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RESEARCH PRODUCTS/Photoactivatable fluorescent proteins:

    PS-CFP2

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References citing PS-CFP2:
  • Kopek BG, Shtengel G, Grimm JB, Clayton DA, Hess HF.
    Correlative photoactivated localization and scanning electron microscopy.
    PLoS One. 2013 Oct 25;8(10):e77209 doi: 10.1371/journal.pone.0077209
    pmid: 24204771
     
  • Koga H, Martinez-Vicente M, Macian F, Verkhusha VV, Cuervo AM.
    A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.
    Nat Commun. 2011 Jul 12;2:386 doi: 10.1038/ncomms1393
    pmid: 21750540
  1. Constructs used: mammalian expression vector pPS-CFP2-N and mammalian expression vector encoding PS-CFP2 fused with CMA-targeting motif (pKFERQ-PS-CFP2).
  2. Expression system: NIH3T3 (mouse fibroblasts), SH-SY5Y (human neuroblastoma), HuH-7 (hepatoma) and MCF-7 (breast adenocarcinoma) cell lines transiently transfected with pPS-CFP2-N or pKFERQ-PS-CFP2 plasmids with Lipofectamine 2000; NIH3T3 cell line stably transfected with pPS-CFP2-N or pKFERQ-PS-CFP2 plasmids (selected with neomycin); primary mouse skin fibroblasts transduced with a lentiviral particles carrying KFERQ-PS-CFP2.
  3. Detection system: photoswitching of PS-CFP2 was performed with a 405/20nm LED array (Norlux) for 10 min using 50mW/cm2 (more than 90% of the cells were viable after the photoconversion). PS-CFP2 imaging was performed with an Axiovert 200 fluorescent microscope (Zeiss). Vizualization of PS-CFP2 initial cyan and activated green signals was performed with (ex. 365/50nm and em. 530/45 nm) and (ex. 475/40nm and em. 535/45nm) filter sets (Chroma) respectively. High-content microscopy analysis was performed in cells in 384-well plates using ArrayScan HCS Reader (Thermo Scientific Cellomics). Images in the green channel were produced using an ex. 475/40 nm, em. 535/45 nm filter set.
  • Owen DM, Rentero C, Rossy J, Magenau A, Williamson D, Rodriguez M, Gaus K.
    PALM imaging and cluster analysis of protein heterogeneity at the cell surface.
    J Biophotonics. 2010 Jul;3(7):446-54 doi: 10.1002/jbio.200900089
    pmid: 20148419
  1. Constructs used: mammalian expression vectors encoding plasma membrane targeted tdEos and PS-CFP2 (LckN10 -tdEos and SrcN15-PS-CFP2 fusions).
  2. Expression system: HeLa cells transiently transfected with Lipofectamine LTX reagent 24hours prior to imaging.
  3. Detection system: Photoactivatable localization microscopy (PALM) with a prototype PALM fluorescence miscroscope (Carl Zeiss) with TIRF illumination.
  • Kulesa PM, Stark DA, Steen J, Lansford R, Kasemeier-Kulesa JC.
    Watching the assembly of an organ a single cell at a time using confocal multi-position photoactivation and multi-time acquisition.
    Organogenesis. 2009 Oct;5(4):238-47
    pmid: 20539744
  1. Constructs used: mammalian expression vector pPS-CFP2-N and mammalian expression vector encoding cell nuclei targeted PS-CFP2 (H2B-PS-CFP2).
  2. Expression system: chick embryo; expression vectors delivered into cells by injection into the neural tube followed by electroporation.
  3. Detection system: Carl Zeiss LSM 5 Pascal microscope.
  • Nowotschin S, Hadjantonakis AK.
    Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos.
    BMC Dev Biol. 2009 Sep 9;9:49 doi: 10.1186/1471-213X-9-49
    pmid: 19740427
     
