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https://www.readbyqxmd.com/read/29321280/analysis-of-mitotic-checkpoint-function-in-xenopus-egg-extracts
#1
Yinghui Mao
Accurate sister chromatid segregation is pivotal in the faithful transmission of genetic information during each cell division. To ensure accurate segregation, eukaryotic organisms have evolved a "mitotic (or spindle assembly) checkpoint" to prevent premature advance to anaphase before successful attachment of every chromosome to the microtubules of the mitotic spindle. An unattached kinetochore generates a diffusible signal that inhibits ubiquitination of substrates such as cyclin B and securins. This protocol presents an in vitro assay for studying the mitotic checkpoint using Xenopus laevis egg extracts...
January 10, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29320686/dynamic-model-for-characterizing-contractile-behaviors-and-mechanical-properties-of-a-cardiomyocyte
#2
Chuang Zhang, Wenxue Wang, Wenhui He, Ning Xi, Yuechao Wang, Lianqing Liu
Studies on the contractile dynamics of heart cells have attracted broad attention for the development of both heart disease therapies and cardiomyocyte-actuated micro-robotics. In this study, a linear dynamic model of a single cardiomyocyte cell was proposed at the subcellular scale to characterize the contractile behaviors of heart cells, with system parameters representing the mechanical properties of the subcellular components of living cardiomyocytes. The system parameters of the dynamic model were identified with the cellular beating pattern measured by a scanning ion conductance microscope...
January 9, 2018: Biophysical Journal
https://www.readbyqxmd.com/read/29286363/mammalian-cell-division-in-3d-matrices-via-quantitative-confocal-reflection-microscopy
#3
Lijuan He, Alexandra Sneider, Weitong Chen, Michelle Karl, Vishnu Prasath, Pei-Hsun Wu, Gunnar Mattson, Denis Wirtz
The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique...
November 29, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29279314/microtubule-regulation-of-kv7-channels-orchestrates-camp-mediated-vasorelaxations-in-rat-arterial-smooth-muscle
#4
Johanna Lindman, Makhala M Khammy, Pia R Lundegaard, Christian Aalkjær, Thomas A Jepps
Microtubules can regulate GPCR (G protein-coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance of this pathway in vascular smooth muscle cells, we investigated the role of microtubule stability on β-adrenoceptor signaling in rat renal and mesenteric arteries. In isometric tension experiments, incubation with the microtubule inhibitors colchicine and nocodazole enhanced isoprenaline-mediated relaxations of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented...
December 26, 2017: Hypertension
https://www.readbyqxmd.com/read/29190756/visualizing-the-spatiotemporal-map-of-rac-activation-in-bovine-aortic-endothelial-cells-under-laminar-and-disturbed-flows
#5
Shuai Shao, Cheng Xiang, Kairong Qin, Aziz Ur Rehman Aziz, Xiaoling Liao, Bo Liu
Disturbed flow can eliminate the alignment of endothelial cells in the direction of laminar flow, and significantly impacts on atherosclerosis in collateral arteries near the bifurcation and high curvature regions. While shear stress induced Rac polarity has been shown to play crucial roles in cell polarity and migration, little is known about the spatiotemporal map of Rac under disturbed flow, and the mechanism of flow-induced cell polarity still needs to be elucidated. In this paper, disturbed flow or laminar flow with 15 dyn/cm2 of average shear stress was applied on bovine aortic endothelial cells (BAECs) for 30 minutes...
