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https://www.readbyqxmd.com/read/28635963/single-molecule-analysis-of-steroid-receptor-and-cofactor-action-in-living-cells
#1
Ville Paakinaho, Diego M Presman, David A Ball, Thomas A Johnson, R Louis Schiltz, Peter Levitt, Davide Mazza, Tatsuya Morisaki, Tatiana S Karpova, Gordon L Hager
Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors...
June 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/28627118/optical-microresonators-for-sensing-and-transduction-a-materials-perspective
#2
REVIEW
Kevin D Heylman, Kassandra A Knapper, Erik H Horak, Morgan T Rea, Sudheer K Vanga, Randall H Goldsmith
Optical microresonators confine light to a particular microscale trajectory, are exquisitely sensitive to their microenvironment, and offer convenient readout of their optical properties. Taken together, this is an immensely attractive combination that makes optical microresonators highly effective as sensors and transducers. Meanwhile, advances in material science, fabrication techniques, and photonic sensing strategies endow optical microresonators with new functionalities, unique transduction mechanisms, and in some cases, unparalleled sensitivities...
June 19, 2017: Advanced Materials
https://www.readbyqxmd.com/read/28618223/covalent-protein-labeling-and-improved-single-molecule-optical-properties-of-aqueous-cdse-cds-quantum-dots
#3
Sara M Wichner, Victor R Mann, Alexander S Powers, Maya A Segal, Mustafa Mir, Jigar N Bandaria, Mark A DeWitt, Xavier Darzacq, Ahmet Yildiz, Bruce E Cohen
Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands...
June 21, 2017: ACS Nano
https://www.readbyqxmd.com/read/28607072/adaptor-protein-mediates-dynamic-pump-assembly-for-bacterial-metal-efflux
#4
Ace George Santiago, Tai-Yen Chen, Lauren A Genova, Won Jung, Alayna M George Thompson, Megan M McEvoy, Peng Chen
Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living Escherichia coli cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals...
June 12, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28586196/real-time-imaging-of-endocytosis-and-intracellular-trafficking-of-semiconducting-polymer-dots
#5
Yuping Han, Xiaoming Li, Haobin Chen, Xingjie Hu, Yao Luo, Ting Wang, Zejun Wang, Qian Li, Chunhai Fan, Jiye Shi, Lihua Wang, Yun Zhao, Changfeng Wu, Nan Chen
Semiconducting polymer dots (Pdots) have shown great promise in biomedical applications, including biosensing, drug delivery, and live imaging of cells and biomolecules. Insight into the mechanism and regulation of cellular uptake and intracellular metabolism of Pdots is important for the development of superior Pdots-based theranostic nanoconjugates. Herein, we performed real-time imaging of endocytosis and intracellular trafficking of a type of fluorescent Pdots that showed excellent biocompatibility in various types of cells...
June 13, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28580376/reversible-cryo-arrests-of-living-cells-to-pause-molecular-movements-for-high-resolution-imaging
#6
Jan Huebinger, Martin E Masip, Jens Christmann, Frank Wehner, Philippe I H Bastiaens
Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells...
April 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28574633/a-general-mechanism-of-photoconversion-of-green-to-red-fluorescent-proteins-based-on-blue-and-infrared-light-reduces-phototoxicity-in-live-cell-single-molecule-imaging
#7
Bartosz Turkowyd, Alexander Balinovic, David Virant, Haruko Gladys Gölz Carnero, Fabienne Caldana, Ulrike Endesfelder
Photoconversion of fluorescent proteins by blue and complementary near-infrared light, termed primed conversion (PC), is a mechanism recently discovered for Dendra2. We demonstrate that controlling the conformation of arginine at residue 66 by threonine at residue 69 of fluorescent proteins from Anthozoan families (Dendra2, mMaple, Eos, mKikGR, pcDronpa protein families) represents a general route to facilitate PC. Mutations of alanine 159 or serine 173, which are known to influence chromophore flexibility and allow for reversible photoswitching, prevent PC...