  • Zakharova MY, Kuznetsov NA, Dubiley SA, Kozyr AV, Fedorova OS, Chudakov DM, Knorre DG, Shemyakin IG, Gabibov AG, Kolesnikov AV.
    Substrate recognition of anthrax lethal factor examined by combinatorial and pre-steady-state kinetic approaches.
    J Biol Chem. 2009 Jul 3;284(27):17902-13 doi: 10.1074/jbc.M807510200
    pmid: 19359249
  1. Constructs used: pQE30-based bacterial expression vector encoding PS-CFP2 fused with TurboYFP.
  2. Expression system: recombinant protein FRET substrate (PS-CFP2 fused with TurboYFP) purified from E. coli strain.
  3. Detection system: Applied Photophysics SX.18MV stopped-flow spectrofluorimeter, protein FRET substrate excited at 405nm and emission detected at 530 nm.
  • Falk MM, Baker SM, Gumpert AM, Segretain D, Buckheit RW 3rd.
    Gap junction turnover is achieved by the internalization of small endocytic double-membrane vesicles.
    Mol Biol Cell. 2009 Jul;20(14):3342-52 doi: 10.1091/mbc.E09-04-0288
    pmid: 19458184
     
  • Nowotschin S, Eakin GS, Hadjantonakis AK.
    Live-imaging fluorescent proteins in mouse embryos: multi-dimensional, multi-spectral perspectives.
    Trends Biotechnol. 2009 May;27(5):266-76 doi: 10.1016/j.tibtech.2009.02.006
    pmid: 19339068
     
  • Hess ST, Gould TJ, Gunewardene M, Bewersdorf J, Mason MD.
    Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy.
    Methods Mol Biol. 2009;544:483-522 doi: 10.1007/978-1-59745-483-4_32
    pmid: 19488720
     
  • Adam V, Lelimousin M, Boehme S, Desfonds G, Nienhaus K, Field MJ, Wiedenmann J, McSweeney S, Nienhaus GU, Bourgeois D.
    Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations.
    Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18343-8 doi: 10.1073/pnas.0805949105
    pmid: 19017808
     
  • Held MA, Boulaflous A, Brandizzi F.
    Advances in fluorescent protein-based imaging for the analysis of plant endomembranes.
    Plant Physiol. 2008 Aug;147(4):1469-81 doi: 10.1104/pp.108.120147
    pmid: 18678739
     
  • Holmstrom TH, Mialon A, Kallio M, Nymalm Y, Mannermaa L, Holm T, Johansson H, Black E, Gillespie D, Salminen TA, Langel U, Valdez BC, Westermarck J.
    c-Jun Supports Ribosomal RNA Processing and Nucleolar Localization of RNA Helicase DDX21.
    J Biol Chem. 2008 Mar 14;283(11):7046-53 doi: 10.1074/jbc.M709613200
    pmid: 18180292
  1. Constructs used: mammalian expression vector encoding PS-CFP2 fused with RNA helicase DDX21.
  2. Expression system: mouse embryo fibroblasts transiently transfected with expression vector using FuGENE reagent.
  3. Detection system: Zeiss LSM510 META laser scanning confocal microscope. Photoswithching of PS-CFP2 was performed with 405-nm laser line.
    Visualization of photoactivated green signal was performed in FITC channel (excitation 490nm, emission 528 nm).
  • Belogurov AA Jr, Kurkova IN, Friboulet A, Thomas D, Misikov VK, Zakharova MY, Suchkov SV, Kotov SV, Alehin AI, Avalle B, Souslova EA, Morse HC 3rd, Gabibov AG, Ponomarenko NA.
    Recognition and degradation of myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis.
    J Immunol. 2008 Jan 15;180(2):1258-67
    pmid: 18178866
  1. Constructs used: pQE30-based bacterial expression vector encoding PS-CFP2 fused with TurboYFP.
  2. Expression system: recombinant EPeFRET protein (PS-CFP2 fused with TurboYFP) purified from BL21 E. coli strain was used for analysis of enzyme and abzyme-mediated cleavage of encephalitogenic peptide by FRET technique.
  3. Detection system: Genius microplate reader (Tecan) with a 405-nm excitation filter and a 535-nm emission filter.
  • Bhattacharyya S, Kulesa PM, Fraser SE.
    Vital labeling of embryonic cells using fluorescent dyes and proteins.
    Methods Cell Biol. 2008;87:187-210 doi: 10.1016/S0091-679X(08)00210-0
    pmid: 18485298
     
  • Stark DA, Kasemeier-Kulesa JC, Kulesa PM.
    Photoactivation cell labeling for cell tracing in avian development.
    CSH Protocols. 2008; pdb.prot4975
     
  • Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J, Olenych S, Davidson MW, Betzig E.
    Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes.
    Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20308-13
    pmid: 18077327
  1. Constructs used: mammalian expression vectors encoding PS-CFP2 -tagged zyxin (constructed based on pPS-CFP2 vector) and EosFP-tagged paxillin.
  2. Expression system: HFF-1 cell line transfected with plasmid using Nucleofector system (Amaxa).
  3. Detection system: two-color photoactivated localization microscopy (PALM) with Olympus IX81 inverted microscope.
  • Shaner NC, Patterson GH, Davidson MW.
    Advances in fluorescent protein technology.
    J Cell Sci. 2007 Dec 15;120(Pt 24):4247-60
    pmid: 18057027
     
  • Olenych SG, Claxton NS, Ottenberg GK, Davidson MW.
    The fluorescent protein color palette.
    Curr Protoc Cell Biol. 2007 Sep;Chapter 21:Unit 21.5 doi: 10.1002/0471143030.cb2105s36
    pmid: 18228502
     
  • Stark DA, Kulesa PM.
    An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model.
    Dev Dyn. 2007 Jun;236(6):1583-94
    pmid: 17486622
  1. Constructs used: mammalian expression vector pPS-CFP2-N.
  2. Expression system: chick embryo; expression vectors delivered into cells by injection into the neural tube followed by electroporation.
  3. Detection system: Carl Zeiss LSM 5 Pascal microscope. Initial visualization of PS-CFP2 cyan signal was performed by excitation with 1-3% laser power (405-nm laser) and detection of signal at 420-480nm. Photoswitching of PS-CFP2 was performed with 5-30% laser power (405-nm laser). Visualization of photoactivated green signal was performed by excitation with 488-nm laser and detection of emission at 505-530nm.
  • Baltrusch S, Lenzen S.
    Novel insights into the regulation of the bound and diffusible glucokinase in MIN6 β-cells.
    Diabetes. 2007 May;56(5):1305-15
    pmid: 17287461
  1. Constructs used: mammalian expression vector encoding PS-CFP2 fused to β-cell dlucokinase (constructed based on pPS-CFP2-C vector).
  2. Expression system: MIN6 cell line (pancreatic β-cells) transiently transfected with plasmid using jetPEI reagent (Qbiogene).
  3. Detection system: redistribution of fusion protein after photoswitching of PS-CFP2 was vizualized with the help of Olympus fluoview 1000 confocal microscope. Photoswitching of PS-CFP2 was performed in ROI mode (20% laser intensity) with 405nm laser diode (25mW).
  • Zhang L, Gurskaya NG, Merzlyak EM, Staroverov DB, Mudrik NN, Samarkina ON, Vinokurov LM, Lukyanov S, Lukyanov KA.
    Method for real-time monitoring of protein degradation at the single cell level.
    Biotechniques. 2007 Apr;42(4):446, 448, 450
    pmid: 17489230
     
  • Chudakov DM, Lukyanov S, Lukyanov KA.
    Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.
    Nat Protoc. 2007;2(8):2024-32
    pmid: 17703215
  1. Constructs used: mammalian expression vectors pPS-CFP2-C and pPS-CFP2-N and Dendra2.
  2. Expression system: HeLa cell line transiently transfected with plasmid encoding PS-CFP2 using Lipofectamine reagent (Invitrogen).
  3. Detection system: used Leica SP5 system to monitor cells 24-48 hours after transfection. Initial visualization of PS-CFP2 cyan signal was performed by scanning (400Hz) in confocal mode using 3% of 405-nm laser line intensity, emission collected at 410-500 nm. Photoswitching of PS-CFP2 was achieved by applying 30% power 405-nm laser, four scans in 'zoom to region of interest' mode (providing continuity of irradiation). Vizualization of activated PS-CFP2 green signal was performed using 4% of 488-nm laser line intensity, emission collected at 500-550 nm.
    Due to the strong emission crosstalk, the simultaneous excitation by 405-nm and 488-nm laser lines to monitor at once the initial cyan and photoactivated green forms of PS-CFP2 is inapplicable. To achieve the dual color imaging of PS-CFP2, use sequential excitation by 405-nm and 488-nm laser lines.
     