2017: PloS One
https://www.readbyqxmd.com/read/29184543/modulation-of-microtubule-dynamics-affects-brucella-abortus-intracellular-survival-pathogen-containing-vacuole-maturation-and-pro-inflammatory-cytokine-production-in-infected-macrophages
#6
Juliana Alves-Silva, Isabela P Tavares, Erika S Guimarães, Miriam M Costa Franco, Barbara C Figueiredo, João T Marques, Gary Splitter, Sergio C Oliveira
The microtubule (MT) cytoskeleton regulates several cellular processes related to the immune system. For instance, an intricate intracellular transport mediated by MTs is responsible for the proper localization of vesicular receptors of innate immunity and its adaptor proteins. In the present study, we used nocodazole to induce MTs depolymerization and paclitaxel or recombinant (r) TIR (Toll/interleukin-1 receptor) domain containing protein (TcpB) to induce MT stabilization in bone marrow-derived macrophages infected with Brucella abortus...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29165592/folate-deficiency-induces-mitotic-aberrations-and-chromosomal-instability-by-compromising-the-spindle-assembly-checkpoint-in-cultured-human-colon-cells
#7
Xihan Guo, Juan Ni, Yuqian Zhu, Tao Zhou, Xiaoling Ma, Jinglun Xue, Xu Wang
Folates comprise the essential B9 vitamin that act as cofactors and cosubstrates in one-carbon metabolism for both biosynthesis and methylation of DNA and RNA. Folate deficiency (FD) has been shown to induce chromosomal instability (CIN), yet the underlying mechanisms are poorly understood. Here, we used human NCM460 colon mucosal cells as a model to investigate the effect of FD on spindle assembly checkpoint (SAC), a cell-cycle regulatory pathway preventing CIN during mitosis. Cells were maintained in medium containing 1...
November 19, 2017: Mutagenesis
https://www.readbyqxmd.com/read/29136577/atomic-force-microscopy-study-revealed-velocity-dependence-and-nonlinearity-of-nanoscale-poroelasticity-of-eukaryotic-cells
#8
Keyvan Mollaeian, Yi Liu, Siyu Bi, Juan Ren
Intracellular network deformation of the cell plays an important role in cellular shape formation. Recent studies suggest that cell reshaping and deformation due to external forces involve cellular volume, pore size, elasticity, and intracellular filaments polymerization degree change. This cell behavior can be described by poroelastic models due to the porous structure of the cytoplasm. In this study, the nanoscale poroelasticity of human mammary basal/claudin low carcinoma cell (MDA-MB-231) was investigated using indentation-based atomic force microscopy...
November 11, 2017: Journal of the Mechanical Behavior of Biomedical Materials
https://www.readbyqxmd.com/read/29134199/rapid-directed-transport-of-dc-sign-clusters-in-the-plasma-membrane
#9
Ping Liu, Violetta Weinreb, Marc Ridilla, Laurie Betts, Pratik Patel, Aravinda M de Silva, Nancy L Thompson, Ken Jacobson
C-type lectins, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), are all-purpose pathogen receptors that exist in nanoclusters in plasma membranes of dendritic cells. A small fraction of these clusters, obvious from the videos, can undergo rapid, directed transport in the plane of the plasma membrane at average speeds of more than 1 μm/s in both dendritic cells and MX DC-SIGN murine fibroblasts ectopically expressing DC-SIGN. Surprisingly, instantaneous speeds can be considerably greater...
November 2017: Science Advances
https://www.readbyqxmd.com/read/29120369/begomoviral-movement-protein-effects-in-human-and-plant-cells-towards-new-potential-interaction-partners
#10
Susanna Krapp, Christian Schuy, Eva Greiner, Irina Stephan, Barbara Alberter, Christina Funk, Manfred Marschall, Christina Wege, Susanne M Bailer, Tatjana Kleinow, Björn Krenz
Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MP(AbMV)), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells...
November 9, 2017: Viruses
https://www.readbyqxmd.com/read/29099266/calpain2-mediates-rab5-driven-focal-adhesion-disassembly-and-cell-migration
#11
Pablo A Mendoza, Patricio Silva, Jorge Díaz, Cecilia Arriagada, Jimena Canales, Oscar Cerda, Vicente A Torres
The early endosome protein Rab5 was recently shown to promote cell migration by enhancing focal adhesion disassembly through mechanisms that remain elusive. Focal adhesion disassembly is associated to proteolysis of talin, in a process that requires calpain2. Since calpain2 has been found at vesicles and endosomal compartments, we hypothesized that Rab5 stimulates calpain2 activity, leading to enhanced focal adhesion disassembly in migrating cells. We observed that calpain2 co-localizes with EEA1-positive early endosomes and co-immunoprecipitate with EEA1 and Rab5 in A549 lung carcinoma cells undergoing spreading, whereas Rab5 knock-down decreased the accumulation of calpain2 at early endosomal-enriched fractions...
