лекция 3 фотоактивируемые белки

891 views
676 views

Published on

Published in: Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
891
On SlideShare
0
From Embeds
0
Number of Embeds
474
Actions
Shares
0
Downloads
0
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

лекция 3 фотоактивируемые белки

  1. 1. Photoconversions of fluorescent proteins
  2. 2. Visualization of protein dynamics in live cells Lippincott-Schwartz & Patterson, Science 2003
  3. 3. FRAP experiments showing the properties of each of the major somatic histone H1 subtypes. Th’ng et al, JBC 2005
  4. 4. Photoactivatable GFPlike proteins
  5. 5. None-to-green photoactivatable fluorescent protein PA-GFP (Patterson & Lippincott-Schwartz, 2002) 300 500 600 400 Wavelength, nm 700
  6. 6. Cyan-to-green photoswitchable PS-CFP Chudakov et al. Nat. Biotech. 2004
  7. 7. None-to-red photoactivatable PA-mCherry, PA-TagRFP V. Verkhusha lab: Chem.Biol. 2005, Nat.Meth. 2009, PNAS 2009, JACS 2010
  8. 8. Green-to-red photoconvertible fluorescent proteins - Kaede (Ando et al., 2002), EosFP, KikGR, Dendra 300 500 600 400 Wavelength, nm 700
  9. 9. 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
  10. 10. Mechanism of reversible photoswitching – cis-trans isomerization and protonation-deprotonation of the chromophore Andersen et al., PNAS 2005
  11. 11. None-to-green reversibly photoswitchable fluorescent proteins – Dronpa (Ando et al., Science 2004), Padron, rsFastLime, mTFP0.7 300 400 500 600 Wavelength, nm 700
  12. 12. Reversibly photoswitchable EGFP (Grotjohann et al, Nature 2011)
  13. 13. rsEGFP photoswitching in cells
  14. 14. Dreiklang - reversibly photoswitchable protein with fluorescence excitation decoupled from switching (Brakemann et al, Nat. Biotech. 2011) 365nm 405nm
  15. 15. IrisFP – combination of reversible and irreversible photoconversions Adam et al., PNAS 2008
  16. 16. Fibrillarin tracking with Dendra2 (point activation by 488 nm laser)
  17. 17. Fibrillarin tracking
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. 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)
  22. 22. 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
  23. 23. Stimulated emission depletion (STED) microscopy Huang et al. Ann. Rev. Biochem. 2009. 78:993–1016 100 – 1000 MW/cm2
  24. 24. 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
  25. 25. Light-induced formation of filopodia via caged intersectin
  26. 26. Phototoxic fluorescent protein Bulina et al. Nat. Biotech. 2006; Nat. Protocols 2006
  27. 27. Search for phototoxic proteins KillerRed Different GFP-like proteins
  28. 28. KillerRed phototoxicity in mammalian cells, localization to: Mitochondria Membrane
  29. 29. KillerRed-mediated CALI of Phospholipase C1-δ δ Pleckstrin Homology (PH) domain Before irradiation After irradiation
  30. 30. KillerRed-histone – temporal blockage of cell cycle or cell death Serebrovskaya et al, Biochem. J. 2011
  31. 31. Photodynamic treatment of KillerRed-expressing tumors in vivo Shirmanova et al, J. Biophotonics 2012
  32. 32. 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
  33. 33. 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

×