The simulation pipeline provides insights into the entangled chromosome milieu in the nucleus and creates simulated chromosome data, both structural and dynamic, that can be directly compared to experimental observations of live cells in interphase and mitosis.
    Keywords:  All chromosome simulation; Chromatin; Chromosomal cross-links; Data visualization; DataTank; Nucleolus; Polymer physics model
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_16
  20. Methods Mol Biol. 2022 ;2415 183-197
    Degron Tagging Using mAID and SMASh Tags in RPE-1 Cells.
    Adrijana Crncec, Helfrid Hochegger.
      Degron tags allow the precise and well-controlled analysis of essential genes by rapidly inducing degradation of the protein of interest. This is critical when the consequences of loss of gene function needs to be analyzed in a strictly defined time window such as a specific cell cycle phase. We have recently published the successful application of degron tags to analyze cell cycle genes such as CDC6, CCNA2, and CCNB1. A critical aspect of our approach was to combine two tags to generate a synergy in the degradation dynamics. Here we outline our approach and describe some of the essential steps to generate double-degron-tagged genes in RPE-1 cells. Similar procedures can easily be applied to other cell lines.
    Keywords:  Auxin-inducible degron (AID); Cyclin; Degron; RPE-1; SMASh
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_14
  21. J Mol Cell Biol. 2021 Dec 27. pii: mjab081. [Epub ahead of print]
    NuMA forms condensates through phase separation to drive spindle pole assembly.
    Huixian Ma, Feifei Qi, Li Ji, Songbo Xie, Jie Ran, Min Liu, Jinmin Gao, Jun Zhou.
      
    DOI:  https://doi.org/10.1093/jmcb/mjab081
  22. Pathogens. 2021 Dec 15. pii: 1625. [Epub ahead of print]10(12):
    Differential Expression of Mitosis and Cell Cycle Regulatory Genes during Recovery from an Acute Respiratory Virus Infection.
    Ajinkya R Limkar, Justin B Lack, Albert C Sek, Caroline M Percopo, Kirk M Druey, Helene F Rosenberg.
      Acute respiratory virus infections can have profound and long-term effects on lung function that persist even after the acute responses have fully resolved. In this study, we examined gene expression by RNA sequencing in the lung tissue of wild-type BALB/c mice that were recovering from a sublethal infection with the pneumonia virus of mice (PVM), a natural rodent pathogen of the same virus family and genus as the human respiratory syncytial virus. We compared these responses to gene expression in PVM-infected mice treated with Lactobacillus plantarum, an immunobiotic agent that limits inflammation and averts the negative clinical sequelae typically observed in response to acute infection with this pathogen. Our findings revealed prominent differential expression of inflammation-associated genes as well as numerous genes and gene families implicated in mitosis and cell-cycle regulation, including cyclins, cyclin-dependent kinases, cell division cycle genes, E2F transcription factors, kinesins, centromere proteins, and aurora kinases, among others. Of particular note was the differential expression of the cell division cycle gene Cdc20b, which was previously identified as critical for the ex vivo differentiation of multi-ciliated cells. Collectively, these findings provided us with substantial insight into post-viral repair processes and broadened our understanding of the mechanisms underlying Lactobacillus-mediated protection.
    Keywords:  RNA sequencing; cell cycle regulation; differential expression; mitosis; pneumonia virus of mice; respiratory virus
    DOI:  https://doi.org/10.3390/pathogens10121625
  23. Methods Mol Biol. 2022 ;2415 87-94
    Staining of the Actin Cytoskeleton During Cell Division in Budding Yeast and Mammalian Cells.
    Katie B Shannon.
      Observation of actin at the cortex in dividing cells can be accomplished using the fungal toxin phalloidin conjugated to fluorophores. Protocols for staining both budding yeast and cultured mammalian cells with fluorescent phalloidin are described. This technique can be combined with immunofluorescence to image actin filaments and other proteins involved in cell division simultaneously.
    Keywords:  Actin; Cytokinesis; Fixation; Fluorescence microscopy; Formaldehyde; Phalloidin
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_6
  24. Methods Mol Biol. 2022 ;2415 95-103
    Synchronizing Mammalian Cells for Mitotic Analysis of the Localization of Survivin.
    Sally P Wheatley.
      This paper describes an easy method to enrich the harvest of adherent mammalian cells at each stage of mitosis (from prometaphase to cytokinesis) by combining Eg5 inhibition using dimethylenastron (DMA) with mitotic shake-off, followed by timed release from the drug.