June 2, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28534613/flashbody-a-next-generation-fluobody-with-fluorescence-intensity-enhanced-by-antigen-binding
#8
Devina Wongso, Jinhua Dong, Hiroshi Ueda, Tetsuya Kitaguchi
Fluorescent probes are valuable tools for visualizing the spatiotemporal dynamics of molecules in living cells. Here we developed a genetically encoded antibody probe with antigen-dependent fluorescence intensity called "Flashbody". We first created a fusion of EGFP to the single chain variable region fragment (scFv) of antibody against seven amino acids of the bone Gla protein C-terminus (BGPC7) called BGP Fluobody, which successfully showed the intracellular localization of BGPC7-tagged protein. To generate BGP Flashbody, circularly permuted GFP was inserted in between two variable region fragments, and the linkers were optimized, resulting in fluorescence intensity increase of 300% upon binding with BGPC7 in a dose-dependent manner...
June 6, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28528628/millisecond-time-resolution-correlative-light-and-electron-microscopy-for-dynamic-cellular-processes
#9
Ludek Stepanek, Gaia Pigino
Molecular motors propel cellular components at velocities up to microns per second with nanometer precision. Imaging techniques combining high temporal and spatial resolution are therefore indispensable to understand the cellular mechanics at the molecular level. For example, intraflagellar transport (IFT) trains constantly shuttle ciliary components between the base and tip of the eukaryotic cilium. 3-D electron microscopy has revealed IFT train morphology and position, but was unable to correlate these features with the direction of train movement...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28523559/techniques-for-single-molecule-mrna-imaging-in-living-cells
#10
Kevin Czaplinski
Typical measurement of macromolecules in a biological sample typically averages the result over all the cells or molecules within the sample, and while these types of measurements provide very useful information, they completely miss heterogeneity among the components within the sample that could be a very important aspect of the sample's function. These techniques are also limited in their ability to examine intracellular spatial orientation of molecular activity, which is often a critical component to the regulation of biological processes, particularly in cells with unique spatial relationships, such as neurons...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/28495969/fold-change-detection-and-scale-invariance-of-cell-cell-signaling-in-social-amoeba
#11
Keita Kamino, Yohei Kondo, Akihiko Nakajima, Mai Honda-Kitahara, Kunihiko Kaneko, Satoshi Sawai
Cell-cell signaling is subject to variability in the extracellular volume, cell number, and dilution that potentially increase uncertainty in the absolute concentrations of the extracellular signaling molecules. To direct cell aggregation, the social amoebae Dictyostelium discoideum collectively give rise to oscillations and waves of cyclic adenosine 3',5'-monophosphate (cAMP) under a wide range of cell density. To date, the systems-level mechanism underlying the robustness is unclear. By using quantitative live-cell imaging, here we show that the magnitude of the cAMP relay response of individual cells is determined by fold change in the extracellular cAMP concentrations...
May 23, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28494964/near-membrane-refractometry-using-supercritical-angle-fluorescence
#12
Maia Brunstein, Lopamudra Roy, Martin Oheim
Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the "footprint" region of the cell during image acquisition...
May 9, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28493880/identifying-stochastic-oscillations-in-single-cell-live-imaging-time-series-using-gaussian-processes
#13
Nick E Phillips, Cerys Manning, Nancy Papalopulu, Magnus Rattray
Multiple biological processes are driven by oscillatory gene expression at different time scales. Pulsatile dynamics are thought to be widespread, and single-cell live imaging of gene expression has lead to a surge of dynamic, possibly oscillatory, data for different gene networks. However, the regulation of gene expression at the level of an individual cell involves reactions between finite numbers of molecules, and this can result in inherent randomness in expression dynamics, which blurs the boundaries between aperiodic fluctuations and noisy oscillators...