  • Wolff M, Wiedenmann J, Nienhaus GU, Valler M, Heilker R.
    Novel fluorescent proteins for high-content screening.
    Drug Discov Today. 2006 Dec;11(23-24):1054-60
    pmid: 17129823
     
  • Chudakov DM, Chepurnykh TV, Belousov VV, Lukyanov S, Lukyanov KA.
    Fast and precise protein tracking using repeated reversible photoactivation.
    Traffic. 2006 Oct;7(10):1304-10
    pmid: 16889652
  1. Constructs used: mammalian expression vector encoding PS-CFP2.
  2. Expression system: HeLa cell line transiently transfected with plasmid encoding PS-CFP2 using Lipofectamine reagent (Invitrogen).
  3. Detection system: Leica confocal inverted microscope TCS SP2 . Photoswitching of PS-CFP2 was achieved by 405-nm laser (50% power, 25-mW laser, 100-millisecond point irradiation in bleach mode). Vizualization of PS-CFP2 was performed with 488-nm laser.
  • Henderson JN, Remington SJ.
    The kindling fluorescent protein: a transient photoswitchable marker.
    Physiology (Bethesda). 2006 Jun;21:162-70.
    pmid: 16714474
     
  • Gurskaya NG, Verkhusha VV, Shcheglov AS, Staroverov DB, Chepurnykh TV, Fradkov AF, Lukyanov S, Lukyanov KA.
    Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.
    Nat Biotechnol. 2006 Apr;24(4):461-5
    pmid: 16550175
     
  • Souslova EA, Chudakov DM.
    Photoswitchable cyan fluorescent protein as a FRET donor.
    Microsc Res Tech. 2006 Mar;69(3):207-9
    pmid: 16538627
     
  • Xia J, Kim SH, Macmillan S, Truant R.
    Practical three color live cell imaging by widefield microscopy.
    Biol Proced Online. 2006;8:63-8
    pmid: 16909160
     
  • Chudakov DM, Lukyanov S, Lukyanov KA.
    Fluorescent proteins as a toolkit for in vivo imaging.
    Trends Biotechnol. 2005 Dec;23(12):605-13
    pmid: 16269193
     
  • Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha VV.
    Innovation: Photoactivatable fluorescent proteins.
    Nat Rev Mol Cell Biol. 2005 Nov;6(11):885-91
    pmid: 16167053
     
  • Melton L.
    Imaging: the big picture.
    Nature. 2005 Sep 29;437(7059):775-9
    pmid: 16193058
     
  • Comley J.
    HIGH CONTENT SCREENING emerging importance of novel reagents/probes and pathway analysis.
    Drug Discovery World Summer. 2005;31-53 http://clients.parabolasoft.co.uk
     
  • Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA.
    Photoswitchable cyan fluorescent protein for protein tracking.
    Nat Biotechnol. 2004 Nov;22(11):1435-9
    pmid: 15502815
  1. Constructs used: pQE30-based bacterial expression vector encoding PS-CFP (derived from a monomeric green fluorescent protein aceGFP);
    mammalian expression vectors encoding PS-CFP alone or fused with cytoplamic β-actin (PS-CFP-actin) and human dopamine transporter (PS-CFP-hDAT).
  2. Expression system: E. coli; HEK 293 cells and L929 cells transiently transfected with expression vectors using Effectene reagent (Qiagen).
  3. Detection system: PS-CFP photoswitching in E. coli colonies was done using Nikon Optiphot fluorescent microscope (3 min irradiation through the violet filter Ex. 405BP10, 100-W mercury lamp, 40x objective). Images of living eukaryotic cells were obtained using digital microscope workstation based on Carl Zeiss Axiovert 200M inverted microscope equipped with 175 W Xenon lamp source and Ablation tunable dye laser system. PS-CFP photoswitching was done by 404 nm dye laser (10-Hz pulse rate, 15-micro Joules output laser power, 5-sec in 2x2 binning mode). PS-CFP imaging was performed through ECFP (ex. 436BP10 nm, Em. 480BP20 nm) and FITC (Ex. 490BP10 nm, Em. 528BP19 nm ) standard Chroma filters.

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