scholarly journals BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy

2015 ◽  
Vol 112 (22) ◽  
pp. 7027-7032 ◽  
Author(s):  
Shouhei Kobayashi ◽  
Takako Koujin ◽  
Tomoko Kojidani ◽  
Hiroko Osakada ◽  
Chie Mori ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cellular Response ◽  
Living Cells ◽  
Dna Sensor ◽  
Exogenous Dna ◽  
Polystyrene Beads ◽  
Pathogen Invasion ◽  
Spatiotemporal Information ◽  
Double Stranded Dna ◽  
A Cell

Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell’s response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF+ DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

scholarly journals CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

2021 ◽  
Author(s):  
Long Jiang ◽  
Katrine Ingelshed ◽  
Yunbing Shen ◽  
Sanjaykumar V. Boddul ◽  
Vaishnavi Srinivasan Iyer ◽  
...  
Keyword(s):  
Cellular Response ◽  
Human Cancer ◽  
Genetic Alterations ◽  
Dna Breaks ◽  
Data Set ◽  
Human Cancer Cell Lines ◽  
Cycle Arrest ◽  
Double Stranded Dna ◽  
A Cell

CRISPR/Cas9 can be used to inactivate or modify genes by inducing double-stranded DNA breaks1–3. As a protective cellular response, DNA breaks result in p53-mediated cell cycle arrest and activation of cell death programs4,5. Inactivating p53 mutations are the most commonly found genetic alterations in cancer, highlighting the important role of the gene6–8. Here, we show that cells deficient in p53, as well as in genes of a core CRISPR-p53 tumor suppressor interactome, are enriched in a cell population when CRISPR is applied. Such enrichment could pose a challenge for clinical CRISPR use. Importantly, we identify that transient p53 inhibition suppresses the enrichment of cells with these mutations. Furthermore, in a data set of >800 human cancer cell lines, we identify parameters influencing the enrichment of p53 mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, our data identify strategies enabling safe CRISPR use.

scholarly journals Mechanistic studies of gene delivery into mammalian cells by electrical short-circuiting via an aqueous droplet in dielectric oil

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243361
Author(s):  
Hirofumi Kurita ◽  
Hirohito Nihonyanagi ◽  
Yuki Watanabe ◽  
Kenta Sugano ◽  
Ryuto Shinozaki ◽  
...  
Keyword(s):  
Electric Field ◽  
Mammalian Cells ◽  
Short Circuit ◽  
Gene Transfection ◽  
Jurkat Cells ◽  
Dependent Manner ◽  
Exogenous Dna ◽  
Membrane Pore ◽  
A Cell ◽  
Aqueous Droplet

We have developed a novel methodology for the delivery of cell-impermeable molecules, based on electrical short-circuiting via a water droplet in dielectric oil. When a cell suspension droplet is placed between a pair of electrodes with an intense DC electric field, droplet bouncing and droplet deformation, which results in an instantaneous short-circuit, can be induced, depending on the electric field strength. We have demonstrated successful transfection of various mammalian cells using the short-circuiting; however, the molecular mechanism remains to be elucidated. In this study, flow cytometric assays were performed with Jurkat cells. An aqueous droplet containing Jurkat cells and plasmids carrying fluorescent proteins was treated with droplet bouncing or short-circuiting. The short-circuiting resulted in sufficient cell viability and fluorescent protein expression after 24 hours’ incubation. In contrast, droplet bouncing did not result in successful gene transfection. Transient membrane pore formation was investigated by uptake of a cell-impermeable fluorescence dye YO-PRO-1 and the influx of calcium ions. As a result, short-circuiting increased YO-PRO-1 fluorescence intensity and intracellular calcium ion concentration, but droplet bouncing did not. We also investigated the contribution of endocytosis to the transfection. The pre-treatment of cells with endocytosis inhibitors decreased the efficiency of gene transfection in a concentration-dependent manner. Besides, the use of pH-sensitive dye conjugates indicated the formation of an acidic environment in the endosomes after the short-circuiting. Endocytosis is a possible mechanism for the intracellular delivery of exogenous DNA.

