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https://www.readbyqxmd.com/read/28722423/generation-of-a-cgmp-indicator-with-an-expanded-dynamic-range-by-optimization-of-amino-acid-linkers-between-a-fluorescent-protein-and-pde5%C3%AE
#1
Shogo Matsuda, Kazuki Harada, Motoki Ito, Mai Takizawa, Devina Wongso, Takashi Tsuboi, Tetsuya Kitaguchi
Here we describe the development of a single fluorescent protein (FP)-based cGMP indicator, Green cGull, based on the cGMP binding domain from mouse phosphodiesterase 5α. The dynamic range of Green cGull was enhanced to a 7.5-fold fluorescence change upon cGMP binding by optimization of the amino acid linkers between the cGMP binding domain and FP. Green cGull has excitation and emission peaks at 498 and 522 nm, respectively, and specifically responds to cGMP in a dose-dependent manner. Live cell imaging analysis revealed that addition of a nitric oxide (NO) donor induced different cGMP kinetics and was cell-type dependent...
January 27, 2017: ACS Sensors
https://www.readbyqxmd.com/read/28716648/applications-of-high-speed-atomic-force-microscopy-to-real-time-visualization-of-dynamic-biomolecular-processes
#2
REVIEW
Takayuki Uchihashi, Simon Scheuring
BACKGROUND: Many biological processes in a living cell are consequences of sequential and hierarchical dynamic events of biological macromolecules such as molecular interactions and conformational changes. Hence, knowledge of structures, assembly and dynamics of proteins is the foundation for understanding how biological molecules work. Among several techniques to analyze dynamics of proteins, high-speed atomic force microscopy (HS-AFM) is unique to provide direct information about both structure and dynamics of single proteins at work...
July 14, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28710754/the-fast-halo-assay-for-the-detection-of-dna-damage
#3
Piero Sestili, Cinzia Calcabrini, Anna Rita Diaz, Carmela Fimognari, Vilberto Stocchi
The need for express screening of the DNA damaging potential of chemicals has progressively increased over the past 20 years due to the wide number of new synthetic molecules to be evaluated, as well as the adoption of more stringent chemical regulations such as the EU REACH and risk reduction politics. In this regard, DNA diffusion assays such as the microelectrophoretic comet assay paved the way for rapid genotoxicity testing. A more significant simplification and speeding up of the experimental processes was achieved with the fast halo assay (FHA) described in the present chapter...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28708098/combining-primed-photoconversion-and-uv-photoactivation-for-aberration-free-live-cell-compliant-multi-color-single-molecule-localization-microscopy-imaging
#4
David Virant, Bartosz Turkowyd, Alexander Balinovic, Ulrike Endesfelder
Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel...
July 14, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28694303/single-molecule-fluorescence-microscopy-review-shedding-new-light-on-old-problems
#5
Sviatlana Shashkova, Mark C Leake
Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared to many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilise light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples...
July 10, 2017: Bioscience Reports
https://www.readbyqxmd.com/read/28690601/illuminating-messengers-an-update-and-outlook-on-rna-visualization-in-bacteria
#6
REVIEW
Lieke A van Gijtenbeek, Jan Kok
To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by Bertrand et al. (1998) and Femino et al. (1998). Since then, a plethora of techniques to image RNA molecules with the aid of fluorescence microscopy has emerged. Many of these approaches are useful for the large eukaryotic cells but their adaptation to study RNA, specifically mRNA molecules, in bacterial cells progressed relatively slow...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28684722/overview-of-single-molecule-speckle-sims-microscopy-and-its-electroporation-based-version-with-efficient-labeling-and-improved-spatiotemporal-resolution
#7
REVIEW
Sawako Yamashiro, Naoki Watanabe
Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy...
July 6, 2017: Sensors
https://www.readbyqxmd.com/read/28671584/an-infrared-actin-probe-for-deep-cell-electroporation-based-single-molecule-speckle-esims-microscopy
#8
Sawako Yamashiro, Naoki Watanabe
Single-molecule speckle (SiMS) microscopy is a powerful method to directly elucidate biochemical reactions in live cells. However, since the signal from an individual fluorophore is extremely faint, the observation area by epi-fluorescence microscopy is restricted to the thin cell periphery to reduce autofluorescence, or only molecules near the plasma membrane are visualized by total internal reflection fluorescence (TIRF) microscopy. Here, we introduce a new actin probe labeled with near infrared (NIR) emissive CF680R dye for easy-to-use, electroporation-based SiMS microscopy (eSiMS) for deep-cell observation...
July 1, 2017: Sensors
https://www.readbyqxmd.com/read/28639395/spatiotemporally-controllable-peptide-based-nanoassembly-in-single-living-cells-for-a-biological-self-portrait
#9
Yuanyuan Zhao, Xu Zhang, Zhipeng Li, Shuaidong Huo, Ke Zhang, Juntao Gao, Hao Wang, Xing-Jie Liang
Simultaneous precise localization and activity evaluation of a biomolecule in a single living cell is through an enzyme-specific signal-amplification process, which involves the localized, site-specific self-assembly, and activation of a presignaling molecule. The inactive presignaling tetraphenylethylene (TPE)-peptide derivative, TPE-YpYY, is nondetectable and highly biocompatible and these small molecules rapidly diffuse into living cells. Upon safely arriving at an active site, and accessing the catalytic pocket of an enzyme, TPE-YpYY immediately and quantitatively accumulates in situ in response to enzymatic activity, forms an enzyme anchor TPE-YYY nanoassembly, displays aggregation-induced emission behavior, and finally lights up the active enzyme, indicating its activity, and allowing its status in living cells to be tracked...
June 22, 2017: Advanced Materials
https://www.readbyqxmd.com/read/28635963/single-molecule-analysis-of-steroid-receptor-and-cofactor-action-in-living-cells
#10
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
#11
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
#12
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
#13
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 27, 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
#14
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
#15
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
#16
Bartosz Turkowyd, Alexander Balinovic, David Virant, Haruko G Gölz Carnero, Fabienne Caldana, Ma Marc Endesfelder, Dominique Bourgeois, 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
#17
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
#18
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
#19
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
#20
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
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