scholarly journals Fabricating polyacrylamide microbeads by inverse emulsification to mimic the size and elasticity of living cells

2017 ◽  
Vol 5 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Nicholas R. Labriola ◽  
Edith Mathiowitz ◽  
Eric M. Darling
Keyword(s):  
Elastic Properties ◽  
Mammalian Cells ◽  
Living Cells

Inverse emulsification was used to fabricate polyacrylamide (PAAm) microbeads with size and elastic properties similar to typical, mammalian cells.

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 Trends in SPR Cytometry: Advances in Label-Free Detection of Cell Parameters

Biosensors ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 102 ◽  
Author(s):  
Richard Schasfoort ◽  
Fikri Abali ◽  
Ivan Stojanovic ◽  
Gestur Vidarsson ◽  
Leon Terstappen
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Mammalian Cells ◽  
Living Cells ◽  
Label Free ◽  
Intact Cells ◽  
Cell Parameters ◽  
Spr Imaging ◽  
Label Free Detection

SPR cytometry entails the measurement of parameters from intact cells using the surface plasmon resonance (SPR) phenomenon. Specific real-time and label-free binding of living cells to sensor surfaces has been made possible through the availability of SPR imaging (SPRi) instruments and researchers have started to explore its potential in the last decade. Here we will discuss the mechanisms of detection and additionally describe the problems and issues of mammalian cells in SPR biosensing, both from our own experience and with information from the literature. Finally, we build on the knowledge and applications that has already materialized in this field to give a forecast of some exciting applications for SPRi cytometry.

scholarly journals Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Biology ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 430
Author(s):  
Amreen ◽  
Hayssam M. Ali ◽  
Mohammad Ahmad ◽  
Mohamed Z. M. Salem ◽  
Altaf Ahmad
Keyword(s):  
Hydrogen Peroxide ◽  
Real Time ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Resonance Energy ◽  
Living Cells ◽  
Stress Conditions ◽  
Live Cells ◽  
Cell Physiology ◽  
Broad Dynamic Range

Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.

Compartmentalized signalling of Ras

2005 ◽  
Vol 33 (4) ◽  
pp. 657-661 ◽  
Author(s):  
M.R. Philips
Keyword(s):  
Subcellular Localization ◽  
Mammalian Cells ◽  
Hypervariable Region ◽  
Living Cells ◽  
Signalling Pathways ◽  
Outer Mitochondrial Membrane ◽  
Ras Proteins ◽  
Post Translational Modifications ◽  
Transient Activation ◽  
Sustained Activation

Ras proteins associate with cellular membranes by virtue of a series of post-translational modifications of their C-terminal CAAX sequences. The discovery that two of the three enzymes that modify CAAX proteins are restricted to the endoplasmic reticulum led to the recognition that all nascent Ras proteins transit endomembranes en route to the PM (plasma membrane) and that at steady-state N-Ras and H-Ras are highly expressed on the Golgi apparatus. To test the hypothesis that Ras proteins on internal membranes can signal, we developed a fluorescent probe that reports when and where in living cells Ras becomes active. We found that growth factors stimulated rapid and transient activation of Ras on the PM followed by delayed and sustained activation on the Golgi. We mapped one pathway responsible for this activity as involving PLCγ (phospholipase Cγ)/DAG (diacylglycerol)+Ca2+/RasGRP1. Using mammalian cells and fission yeast, we have shown that differential localization of activated Ras preferentially activates distinct signalling pathways. In very recent work, we have found that (i) the subcellular localization of K-Ras can be acutely modulated by phosphorylation of its C-terminal hypervariable region by PKC, (ii) among the membranes upon which phosphorylated K-Ras accumulates is the outer mitochondrial membrane and (iii) phosphorylated, internalized K-Ras promotes apoptosis. Thus the signalling output of Ras depends on its subcellular localization.

scholarly journals Mass spectrometry-based methods for analysing the mitochondrial interactome in mammalian cells

2019 ◽  
Vol 167 (3) ◽  
pp. 225-231 ◽  
Author(s):  
Takumi Koshiba ◽  
Hidetaka Kosako
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Living Cells ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Protein Protein Interaction ◽  
Membrane Protein Complexes ◽  
Protein Protein Interaction Networks