scholarly journals Ensuring the Stability of the Genome: DNA Damage Checkpoints

2001 ◽  
Vol 1 ◽  
pp. 684-702 ◽  
Author(s):  
Christine Latif ◽  
Susan H. Harvey ◽  
Susan J. O'Connell
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Model Systems ◽  
Checkpoint Proteins ◽  
Functional Conservation ◽  
Cycle Arrest ◽  
A Cell ◽  
The Stability

The cellular response to DNA damage is vital for the cell�s ability to maintain genomic integrity. Checkpoint signalling pathways, which induce a cell cycle arrest in response to DNA damage, are an essential component of this process. This is reflected by the functional conservation of these pathways in all eukaryotes from yeast to mammalian cells. This review will examine the cellular response to DNA damage throughout the cell cycle. A key component of the DNA damage response is checkpoint signalling, which monitors the state of the genome prior to DNA replication (G1/S) and chromosome segregation (G2/M). Checkpoint signalling in model systems including mice, Xenopus laevis, Drosophila melanogaster, and the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been useful in elucidating these pathways in mammalian cells. An examination of this research, with emphasis on the function of checkpoint proteins, their relationship to DNA repair, and their involvement in oncogenesis is undertaken here.

Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells

Science ◽  
2011 ◽  
Vol 333 (6047) ◽  
pp. 1307-1311 ◽  
Author(s):  
Zhen Xie ◽  
Liliana Wroblewska ◽  
Laura Prochazka ◽  
Ron Weiss ◽  
Yaakov Benenson
Keyword(s):  
Cellular Response ◽  
High Specificity ◽  
Cell Types ◽  
Living Cells ◽  
Logic Circuit ◽  
Cell Type ◽  
Expression Levels ◽  
Regulatory Circuit ◽  
Specific Cancer ◽  
A Cell

Engineered biological systems that integrate multi-input sensing, sophisticated information processing, and precisely regulated actuation in living cells could be useful in a variety of applications. For example, anticancer therapies could be engineered to detect and respond to complex cellular conditions in individual cells with high specificity. Here, we show a scalable transcriptional/posttranscriptional synthetic regulatory circuit—a cell-type “classifier”—that senses expression levels of a customizable set of endogenous microRNAs and triggers a cellular response only if the expression levels match a predetermined profile of interest. We demonstrate that a HeLa cancer cell classifier selectively identifies HeLa cells and triggers apoptosis without affecting non-HeLa cell types. This approach also provides a general platform for programmed responses to other complex cell states.

scholarly journals The DNA Sensor IFIX Drives Proteome Alterations To Mobilize Nuclear and Cytoplasmic Antiviral Responses, with Its Acetylation Acting as a Localization Toggle

mSystems ◽  
2021 ◽  
Author(s):  
Timothy R. Howard ◽  
Marni S. Crow ◽  
Todd M. Greco ◽  
Krystal K. Lum ◽  
Tuo Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cytokine Expression ◽  
Dna Sensor ◽  
Dna Sensors ◽  
Antiviral Responses ◽  
Double Stranded Dna ◽  
Spread Of Infection

Mammalian cells must be able to detect and respond to invading pathogens to prevent the spread of infection. DNA sensors, such as IFIX, are proteins that bind to pathogen-derived double-stranded DNA and induce antiviral cytokine expression.

scholarly journals STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

2020 ◽  
Vol 219 (12) ◽  
Author(s):  
Tara D. Fischer ◽  
Chunxin Wang ◽  
Benjamin S. Padman ◽  
Michael Lazarou ◽  
Richard J. Youle
Keyword(s):  
Mammalian Cells ◽  
Membrane Vesicles ◽  
Double Stranded Dna ◽  
Single Membrane ◽  
Interferon Pathway ◽  
A Cell ◽  
Wd40 Domain ◽  
Mediate Cell ◽  
Sting Pathway ◽  
Cyclic Dinucleotide

Following the detection of cytosolic double-stranded DNA from viral or bacterial infection in mammalian cells, cyclic dinucleotide activation of STING induces interferon β expression to initiate innate immune defenses. STING activation also induces LC3B lipidation, a classical but equivocal marker of autophagy, that promotes a cell-autonomous antiviral response that arose before evolution of the interferon pathway. We report that STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles mediated by ATG16L1 via its WD40 domain, bypassing the requirement of canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 that binds and inhibits the vacuolar ATPase (V-ATPase) and by SopF, a bacterial effector that catalytically modifies the V-ATPase to inhibit LC3B lipidation via ATG16L1. These results indicate that activation of the cGAS-STING pathway induces V-ATPase–dependent LC3B lipidation that may mediate cell-autonomous host defense, an unanticipated mechanism that is distinct from LC3B lipidation onto double-membrane autophagosomes.

