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лекция 3 фотоактивируемые белки лекция 3 фотоактивируемые белки Presentation Transcript

  • Photoconversions of fluorescent proteins
  • Visualization of protein dynamics in live cells Lippincott-Schwartz & Patterson, Science 2003
  • FRAP experiments showing the properties of each of the major somatic histone H1 subtypes. Th’ng et al, JBC 2005
  • Photoactivatable GFPlike proteins
  • None-to-green photoactivatable fluorescent protein PA-GFP (Patterson & Lippincott-Schwartz, 2002) 300 500 600 400 Wavelength, nm 700
  • Cyan-to-green photoswitchable PS-CFP Chudakov et al. Nat. Biotech. 2004
  • None-to-red photoactivatable PA-mCherry, PA-TagRFP V. Verkhusha lab: Chem.Biol. 2005, Nat.Meth. 2009, PNAS 2009, JACS 2010
  • Green-to-red photoconvertible fluorescent proteins - Kaede (Ando et al., 2002), EosFP, KikGR, Dendra 300 500 600 400 Wavelength, nm 700
  • None-to-red reversibly photoswitchable fluorescent proteins - Kindling Fluorescent Protein KFP1 (Chudakov et al., Nat. Biotech. 2003), rsCherry, rsCherryRev, rsTagRFP 300 400 500 600 Wavelength, nm 700
  • Mechanism of reversible photoswitching – cis-trans isomerization and protonation-deprotonation of the chromophore Andersen et al., PNAS 2005
  • None-to-green reversibly photoswitchable fluorescent proteins – Dronpa (Ando et al., Science 2004), Padron, rsFastLime, mTFP0.7 300 400 500 600 Wavelength, nm 700
  • Reversibly photoswitchable EGFP (Grotjohann et al, Nature 2011)
  • rsEGFP photoswitching in cells
  • Dreiklang - reversibly photoswitchable protein with fluorescence excitation decoupled from switching (Brakemann et al, Nat. Biotech. 2011) 365nm 405nm
  • IrisFP – combination of reversible and irreversible photoconversions Adam et al., PNAS 2008
  • Fibrillarin tracking with Dendra2 (point activation by 488 nm laser)
  • Fibrillarin tracking
  • Visualization of target protein degradation in real time at single cell level using PAFPs Green fluorescence intensity depends on both protein synthesis and degradation Expression of Dendra2-tagged protein Dendra2 photoconversion in whole cell Time-lapse Red fluorescence intensity depends only on protein degradation t0 t1 ... tn Quantification Red 1 fluorescence 0.5 Zhang et al. Biotechniques 2007 τ1/2 Time
  • rsTagRFP – the first RFP with reversibly photoswitchable absorbance for photochromic FRET ON: EC = 37,000 M-1cm-1 QY = 0.11 OFF: EC = 1,600 M-1cm-1 QY = 0.11 Subach et al, Chem. Biol. 2010
  • Interaction between EGFR and Grb2 upon EGF stimulation visualized by pcFRET between EYFP and rsTagRFP EGFR-EYFP Grb2-rsTagRFP Endosomes 120 + EGF 25 EYFP fluorescence Normalized fluorescence increase, % 30 min 2 min + EGF - 2 min Membrane 100 20 EGFREYFP 15 80 10 60 + EGF 5 40 0 20 -5 0 -5 0 Grb2-rsTagRFP 0 5 10 15 20 25 30 35 2 4 6 8 10 12 Time, min cycles Photoswitching
  • Super-resolution imaging (nanoscopy) PALM - photoactivated localization microscopy (Betzig et al, Science 2006) STORM - stochastic optical reconstruction microscopy (Rust et al, Nat.Meth. 2006) Zyxin Paxillin Spatial resolution – 15-50 nm (conventional light microscopy >200 nm)
  • PALM-STORM principle The method is based on repeated photactivation/localization/bleaching cycles, allowing sequential imaging at single molecule scale. Bates, Huang, Zhuang. Curr. Opin. Chem. Biol. 2008
  • Stimulated emission depletion (STED) microscopy Huang et al. Ann. Rev. Biochem. 2009. 78:993–1016 100 – 1000 MW/cm2
  • RESOLFT: reversible saturable optically linear fluorescence transition STED-like laser scheme, but the use of reversibly switchable FPs allows to decrease light intensity 106-fold, down to 100-1000 W/cm2 DreiklangMap2 Keratin19Dreiklang Brakemann et al, Nat. Biotech. 2011
  • Light-induced formation of filopodia via caged intersectin
  • Phototoxic fluorescent protein Bulina et al. Nat. Biotech. 2006; Nat. Protocols 2006
  • Search for phototoxic proteins KillerRed Different GFP-like proteins
  • KillerRed phototoxicity in mammalian cells, localization to: Mitochondria Membrane
  • KillerRed-mediated CALI of Phospholipase C1-δ δ Pleckstrin Homology (PH) domain Before irradiation After irradiation
  • KillerRed-histone – temporal blockage of cell cycle or cell death Serebrovskaya et al, Biochem. J. 2011
  • Photodynamic treatment of KillerRed-expressing tumors in vivo Shirmanova et al, J. Biophotonics 2012
  • KillerRed structure: water channel to the DsRed-like chromophore KillerRed produces no singlet oxygen; Radicals/H2O2 can be detected KillerRed is probably a Type I photosensitizer: Chr* + DH → Chr•- + D•+ Chr•- + O2 → Chr + O2•- Possible role of the water channel: • O2 access • Proton wire Pletnev et al JBC 2009
  • Potential applications of KillerRed Phototoxic fluorescent proteins ROS signaling Cell killing Embryology Immunology Oncology Photodynamic therapy Fluorescence/Electron correlating microscopy CALI of proteins CALI of RNA/DNA Protein interactions