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Johanna M Buschhaus, Brock Humphries, Kathryn E Luker, Gary D Luker
Fluorescence lifetime imaging (FLIM) is a powerful imaging modality used to gather fluorescent reporter data independent of intracellular reporter intensity or imaging depth. We applied this technique to image real-time activation of a reporter for the proteolytic enzyme, caspase-3, in response to apoptotic cell death. This caspase-3 reporter activity provides valuable insight into cancer cell responsiveness to therapy and overall viability at a single-cell scale. Cleavage of a aspartate-glutamate-valine-aspartate (DEVD) linkage sequence alters Förster resonance energy transfer (FRET) within the reporter, affecting its lifetime...
June 13, 2018: Cells
Herbert Schneckenburger, Petra Weber, Michael Wagner, Sandra Enderle, Bernd Kalthof, Linn Schneider, Claudia Herzog, Julian Weghuber, Peter Lanzerstorfer
Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam...
February 2, 2019: International Journal of Molecular Sciences
Trillian Gregg, Sophia M Sdao, Rashpal S Dhillon, Jarred W Rensvold, Sophie L Lewandowski, David J Pagliarini, John M Denu, Matthew J Merrins
β-cell mitochondria play a central role in coupling glucose metabolism with insulin secretion. Here, we identified a metabolic function of cyclin-dependent kinase 1 (CDK1)/cyclin B1 - the activation of mitochondrial respiratory complex I - that is active in quiescent adult β-cells and hyperactive in β-cells from obese ( ob/ob ) mice. In wild-type islets, respirometry revealed that 65% of complex I flux and 49% of state 3 respiration is sensitive to CDK1 inhibition. Islets from ob/ob mice expressed more cyclin B1 and exhibited a higher sensitivity to CDK1 blockade, which reduced complex I flux by 76% and state 3 respiration by 79%...
January 30, 2019: Journal of Biological Chemistry
Wenjie Liu, Yi Cui, Wen Ren, Joseph Irudayaraj
Hormone-dependent gene expression involves dynamic and orchestrated regulation of epigenome leading to a cancerous state. Estrogen receptor (ER)-positive breast cancer rely on chromatin remodeling and association with epigenetic factors in inducing ER-dependent oncogenesis and thus cell over-proliferation. The mechanistic differences between epigenetic regulation and hormone signaling provide an avenue for combination therapy of ER-positive breast cancer. We hypothesized that epigenetic biomarkers within single nucleosome proximity of ER-dependent genes could serve as potential therapeutic targets...
January 30, 2019: Clinical Epigenetics
Jacob Schwarz, Hannah J Leopold, Ryan Leighton, Robert C Miller, Cody P Aplin, Boersma J Arnold, Ahmed A Heikal, Erin D Sheets
Living cells are crowded with macromolecules and organelles, which affect a myriad of biochemical processes. As a result, there is a need for sensitive molecular sensors for quantitative, site-specific assessment of macromolecular crowding. Here, we investigated the excited-state dynamics of recently developed hetero-FRET sensors (mCerulean3-linker-mCitrine) in homogeneous and heterogeneous environments using time-resolved fluorescence measurements, which are compatible with fluorescence lifetime imaging microscopy (FLIM)...
January 28, 2019: Methods and Applications in Fluorescence
Rossella Venditti, Laura Rita Rega, Maria Chiara Masone, Michele Santoro, Elena Polishchuk, Daniela Sarnataro, Simona Paladino, Sabato D'Auria, Antonio Varriale, Vesa M Olkkonen, Giuseppe Di Tullio, Roman Polishchuk, Maria Antonietta De Matteis
ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins...
January 18, 2019: Journal of Cell Biology
Rebecca S Saleeb, Deirdre M Kavanagh, Alison R Dun, Paul A Dalgarno, Rory R Duncan
Autophagy is an intracellular degradation pathway that transports cytoplasmic material to the lysosome for hydrolysis. It is completed by SNARE-mediated fusion of the autophagosome and endolysosome membranes. This process must be carefully regulated to maintain the organization of the membrane system and prevent mistargeted degradation. As yet, models of autophagosomal fusion have not been verified within a cellular context because of difficulties with assessing protein interactions in situ. Here, we used high-resolution fluorescence lifetime imaging (FLIM)-FRET of HeLa cells to identify protein interactions within the spatiotemporal framework of the cell...
January 17, 2019: Journal of Biological Chemistry
Franziska Doll, Raphael Steimbach, Andreas Zumbusch
The abundant post-translational modification methylation alters a protein's function, stability, and/or localization. Its malfunctions are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now their usage was limited to cell lysates. Here, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and Förster resonance energy transfer (FRET) detected by fluorescence lifetime imaging (FLIM) microscopy...
January 15, 2019: Chembiochem: a European Journal of Chemical Biology
Cornelia S Ziegler, Leila Bouchab, Marc Tramier, Dominique Durand, Franck Fieschi, Sophie Dupré-Crochet, Fabienne Mérola, Oliver Nüße, Marie Erard
Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22phox) and three cytosolic subunits (p40phox , p47phox , and p67phox ) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites...
January 10, 2019: Journal of Biological Chemistry
Wenjun Guo, Sunil Kumar, Frederik Görlitz, Edwin Garcia, Yuriy Alexandrov, Ian Munro, Douglas J Kelly, Sean Warren, Peter Thorpe, Christopher Dunsby, Paul French
We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence...
January 10, 2019: SLAS Technology
Moshe Levi, Enrico Gratton
The cloning of the renal NaPi-2a (SLC34A1) and NaPi-2c (SLC34A3) phosphate transporters has made it possible to characterize the molecular and biophysical regulation of renal proximal tubular reabsorption of inorganic phosphate (Pi). Dietary factors, such as Pi and K, and several hormones and phosphatonins, including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and glucocorticoids, regulate the transporters through various transcriptional, translational, and post-translational mechanisms that involve acute trafficking via endocytosis or exocytosis, interactions with PDZ domain proteins, lipid microdomains, and diffusion and clustering in the apical brush border membrane...
January 6, 2019: Pflügers Archiv: European Journal of Physiology
Ruofan Cao, Horst Wallrabe, Karsten Siller, Shagufta Rehman Alam, Ammasi Periasamy
Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin...
January 2, 2019: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Kristin H Rainey, George H Patterson
FRET is a powerful approach to study the interactions of fluorescent molecules, and numerous methods have been developed to measure FRET in cells. Here, we present a method based on a donor molecule's photoswitching properties, which are slower in the presence vs. the absence of an acceptor. The technique, photoswitching FRET (psFRET), is similar to an established but underutilized method called photobleaching FRET (pbFRET), with the major difference being that the molecules are switched "off" rather than photobleached...
December 31, 2018: Proceedings of the National Academy of Sciences of the United States of America
Elizabeth J Osterlund, Nehad Hirmiz, Christian Tardif, David W Andrews
The Bcl-2 proteins control cell death via interchanging interactions within the Bcl-2 family. Fluorescence lifetime imaging microscopy (FLIM) is used to detect Förster resonance energy transfer (FRET) between two fluorescent-fusion proteins in live cells. FLIM-FRET has been used to detect specific interactions and their disruption, for Bcl-2 family proteins. To date, this has been possible only in low throughput, due to the time required for serial data acquisition. We developed an automated optical system with eight parallel detectors for rapid and efficient data collection...
2019: Methods in Molecular Biology
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
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