Use of Flexible Materials with a Novel Pressure Driven Equibiaxial Cell Stretching Device for Mechanical Stimulation of Single Mammalian Cells

2003 ◽  
Vol 773 ◽  
Author(s):  
James D. Kubicek ◽  
Stephanie Brelsford ◽  
Philip R. LeDuc
Keyword(s):  
Cell Fate ◽  
Mechanical Stimulation ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Single Cells ◽  
Complex Nature ◽  
Cellular Behavior ◽  
Cell Stretching ◽  
Stimulation Of ◽  
Pressure Driven

AbstractMechanical stimulation of single cells has been shown to affect cellular behavior from the molecular scale to ultimate cell fate including apoptosis and proliferation. In this, the ability to control the spatiotemporal application of force on cells through their extracellular matrix connections is critical to understand the cellular response of mechanotransduction. Here, we develop and utilize a novel pressure-driven equibiaxial cell stretching device (PECS) combined with an elastomeric material to control specifically the mechanical stimulation on single cells. Cells were cultured on silicone membranes coated with molecular matrices and then a uniform pressure was introduced to the opposite surface of the membrane to stretch single cells equibiaxially. This allowed us to apply mechanical deformation to investigate the complex nature of cell shape and structure. These results will enhance our knowledge of cellular and molecular function as well as provide insights into fields including biomechanics, tissue engineering, and drug discovery.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

scholarly journals Magnetic Nanoparticles Mediated-Gene Delivery for Simpler and More Effective Transformation of Pichia pastoris

2021 ◽  
Author(s):  
Seyda Yildiz ◽  
Kubra Solak ◽  
Melek Acar ◽  
Ahmet Mavi ◽  
Yagmur Unver
Keyword(s):  
Magnetic Nanoparticles ◽  
Pichia Pastoris ◽  
Gene Delivery ◽  
Delivery Method ◽  
Exogenous Dna ◽  
Novel Gene ◽  
A Cell

The introduction of exogenous DNA into a cell can be used to produce large quantities of protein. Here, we describe a novel gene delivery method into Pichia pastoris based on...

scholarly journals xCELLanalyzer: A Framework for the Analysis of Cellular Impedance Measurements for Mode of Action Discovery

2019 ◽  
Vol 24 (3) ◽  
pp. 213-223 ◽  
Author(s):  
Raimo Franke ◽  
Bettina Hinkelmann ◽  
Verena Fetz ◽  
Theresia Stradal ◽  
Florenz Sasse ◽  
...  
Keyword(s):  
Data Analysis ◽  
Bioactive Compounds ◽  
Mode Of Action ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Label Free ◽  
Synthesis Inhibitor ◽  
Analysis Pipeline ◽  
Bioactive Natural Products ◽  
Data Analysis Pipeline

Mode of action (MoA) identification of bioactive compounds is very often a challenging and time-consuming task. We used a label-free kinetic profiling method based on an impedance readout to monitor the time-dependent cellular response profiles for the interaction of bioactive natural products and other small molecules with mammalian cells. Such approaches have been rarely used so far due to the lack of data mining tools to properly capture the characteristics of the impedance curves. We developed a data analysis pipeline for the xCELLigence Real-Time Cell Analysis detection platform to process the data, assess and score their reproducibility, and provide rank-based MoA predictions for a reference set of 60 bioactive compounds. The method can reveal additional, previously unknown targets, as exemplified by the identification of tubulin-destabilizing activities of the RNA synthesis inhibitor actinomycin D and the effects on DNA replication of vioprolide A. The data analysis pipeline is based on the statistical programming language R and is available to the scientific community through a GitHub repository.

Sign in / Sign up

Export Citation Format

Share Document