1. Photoconversions of
fluorescent proteins

2. Visualization of protein dynamics in live cells
Lippincott-Schwartz & Patterson, Science 2003

3. FRAP experiments
showing the properties of
each of the major somatic
histone H1 subtypes.
Th’ng et al, JBC 2005

4. Photoactivatable GFPlike proteins

5. None-to-green photoactivatable
fluorescent protein PA-GFP
(Patterson & Lippincott-Schwartz, 2002)
300
500
600
400
Wavelength, nm
700

6. Cyan-to-green photoswitchable PS-CFP
Chudakov et al. Nat. Biotech. 2004

7. None-to-red photoactivatable
PA-mCherry, PA-TagRFP
V. Verkhusha lab: Chem.Biol. 2005, Nat.Meth. 2009, PNAS 2009, JACS 2010

8. Green-to-red photoconvertible fluorescent proteins
- Kaede (Ando et al., 2002), EosFP, KikGR, Dendra
300
500
600
400
Wavelength, nm
700

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. Mechanism of reversible photoswitching – cis-trans
isomerization and protonation-deprotonation of the
chromophore
Andersen et al., PNAS 2005

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. Reversibly photoswitchable EGFP (Grotjohann et al, Nature 2011)

13. rsEGFP photoswitching in cells

14. Dreiklang - reversibly photoswitchable protein with
fluorescence excitation decoupled from switching
(Brakemann et al, Nat. Biotech. 2011)
365nm
405nm

15. IrisFP – combination of reversible and irreversible
photoconversions
Adam et al., PNAS 2008

17. Fibrillarin tracking with Dendra2
(point activation by 488 nm laser)

18. Fibrillarin tracking

19. 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

20. 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

21. 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

22. 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)

23. 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

24. Stimulated emission depletion (STED)
microscopy
Huang et al. Ann. Rev. Biochem. 2009. 78:993–1016
100 – 1000 MW/cm2

25. 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

27. Light-induced
formation of
filopodia via
caged
intersectin

28. Phototoxic fluorescent
protein
Bulina et al. Nat. Biotech. 2006; Nat. Protocols 2006

29. Search for
phototoxic
proteins
KillerRed
Different GFP-like proteins

30. KillerRed phototoxicity in mammalian cells,
localization to:
Mitochondria
Membrane

31. KillerRed-mediated CALI of Phospholipase C1-δ
δ
Pleckstrin Homology (PH) domain
Before irradiation
After irradiation

32. KillerRed-histone –
temporal blockage of cell cycle or cell death
Serebrovskaya et al, Biochem. J. 2011

33. Photodynamic treatment of KillerRed-expressing tumors
in vivo
Shirmanova et al, J. Biophotonics 2012

34. 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

35. 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