November 3, 2017: Cell Adhesion & Migration
https://www.readbyqxmd.com/read/29092800/in-vivo-imaging-of-mauthner-axon-regeneration-remyelination-and-synapses-re-establishment-after-laser-axotomy-in-zebrafish-larvae
#12
Bing-Bing Hu, Min Chen, Rong-Chen Huang, Yu-Bin Huang, Yang Xu, Wu Yin, Lei Li, Bing Hu
Zebrafish is an excellent model to study central nervous system (CNS) axonal degeneration and regeneration since we can observe these processes in vivo and in real time in transparent larvae. Previous studies have shown that Mauthner cell (M-cell) axon regenerates poorly after mechanical spinal cord injury. Inconsistent with this result, however, we have found that M-cell possesses a great capacity for axon regeneration after two-photon laser ablation. By using ZEISS LSM 710 two-photon microscope, we performed specific unilateral axotomy of GFP labeled M-cells in the Tol-056 enhancer trap line larvae...
October 29, 2017: Experimental Neurology
https://www.readbyqxmd.com/read/29059502/effects-of-the-protonation-state-of-titratable-residues-and-the-presence-of-water-molecules-on-the-nocodazole-binding-to-%C3%AE-tubulin
#13
Dulce Consuelo Guzmán-Ocampo, Rodrigo Aguayo-Ortiz, Lucia Cano-González, Alicia Hernández-Campos, Rafael Castillo, Laura Dominguez
Regulation of microtubule assembly by antimitotic agents is a potential therapeutic strategy for the treatment of cancer, parasite infections, and neurodegenerative diseases. One of these agents is Nocodazole (NZ), which inhibits microtubule polymerization by binding to β-tubulin. NZ was recently co-crystallized in Gallus gallus tubulin, displaying novel information of the interaction features for ligand recognition and stability. In this work, we employed state of the art computational approaches to evaluate the protonation effects of titratable residues and the presence of water molecules in the binding of NZ...
October 23, 2017: ChemMedChem
https://www.readbyqxmd.com/read/29025057/nqo2-inhibition-relieves-reactive-oxygen-species-effects-on-mouse-oocyte-meiotic-maturation-and-embryo-development
#14
Dandan Chen, Xin Li, Xiaoyun Liu, Xiaoyu Liu, Xiuying Jiang, Juan Du, Qian Wang, Yuanjing Liang, Wei Ma
NRH: quinone oxidoreductase 2 (NQO2) is a cytosolic and ubiquitously expressed flavoprotein that catalyzes the two-electron reduction of quinone to hydroquinones. Herein, we assessed the protein expression, subcellular localization, and possible functions of NQO2 in mouse oocyte meiotic maturation and embryo development. Western blot analysis detected high and stable protein expression of NQO2 in mouse oocytes during meiotic progression. Immunofluorescence illustrated NQO2 distribution on nuclear membrane, chromosomes, and meiotic spindles...
October 1, 2017: Biology of Reproduction
https://www.readbyqxmd.com/read/29024608/erastin-like-anti-warburg-agents-prevent-mitochondrial-depolarization-induced-by-free-tubulin-and-decrease-lactate-formation-in-cancer-cells
#15
David N DeHart, John J Lemasters, Eduardo N Maldonado
In Warburg metabolism, suppression of mitochondrial metabolism contributes to a low cytosolic ATP/ADP ratio favoring enhanced aerobic glycolysis. Flux of metabolites across the mitochondrial outer membrane occurs through voltage-dependent anion channels (VDAC). In cancer cells, free dimeric tubulin induces VDAC closure and dynamically regulates mitochondrial membrane potential (ΔΨ). Erastin, a small molecule that binds to VDAC, antagonizes the inhibitory effect of tubulin on VDAC and hyperpolarizes mitochondria in intact cells...