Abstract Protein–protein interactions are essential biologic processes that occur at inter- and intracellular levels. To gain insight into the various complex cellular functions of these interactions, it is necessary to assess them under physiologic conditions. Recent advances in various proteomic technologies allow to investigate protein–protein interaction networks in living cells. The combination of proximity-dependent labelling and chemical cross-linking will greatly enhance our understanding of multi-protein complexes that are difficult to prepare, such as organelle-bound membrane proteins. In this review, we describe our current understanding of mass spectrometry-based proteomics mapping methods for elucidating organelle-bound membrane protein complexes in living cells, with a focus on protein–protein interactions in mitochondrial subcellular compartments.

scholarly journals Influence of the PDMS substrate stiffness on the adhesion of Acanthamoeba castellanii

2014 ◽  
Vol 5 ◽  
pp. 1393-1398 ◽  
Author(s):  
Sören B Gutekunst ◽  
Carsten Grabosch ◽  
Alexander Kovalev ◽  
Stanislav N Gorb ◽  
Christine Selhuber-Unkel
Keyword(s):  
Cell Adhesion ◽  
Elastic Properties ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Acanthamoeba Castellanii ◽  
Substrate Stiffness ◽  
Human Pathogen ◽  
Human Pathogens ◽  
Young’S Moduli ◽  
Influence Of Substrate

Background: Mechanosensing of cells, particularly the cellular response to substrates with different elastic properties, has been discovered in recent years, but almost exclusively in mammalian cells. Much less attention has been paid to mechanosensing in other cell systems, such as in eukaryotic human pathogens. Results: We report here on the influence of substrate stiffness on the adhesion of the human pathogen Acanthamoebae castellanii (A. castellanii). By comparing the cell adhesion area of A. castellanii trophozoites on polydimethylsiloxane (PDMS) substrates with different Young’s moduli (4 kPa, 29 kPa, and 128 kPa), we find significant differences in cell adhesion area as a function of substrate stiffness. In particular, the cell adhesion area of A. castellanii increases with a decreasing Young’s modulus of the substrate. Conclusion: The dependence of A. castellanii adhesion on the elastic properties of the substrate is the first study suggesting a mechanosensory effect for a eukaryotic human pathogen. Interestingly, the main targets of A. castellanii infections in the human body are the eye and the brain, i.e., very soft environments. Thus, our study provides first hints towards the relevance of mechanical aspects for the pathogenicity of eukaryotic parasites.

scholarly journals Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells

2010 ◽  
Vol 107 (5) ◽  
pp. 1870-1875 ◽  
Author(s):  
Alex K. Shalek ◽  
Jacob T. Robinson ◽  
Ethan S. Karp ◽  
Jin Seok Lee ◽  
Dae-Ro Ahn ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Silicon Nanowires ◽  
Mammalian Cells ◽  
Living Cells ◽  
Diverse Range ◽  
Viral Packaging ◽  
Neuronal Progenitor ◽  
Efficient Delivery ◽  
Surface Modified

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell’s membrane and subsequently release surface-bound molecules directly into the cell’s cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology’s potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

scholarly journals Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

2008 ◽  
Vol 410 (3) ◽  
pp. 463-472 ◽  
Author(s):  
Jesper S. Hansen ◽  
Nils J. Færgeman ◽  
Birthe B. Kragelund ◽  
Jens Knudsen
Keyword(s):  
Endoplasmic Reticulum ◽  
Fatty Acid ◽  
Ligand Binding ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Living Cells ◽  
Dependent Manner ◽  
Fold Reduction ◽  
Epithelial Cell Lines ◽  
Mammary Gland Epithelial Cell

In the present study, we microinjected fluorescently labelled liver bovine ACBP (acyl-CoA-binding protein) [FACI-50 (fluorescent acyl-CoA indicator-50)] into HeLa and BMGE (bovine mammary gland epithelial) cell lines to characterize the localization and dynamics of ACBP in living cells. Results showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations. Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved in vesicular trafficking.

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