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Laura Canty, Santosh Hariharan, Qian Liu, Steven A Haney, David W Andrews
The discovery and use of fluorescent proteins revolutionized cell biology by allowing the visualization of proteins in living cells. Advances in fluorescent proteins, primarily through genetic engineering, have enabled more advanced analyses, including Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) and the development of genetically encoded fluorescent biosensors. These fluorescence protein-based sensors are highly effective in cells grown in monolayer cultures...
2018: PloS One
Brandee M S S Goo, Bethany J Sanstrum, Diana Z Y Holden, Yi Yu, Nicholas G James
Activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) is an immediate early gene product that is transcribed in dendritic spines and, to date, has been best characterized as a positive regulator of AMPAR endocytosis during long-term depression (LTD) through interaction with endocytic proteins. Here, we show that protein interacting with C terminal kinase 1 (PICK1), a protein known to bind to the GluA2 subunit of AMPARs and associated with AMPAR trafficking, was pulled-down from brain homogenates and synaptosomes when using Arc as immobilized bait...
October 2, 2018: Scientific Reports
Malgorzata Lichocka, Wojciech Rymaszewski, Karolina Morgiewicz, Izabela Barymow-Filoniuk, Aleksander Chlebowski, Miroslaw Sobczak, Marcus A Samuel, Elmon Schmelzer, Magdalena Krzymowska, Jacek Hennig
BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels...
September 6, 2018: BMC Plant Biology
Aljaž Gaber, Seung Joong Kim, Robyn M Kaake, Mojca Benčina, Nevan Krogan, Andrej Šali, Miha Pavšič, Brigita Lenarčič
Cell-surface tumor marker EpCAM plays a key role in proliferation, differentiation and adhesion processes in stem and epithelial cells. It is established as a cell-cell adhesion molecule, forming intercellular interactions through homophilic association. However, the mechanism by which such interactions arise has not yet been fully elucidated. Here, we first show that EpCAM monomers do not associate into oligomers that would resemble an inter-cellular homo-oligomer, capable of mediating cell-cell adhesion, by using SAXS, XL-MS and bead aggregation assays...
September 5, 2018: Scientific Reports
Eva Bártová, Gabriela Lochmanová, Soňa Legartová, Jana Suchánková, Radek Fedr, Jana Krejčí, Zbyněk Zdráhal
Histone posttranslational modifications regulate diverse nuclear functions, including DNA repair. Here, we use mass spectrometry, western blotting, immunohistochemistry and advanced confocal microscopy in order to show radiation-specific changes in the histone signature. We studied wild-type mouse embryonic stem cells (mESCs) and mESCs with a depletion of histone deacetylase 1 (HDAC1), which plays a role in DNA repair. Irradiation by γ-rays increased the S139 phosphorylation of histone H2AX but reduced the level of the H3K9-R17 peptide, which contains S10 phosphorylation (H3S10ph)...
November 2018: Biochimie
Sean C Warren, Max Nobis, Astrid Magenau, Yousuf H Mohammed, David Herrmann, Imogen Moran, Claire Vennin, James Rw Conway, Pauline Mélénec, Thomas R Cox, Yingxiao Wang, Jennifer P Morton, Heidi Ce Welch, Douglas Strathdee, Kurt I Anderson, Tri Giang Phan, Michael S Roberts, Paul Timpson
Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene , a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions...
July 9, 2018: ELife
Aleš Holoubek, Petr Herman, Jan Sýkora, Barbora Brodská, Jana Humpolíčková, Markéta Kráčmarová, Dana Gášková, Martin Hof, Kateřina Kuželová
Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1...
June 29, 2018: Methods and Applications in Fluorescence
Yuchen Long, Yvonne Stahl, Stefanie Weidtkamp-Peters, Wouter Smet, Yujuan Du, Theodorus W J Gadella, Joachim Goedhart, Ben Scheres, Ikram Blilou
Protein complex formation has been extensively studied using Förster resonance energy transfer (FRET) measured by Fluorescence Lifetime Imaging Microscopy (FLIM). However, implementing this technology to detect protein interactions in living multicellular organism at single-cell resolution and under native condition is still difficult to achieve. Here we describe the optimization of the labeling conditions to detect FRET-FLIM in living plants. This study exemplifies optimization procedure involving the identification of the optimal position for the labels either at the N or C terminal region and the selection of the bright and suitable, fluorescent proteins as donor and acceptor labels for the FRET study...
2018: Frontiers in Plant Science
Yin Yang, Qian Li, Qi-Hua He, Ji-Sheng Han, Li Su, You Wan
Activation of the cholecystokinin type B receptor (CCKBR) by cholecystokinin octapeptide (CCK-8) inhibits opioid analgesia. Chronic opiate treatment leads to an increase in the CCK-8 concentration and thus enhances the antagonism of CCK-8 against opioid analgesia; the underlying molecular mechanisms remain of great interest. In the present study, we validated the colocalization of the μ-opioid receptor (MOR) and CCKBR in pain signal transmission-related spinal cord dorsal horn and dorsal root ganglion neurons of rats...
May 21, 2018: Experimental & Molecular Medicine
Yiqun Xue, Jingjing Xing, Yinglang Wan, Xueqin Lv, Lusheng Fan, Yongdeng Zhang, Kai Song, Li Wang, Xiaohua Wang, Xin Deng, František Baluška, John M Christie, Jinxing Lin
Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of phot1-GFP proteins we demonstrated that in the dark phot1 proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of phot1-GFP increased in a dose-dependent manner in response to BL...