September 1, 2017: SLAS Discovery
https://www.readbyqxmd.com/read/28994750/use-of-immunolabeling-to-analyze-stable-dynamic-and-nascent-microtubules-in-the-zebrafish-embryo
#16
Rebecca J McFarland, Sharlene P Brown, Eudorah Vital, Jonathan M Werner, Rachel M Brewster
Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments...
September 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28985013/hdac2-3-binding-and-deacetylation-of-bubr1-initiates-the-spindle-assembly-checkpoint-silencing
#17
Inai Park, Mi-Sun Kwon, Sangjin Paik, Hyeonjong Kim, Hae-Ock Lee, Eunhee Choi, Hyunsook Lee
BubR1 acetylation is essential in spindle assembly checkpoint (SAC) signaling. Here we show that BubR1 deacetylation is a signal that initiates the mitotic exit. Sustained BubR1 acetylation arrests the cells in metaphase, although chromosome congression is achieved. BubR1 deacetylation was coordinated with dephosphorylation in mitotic exit, suggesting the presence of a coordinated acetylation-phosphorylation code in mitotic signaling. HDAC2 and 3 bound to acetylated BubR1 exclusively in mitosis and led to the polyubiquitination of BubR1...
October 6, 2017: FEBS Journal
https://www.readbyqxmd.com/read/28950916/%C3%AE-carboxymuconolactone-decarboxylase-a-novel-cell-cycle-related-basal-body-protein-in-the-early-branching-eukaryote-trichomonas-vaginalis
#18
Wei-Hung Cheng, Kuo-Yang Huang, Po-Jung Huang, Chi-Ching Lee, Yuan-Ming Yeh, Fu-Man Ku, Rose Lin, Mei-Ling Cheng, Cheng-Hsun Chiu, Petrus Tang
BACKGROUND: γ-Carboxymuconolactone decarboxylase (CMD) participates in the β-ketoadipate pathway, which catalyzes aromatic compounds to produce acetyl- or succinyl-CoA, in prokaryotes and yeast. Our previous study demonstrated that expression of a CMD homologue that contains two signatures (dualCMD) is negatively regulated by iron in Trichomonas vaginalis. However, we were not able to identify the components of the β-ketoadipate pathway in the parasite's genome. These observations prompted us to investigate the biological functions of this novel CMD homologue in T...
September 26, 2017: Parasites & Vectors
https://www.readbyqxmd.com/read/28942021/a-novel-immunofluorescence-method-to-visualize-microtubules-in-the-antiparallel-overlaps-of-microtubule-plus-ends-in-the-anaphase-and-telophase-midzone
#19
Aya Ifuji, Takahisa Kuga, Yuichiro Kaibori, Youhei Saito, Yuji Nakayama
Cell division, in which duplicated chromosomes are separated into two daughter cells, is the most dynamic event during cell proliferation. Chromosome movement is powered mainly by microtubules, which vary in morphology and are organized into characteristic structures according to mitotic progression. During the later stages of mitosis, antiparallel microtubules form the spindle midzone, and the irregular formation of the midzone often leads to failure of cytokinesis, giving rise to the unequal segregation of chromosomes...
November 15, 2017: Experimental Cell Research
https://www.readbyqxmd.com/read/28934466/cohesin-acetyltransferase-esco2-regulates-sac-and-kinetochore-functions-via-maintaining-h4k16-acetylation-during-mouse-oocyte-meiosis
#20
Yajuan Lu, Xiaoxin Dai, Mianqun Zhang, Yilong Miao, Changyin Zhou, Zhaokang Cui, Bo Xiong
Sister chromatid cohesion, mediated by cohesin complex and established by the acetyltransferases Esco1 and Esco2, is essential for faithful chromosome segregation. Mutations in Esco2 cause Roberts syndrome, a developmental disease characterized by severe prenatal retardation as well as limb and facial abnormalities. However, its exact roles during oocyte meiosis have not clearly defined. Here, we report that Esco2 localizes to the chromosomes during oocyte meiotic maturation. Depletion of Esco2 by morpholino microinjection leads to the precocious polar body extrusion, the escape of metaphase I arrest induced by nocodazole treatment and the loss of BubR1 from kinetochores, indicative of inactivated SAC...
September 19, 2017: Nucleic Acids Research
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