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Single molecule microscopy

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https://www.readbyqxmd.com/read/28317888/direct-evidence-of-lack-of-colocalisation-of-fluorescently-labelled-gold-labels-used-in-correlative-light-electron-microscopy
#1
Benjamin T Miles, Alexander B Greenwood, David Benito-Alifonso, Hugh Tanner, M Carmen Galan, Paul Verkade, Henkjan Gersen
Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle...
March 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28315485/quantifying-transcription-factor-binding-dynamics-at-the-single-molecule-level-in-live-cells
#2
Diego M Presman, David A Ball, Ville Paakinaho, Jonathan B Grimm, Luke D Lavis, Tatiana S Karpova, Gordon L Hager
Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data...
March 14, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28303166/single-molecule-studies-of-high-mobility-group-b-architectural-dna-bending-proteins
#3
REVIEW
Divakaran Murugesapillai, Micah J McCauley, L James Maher, Mark C Williams
Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function...
February 2017: Biophysical Reviews
https://www.readbyqxmd.com/read/28300263/size-specific-emission-in-peptide-capped-gold-quantum-clusters-with-tunable-photoswitching-behavior
#4
Abhishek Baral, Kingshuk Basu, Sirshendu Ghosh, Kalishankar Bhattacharyya, Subhasish Roy, Ayan Datta, Arindam Banerjee
Three different types of fluorescent gold clusters (namely blue, green and red emitting) have been prepared from a gold precursor (chloroauric acid) under moderate conditions in aqueous medium. A cysteine containing dipeptide has been used for the formation of these quantum clusters as this peptide molecule contains a thiol group in the side chain to cap these nascently formed clusters and the free amino and carboxylic moieties assist in water solubility. Thus, the clusters are also environmentally friendly as the capped peptide is made up of only naturally occurring protein amino acids...
March 16, 2017: Nanoscale
https://www.readbyqxmd.com/read/28295806/proteins-mediating-dna-loops-effectively-block-transcription
#5
Zsuzsanna Vörös, Yan Yan, Daniel T Kovari, Laura Finzi, David Dunlap
Loops are ubiquitous topological elements formed when proteins simultaneously bind to two non-contiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM)...
March 14, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28294967/single-lipid-molecule-dynamics-on-supported-lipid-bilayers-with-membrane-curvature
#6
Philip P Cheney, Alan W Weisgerber, Alec M Feuerbach, Michelle K Knowles
The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature...
March 15, 2017: Membranes
https://www.readbyqxmd.com/read/28294302/polyclonal-and-monoclonal-igg-binding-on-protein-a-resins-evidence-of-competitive-binding-effects
#7
Justin Weinberg, Shaojie Zhang, Gillian Crews, Edward Healy, Giorgio Carta, Todd Przybycien
Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region...
March 14, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28288800/determining-the-limitations-and-benefits-of-noise-in-gene-regulation-and-signal-transduction-through-single-cell-microscopy-based-analysis
#8
REVIEW
Marie D Harton, Eric Batchelor
Stochastic fluctuations, termed "noise," in the level of biological molecules can greatly impact cellular functions. While biological noise can sometimes be detrimental, recent studies have provided an increasing number of examples in which biological noise can be functionally beneficial. Rather than provide an exhaustive review of the growing literature in this field, in this review we focus on single cell studies based on quantitative microscopy that have generated a deeper understanding of the sources, characteristics, limitations, and benefits of biological noise...
March 10, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/28287952/single-molecule-observation-of-dna-compaction-by-meiotic-protein-sycp3
#9
Johanna L Syrjänen, Iddo Heller, Andrea Candelli, Owen R Davies, Erwin J G Peterman, Gijs J L Wuite, Luca Pellegrini
In a previous paper (Syrjänen et al., 2014), we reported the first structural characterisation of a synaptonemal complex (SC) protein, SYCP3, which led us to propose a model for its role in chromosome compaction during meiosis. As a component of the SC lateral element, SYCP3 has a critical role in defining the specific chromosome architecture required for correct meiotic progression. In the model, the reported compaction of chromosomal DNA caused by SYCP3 would result from its ability to bridge distant sites on a DNA molecule with the DNA-binding domains located at each end of its strut-like structure...
March 13, 2017: ELife
https://www.readbyqxmd.com/read/28287710/single-molecule-localization-microscopy-in-eukaryotes
#10
Markus Sauer, Mike Heilemann
Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures...
March 13, 2017: Chemical Reviews
https://www.readbyqxmd.com/read/28287156/single-molecule-investigation-of-kinesin-1-motility-using-engineered-microtubule-defects
#11
Michael W Gramlich, Leslie Conway, Winnie H Liang, Joelle A Labastide, Stephen J King, Jing Xu, Jennifer L Ross
The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell's control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport...
March 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28287133/activated-full-length-myosin-x-moves-processively-on-filopodia-with-large-steps-toward-diverse-two-dimensional-directions
#12
Osamu Sato, Hyun Suk Jung, Satoshi Komatsu, Yoshikazu Tsukasaki, Tomonobu M Watanabe, Kazuaki Homma, Mitsuo Ikebe
Myosin-X, (Myo 10), is an unconventional myosin that transports the specific cargos to filopodial tips, and is associated with the mechanism underlying filopodia formation and extension. To clarify the innate motor characteristic, we studied the single molecule movement of a full-length myosin-X construct with leucine zipper at the C-terminal end of the tail (M10(Full)LZ) and the tail-truncated myosin-X without artificial dimerization motif (BAP-M10(1-979)HMM). M10(Full)LZ localizes at the tip of filopodia like myosin-X full-length (M10(Full))...