June 4, 2018: Molecular Plant
Wang Li, Chunyang Geng, Bo Liu
Synaptic vesicle (SV) will be transported to the bouton along the axon, once it is formed in a cell body. After docking in the active zone, neurotransmitters will be released upon the stimulation, and then transmission of chemical signals will be initiated. Presently, many advanced technologies and burgeoning molecular sensors are being used to explore the synaptic transmission. These studies provide a new sight into the presynaptic structure and its function. The present review summarizes the application of fluorescent proteins (FPs) for SV tracking and recycling...
2018: Folia Neuropathologica
Salah Edin El Meshri, Emmanuel Boutant, Assia Mouhand, Audrey Thomas, Valéry Larue, Ludovic Richert, Valérie Vivet-Boudou, Yves Mély, Carine Tisné, Delphine Muriaux, Hugues de Rocquigny
BACKGROUND: HIV-1 Gag polyprotein orchestrates the assembly of viral particles. Its C-terminus consists of the nucleocapsid (NC) domain that interacts with RNA, and the p6 domain containing the PTAP motif that binds the cellular ESCRT factor TSG101 and ALIX. Deletion of the NC domain of Gag (GagNC) results in defective Gag assembly, a decrease in virus production and, thus probably affects recruitment of the ESCRT machinery. To investigate the role of GagNC in this recruitment, we analysed its impact on TSG101 and ALIX localisations and interactions in cells expressing Gag...
June 2018: Biochimica et biophysica acta. General subjects
Feng Yi, Linda G Zachariassen, Katherine N Dorsett, Kasper B Hansen
N -Methyl-d-aspartate (NMDA)-type glutamate receptors mediate excitatory synaptic transmission in the central nervous system and play critical roles in many neuronal processes. The physiologic roles of NMDA receptors are shaped by their functional properties, which are highly dependent on subunit composition. Most NMDA receptors are assembled from two GluN1 and two GluN2 subunits, but diversity in subunit composition is made possible by eight GluN1 splice variants (i.e., isoforms) and four distinct GluN2 subunits (GluN2A-D)...
May 2018: Molecular Pharmacology
Anna M Chizhik, Carina Wollnik, Daja Ruhlandt, Narain Karedla, Alexey I Chizhik, Lara Hauke, Dirk Hähnel, Ingo Gregor, Jörg Enderlein, Florian Rehfeldt
We report a novel method, dual color axial nanometric localization by Metal Induced Energy Transfer (dcMIET), and combine it with Förster Resonant Energy Transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells (hMSCs). Our approach is based on Fluorescence-Lifetime-Imaging Microscopy (FLIM), and allows for precise determination of the 3D architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell-matrix mechanics...
February 14, 2018: Molecular Biology of the Cell
Daniëlle R J Verboogen, Maksim V Baranov, Martin Ter Beest, Geert van den Bogaart
Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions...
December 29, 2017: Journal of Visualized Experiments: JoVE
Vera Schützhold, Joachim Fandrey, Katrin Prost-Fingerle
Fluorescence resonance energy transfer (FRET) is widely used as a method to investigate protein-protein interactions in living cells. A FRET pair donor fluorophore in close proximity to an appropriate acceptor fluorophore transfers emission energy to the acceptor, resulting in a shorter lifetime of the donor fluorescence. When the respective FRET donor and acceptor are fused with two proteins of interest, a reduction in donor lifetime, as detected by fluorescence lifetime imaging microscopy (FLIM), can be taken as proof of close proximity between the fluorophores and therefore interaction between the proteins of interest...
2018: Methods in Molecular Biology
Krishna M Padmanabha Das, Lisa Wechselberger, Márton Liziczai, Montserrat De la Rosa Rodriguez, Gernot F Grabner, Christoph Heier, Roland Viertlmayr, Claudia Radler, Jörg Lichtenegger, Robert Zimmermann, Jan Willem Borst, Rudolf Zechner, Sander Kersten, Monika Oberer
Elaborate control mechanisms of intracellular triacylglycerol (TAG) breakdown are critically involved in the maintenance of energy homeostasis. Hypoxia-inducible lipid droplet-associated protein (HILPDA)/hypoxia-inducible gene-2 (Hig-2) has been shown to affect intracellular TAG levels, yet, the underlying molecular mechanisms are unclear. Here, we show that HILPDA inhibits adipose triglyceride lipase (ATGL), the enzyme catalyzing the first step of intracellular TAG hydrolysis. HILPDA shares structural similarity with G0/G1 switch gene 2 (G0S2), an established inhibitor of ATGL...
March 2018: Journal of Lipid Research
Horst Wallrabe, Zdenek Svindrych, Shagufta R Alam, Karsten H Siller, Tianxiong Wang, David Kashatus, Song Hu, Ammasi Periasamy
Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line...
January 8, 2018: Scientific Reports
Franziska Doll, Jessica Hassenrück, Valentin Wittmann, Andreas Zumbusch
Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac4 GlcNCyoc, into the cellular glycome via metabolic engineering. Ac4 GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels-Alder chemistry with fluorophores inside living cells...
2018: Methods in Enzymology
Johanna M Buschhaus, Anne E Gibbons, Kathryn E Luker, Gary D Luker
Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used. Changes in FRET by fluorescence lifetime imaging microscopy (FLIM) were measured. Unlike FRET measurements based on fluorescence intensity, lifetime measurements are independent of reporter concentration and scattering of light in tissue, making FLIM a robust method for imaging in 3D environments...
December 11, 2017: Current Protocols in Cell Biology
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