May 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28488813/imaging-mass-spectrometry-for-metabolites-technical-progress-multimodal-imaging-and-biological-interactions
#14
REVIEW
Ying-Ning Ho, Lin-Jie Shu, Yu-Liang Yang
Imaging mass spectrometry (IMS) allows the study of the spatial distribution of small molecules in biological samples. IMS is able to identify and quantify chemicals in situ from whole tissue sections to single cells. Both vacuum mass spectrometry (MS) and ambient MS systems have advanced considerably over the last decade; however, some limitations are still hard to surmount. Sample pretreatment, matrix or solvent choices, and instrument improvement are the key factors that determine the successful application of IMS to different samples and analytes...
May 10, 2017: Wiley Interdisciplinary Reviews. Systems Biology and Medicine
https://www.readbyqxmd.com/read/28484218/a-molecular-beacon-based-approach-for-live-cell-imaging-of-rna-transcripts-with-minimal-target-engineering-at-the-single-molecule-level
#15
Mingming Chen, Zhao Ma, Xiaotian Wu, Shiqi Mao, Yantao Yang, Jie Tan, Christopher J Krueger, Antony K Chen
Analysis of RNA dynamics and localization at the single-molecule level in living cells has been predominantly achieved by engineering target RNAs with large insertions of tandem repeat sequences that are bound by protein-based or oligonucleotide-based fluorescent probes. Thus, individual RNAs are tagged by multiple fluorescent probes, making them detectable by fluorescence microscopy. Since large insertions may affect RNA processes including trafficking and localization, here we present a strategy to visualize single RNA transcripts in living cells using molecular beacons (MBs) - fluorogenic oligonucleotide probes - with minimal target engineering...
May 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28481515/sequence-directed-covalent-protein-dna-linkages-in-a-single-step-using-huh-tags
#16
Klaus N Lovendahl, Amanda N Hayward, Wendy R Gordon
We present a robust strategy to covalently link proteins and DNA using HUH-endonuclease domains as fusion partners (HUH-tags). We show that HUH-tags react robustly with specific sequences of unmodified single-stranded DNA, and we have identified five tags that react orthogonally with distinct DNA sequences. We demonstrate the versatility of HUH-tags as fusion partners in Cas9-mediated gene editing and the construction of doubly DNA-tethered proteins for single-molecule studies. Finally we demonstrate application to cellular imaging in live and fixed cells...
May 16, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28448050/single-molecule-analysis-of-laser-localized-psoralen-adducts
#17
Jing Huang, Himabindu Gali, Julia Gichimu, Marina A Bellani, Durga Pokharel, Manikandan Paramasivam, Michael M Seidman
The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies...
April 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28426025/modular-low-light-microscope-for-imaging-cellular-bioluminescence-and-radioluminescence
#18
Tae Jin Kim, Silvan Türkcan, Guillem Pratx
Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy techniques, such as bioluminescence, chemiluminescence or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back to back from each other, using standard optomechanical components...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28424513/super-multiplex-vibrational-imaging
#19
Lu Wei, Zhixing Chen, Lixue Shi, Rong Long, Andrew V Anzalone, Luyuan Zhang, Fanghao Hu, Rafael Yuste, Virginia W Cornish, Wei Min
The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions...
April 27, 2017: Nature
https://www.readbyqxmd.com/read/28414097/single-molecule-and-super-resolution-imaging-of-transcription-in-living-bacteria
#20
REVIEW
Mathew Stracy, Achillefs N Kapanidis
In vivo single-molecule and super-resolution techniques are transforming our ability to study transcription as it takes place in its native environment in living cells. This review will detail the methods for imaging single molecules in cells, and the data-analysis tools which can be used to extract quantitative information on the spatial organization, mobility, and kinetics of the transcription machinery from these experiments. Furthermore, we will highlight studies which have applied these techniques to shed new light on bacterial transcription...
April 13, 2017: Methods: a Companion to Methods in Enzymology
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