March 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28277577/preservation-of-electronic-properties-of-double-decker-complexes-on-metallic-supports
#13
B Cirera, J Matarrubia, T Kaposi, N Giménez-Agulló, M Paszkiewicz, F Klappenberger, R Otero, J M Gallego, P Ballester, J V Barth, R Miranda, J R Galán-Mascarós, W Auwärter, D Ecija
Single-molecule magnets based on lanthanide double-deckers are attracting significant attention due to their unrivaled single-ion anisotropy. To exploit their fascinating electronic and magnetic properties in devices for information storage or spin transport, studies on the preservation or variation of electronic and magnetic functionalities upon adsorption on surfaces are necessary. Herein, we introduced a comprehensive scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS) surface science study, complemented by density functional theory (DFT) simulations, of a recently synthesized single-molecule magnet based on porphyrazine deckers, conveniently equipped with ethyl moieties to make them soluble and sublimable...
March 9, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28276317/inhibitors-of-pi3k-erk-1-2-p38-mapk-show-preferential-activity-against-endocrine-resistant-breast-cancer-cells
#14
Maitham A Khajah, Princy M Mathew, Yunus A Luqmani
Current mainstream pharmacological options for treatment of endocrine resistant breast cancer have limitations in termsof side effect profile and lack of discrimination between normal and cancer cells. In the current study, we assessed the responses of normal breast epithelial cells MCF10A, estrogen receptor (ER) positive MCF-7 and ER silenced pII breast cancer cells, to inhibitors (either individually or in combination) of downstream signaling molecules. The expression/activity of ERK1/2, p38 MAPK and Akt was determined by western blotting...
March 8, 2017: Oncology Research
https://www.readbyqxmd.com/read/28276025/single-molecule-tracking-and-localization-of-mitochondrial-protein-complexes-in-live-cells
#15
Timo Appelhans, Karin Busch
Mitochondria are the power plant of most non-green eukaryotic cells. An understanding of their function and regulation is only possible with the knowledge of the spatiotemporal dynamics of their proteins. Mitochondrial membrane proteins involved in diverse functions like protein import, cell respiration, metabolite transport, and mitochondrial morphology are mobile within membranes. Here, we provide a protocol for a superresolution fluorescence microscopy technique named tracking and localization microscopy (TALM) that allows for localization and diffusion analysis of single mitochondrial membrane proteins in situ in cell cultures...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28276020/localizing-mrnas-encoding-mitochondrial-proteins-in-yeast-by-fluorescence-microscopy-and-subcellular-fractionation
#16
Dmitry Zabezhinsky, Hannah Sperber, Jeffrey E Gerst
Mitochondria are thought to have evolved from ancestral proteobacteria and, as a result of symbiosis, became an indispensable organelle in all eukaryotic cells. Mitochondria perform essential functions that provide the cell with ATP, amino acids, phospholipids, and both heme and iron-sulfur clusters. However, only 1% of mitochondrial proteins are encoded by the mitochondrial genome, while the remaining 99% are encoded in the nucleus. This raises a logistical challenge to the cell, as these nuclear-encoded proteins have to be translated, delivered to the mitochondrial surface, and translocated to its various compartments...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28274595/interdependence-of-thyroglobulin-processing-and-thyroid-hormone-export-in-the-mouse-thyroid-gland
#17
Jonas Weber, Joseph McInnes, Cise Kizilirmak, Maren Rehders, Maria Qatato, Eva K Wirth, Ulrich Schweizer, Francois Verrey, Heike Heuer, Klaudia Brix
Thyroid hormone (TH) target cells need to adopt mechanisms to maintain sufficient levels of TH to ensure regular functions. This includes thyroid epithelial cells, which generate TH in addition to being TH-responsive. However, the cellular and molecular pathways underlying thyroid auto-regulation are insufficiently understood. In order to investigate whether thyroglobulin processing and TH export are sensed by thyrocytes, we inactivated thyroglobulin-processing cathepsins and TH-exporting monocarboxylate transporters (Mct) in the mouse...
March 5, 2017: European Journal of Cell Biology
https://www.readbyqxmd.com/read/28272024/multidomain-proteins-under-force
#18
Jessica Valle Orero, Jaime Rivas-Pardo, Ionel Popa
Advancements in single molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigating how domain folding under force can have physiological roles. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91 - two immunoglobulin-like domains from the muscle protein titin, and two α+β fold proteins - ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force...
March 8, 2017: Nanotechnology
https://www.readbyqxmd.com/read/28264566/quantitative-visualization-of-molecular-delivery-and-uptake-at-living-cells-with-self-referencing-scanning-ion-conductance-microscopy-scanning-electrochemical-microscopy
#19
Ashley Page, Minkyung Kang, Alexander Armitstead, David Perry, Patrick R Unwin
A multifunctional dual-channel scanning probe nanopipet that enables simultaneous scanning ion conductance microscopy (SICM) and scanning electrochemical microscopy (SECM) measurements is demonstrated to have powerful new capabilities for spatially mapping the uptake of molecules of interest at living cells. One barrel of the probe is filled with electrolyte and the molecules of interest and is open to the bulk solution for both topographical feedback and local delivery to a target interface, while a solid carbon electrode in the other barrel measures the local concentration and flux of the delivered molecules...
March 7, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28262827/single-molecule-counting-of-point-mutations-by-transient-dna-binding
#20
Xin Su, Lidan Li, Shanshan Wang, Dandan Hao, Lei Wang, Changyuan Yu
High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy...
March 6, 2017: Scientific Reports
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