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
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
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
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
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