    Keywords:  Dimethylenastron (DMA); Eg5; Mammalian; Mitotic synchrony; Survivin
    DOI:  https://doi.org/10.1007/978-1-0716-1904-9_7
  25. Methods Mol Biol. 2022 ;2445 127-137
    Analysis of Autophagic Vesicles in Mitotic Cells.
    Eugenia Almacellas, Marta Garcia-Cajide, Albert Tauler, Caroline Mauvezin.
      The detection of autophagic vesicles in interphase cells is well characterized with markers such as LC3, SQSTM1 (also known as p62) and LAMP2, which are commonly used in immunofluorescence and biochemistry assays to evaluate the status of autophagy in adherent cells. During mitosis, cells undergo important morphological changes which alter the position of the central plane, therefore the imaging of dividing cells has to be specifically designed. Here, we describe a method to label and image autophagic vesicles in mitotic cells to systematically analyze their number, morphology and distribution.
    Keywords:  Adherent cells; Autophagy; Image analysis; Immunofluorescence; Lysosomes; Mitosis; Radial plot
    DOI:  https://doi.org/10.1007/978-1-0716-2071-7_9
  26. Methods Mol Biol. 2022 ;2445 65-74
    A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle.
    Vitaliy O Kaminskyy.
      Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry-based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.
    Keywords:  Autophagic flux; Autophagosome formation; Autophagy; Cell cycle; Cell permeabilization; DNA staining; Flow cytometry; LC3; Quantification
    DOI:  https://doi.org/10.1007/978-1-0716-2071-7_5
  27. ACS Cent Sci. 2021 Dec 22. 7(12): 1986-1995
    A Structural Ensemble of a Tau-Microtubule Complex Reveals Regulatory Tau Phosphorylation and Acetylation Mechanisms.
    Z Faidon Brotzakis, Philip R Lindstedt, Ross J Taylor, Dillon J Rinauro, Nicholas C T Gallagher, Gonçalo J L Bernardes, Michele Vendruscolo.
      Tau is a microtubule-associated protein that regulates the stability of microtubules. We use metainference cryoelectron microscopy, an integrative structural biology approach, to determine an ensemble of conformations representing the structure and dynamics of a tau-microtubule complex comprising the entire microtubule-binding region of tau (residues 202-395). We thus identify the ground state of the complex and a series of excited states of lower populations. A comparison of the interactions in these different states reveals positions along the tau sequence that are important to determine the overall stability of the tau-microtubule complex. This analysis leads to the identification of positions where phosphorylation and acetylation events have destabilizing effects, which we validate by using site-specific post-translationally modified tau variants obtained by chemical mutagenesis. Taken together, these results illustrate how the simultaneous determination of ground and excited states of macromolecular complexes reveals functional and regulatory mechanisms.
    DOI:  https://doi.org/10.1021/acscentsci.1c00585
  28. ACS Nano. 2021 Dec 30.
    Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy.
    Yi-Teng Hsiao, Chia-Ni Tsai, Te-Hsin Chen, Chia-Lung Hsieh.
      Chromatin is a DNA-protein complex that is densely packed in the cell nucleus. The nanoscale chromatin compaction plays critical roles in the modulation of cell nuclear processes. However, little is known about the spatiotemporal dynamics of chromatin compaction states because it remains difficult to quantitatively measure the chromatin compaction level in live cells. Here, we demonstrate a strategy, referenced as DYNAMICS imaging, for mapping chromatin organization in live cell nuclei by analyzing the dynamic scattering signal of molecular fluctuations. Highly sensitive optical interference microscopy, coherent brightfield (COBRI) microscopy, is implemented to detect the linear scattering of unlabeled chromatin at a high speed. A theoretical model is established to determine the local chromatin density from the statistical fluctuation of the measured scattering signal. DYNAMICS imaging allows us to reconstruct a speckle-free nucleus map that is highly correlated to the fluorescence chromatin image. Moreover, together with calibration based on nanoparticle colloids, we show that the DYNAMICS signal is sensitive to the chromatin compaction level at the nanoscale. We confirm the effectiveness of DYNAMICS imaging in detecting the condensation and decondensation of chromatin induced by chemical drug treatments. Importantly, the stable scattering signal supports a continuous observation of the chromatin condensation and decondensation processes for more than 1 h. Using this technique, we detect transient and nanoscopic chromatin condensation events occurring on a time scale of a few seconds. Label-free DYNAMICS imaging offers the opportunity to investigate chromatin conformational dynamics and to explore their significance in various gene activities.
    Keywords:  chromatin; interference microscopy; label-free; live cells; scattering
    DOI:  https://doi.org/10.1021/acsnano.1c09748
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