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Les sondes photo-activables: mécanismes d'action et les principes d'application c o n v e r t i b l e s.

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Presentation on theme: "Les sondes photo-activables: mécanismes d'action et les principes d'application c o n v e r t i b l e s."— Presentation transcript:

1 Les sondes photo-activables: mécanismes d'action et les principes d'application c o n v e r t i b l e s

2 Photobleaching

3 Control of Antibody Specificity

4 Photobleaching Control of Antibody Specificity

5 Fluorescence Recovery After Photobleaching Fluorescencce Loss In Photobleaching Photobleaching proteins mobility in-vivo

6 Goodwin et al, Biophys J. 2005, 89(2): 1398 Diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas are similar to one another and the fluorescent lipid probes DiIC16 and DiIC18 in the plasma membrane of COS-7 cells Fluorescencce Recovery After Photobleaching

7 Rosin-Arbesfeld R. et al. EMBO J. 2003; 22(5): 1101 Nuclear export rates of GFP-tagged APC constructs measured by FLIP. Individual COS cells transfected with GFP-tagged HC construct; graphs and images shown are of a representative cell from each group. Fluorescencce Loss In Photobleaching

8 Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells. Dunn et al. J Microsc Jan;205(Pt 1): Fluorescence localization after photobleaching (FLAP):

9 Photoconversion proteins mobility in-vivo

10 65-67 (Ser - dehydroTyr - Gly) Photoconversion Molecular Basis GFP Cromohore of GFP

11 ~ 400 nm Patterson at al. Science 2002, 297: 1873 Photoconversion in wild-type GFP is thought to involve a shift in the chromophore population from the neutral phenolic form to the anionic phenolate form Photoconversion Molecular Basis

12 Patterson at al. Science 2002, 297: 1873 Native and photoactivated absorbance spectra ofT203H mutant of GFP ~ 400 nm Threonine Histidine Photoconversion Molecular Basis

13 Lippincott-Schwartz et al. Science. 2003, 300: 87 The inset images show the 488-nm excited fluorescence from a cell expressing wtGFP or PA-GFP before and after photoactivation. Photoconversion wtGFP PA-GFP

14 Chudakov et al. Nature Biotechnology 2004; 22: 1435 PS-CFP-hDAT tracking within filopodia of HEK293 cells. (human dopamine transporter) Photoswitchable cyan fluorescent protein Photoconversion

15 Shaner NC et al. Nat Methods. 2005; 12 : 905 A guide to choosing fluorescent proteins. UV Green UV

16 Conventional / Confocal / Biphoton excitation area

17 Luo at al. Cell Structure and Function 2006, 31: 63 Comparison of photoactivation of PA-GFP in vivo with single- photon (405 nm) and multiphoton (790 nm) laser light. Photoconversion Excitation area

18 Luo at al. Cell Structure and Function 2006, 31: 63 TfRs are transported from TGN to early endosomes in HeLa cells Photoconversion Trafficking

19 YFP CFP FRET Acceptor Photobleaching FRET Molecular Basis

20 Before YFP CFP After FRET Acceptor Photobleaching FRET Calculation

21 Keese et al. J Biol Chem 2005; 280: Confocal imaging of EGFR phosphorylation in a SW-480 cell by acceptor photobleaching FRET. a, F4-Cy3 staining before photobleaching. b, Py72-Cy5 staining before photobleaching. c, F4-Cy3 staining after photobleaching of Cy5. The area in which Cy5 was photobleached is marked. Here an increase in the intensity of the Cy3 fluorescence can be observed. d, Py72-Cy5 staining after photobleaching. The area in which Cy5 was photobleached is marked. e, difference image of c and a (I(donor postbleach) - I(donor prebleach)) showing the increase in the Cy3 fluorescence. Acceptor Photobleaching FRET

22 Chromophore-assisted laser inactivation Causing local damage to a protein of interest Rajfur Z. et al.Nat Cell Biol. 2002:4; 286 Figure3 A typical example of ‘slow’ retraction of stress fibre after CALI irradiation at the FA. a–f,The stress fibres are visualized as bright green spots of expressed EGFP– α -actinin. The irradiated spot is indicated by the yellow circle. The yellow arrowhead represents a reliablemark for the initial position of the FA and the red arrowhead follows the position of retracted stress fibre after CALI treatment on the subsequent panels.

23 Uncaging Photorelease of caged Ca++ 2-nitrophenylethyl group NO 2 COO OOC OO N --- COO N - Ca + + Nitrophenylethyl EGTA Fluo-3 loaded cell Ca + +

24 Photoconvertible inhibitor Uncaging Local photorelease of caged biologically active compound

25 Uncaging Local photorelease of caged biologically active compound Photoactivable inhibitor

26 Uncaging Photorelease of caged phosphopeptides 2-nitrophenylethyl protecting group NO 2 1-(2-nitrophenyl)ethyl-caged phospho-serine, -threonine, -tyrosine - P O O OO - Rothman et al. Org Lett. 2002; 17: 2865

27 Uncaging Photoactivable inhibition of proteins Vazquez et al. J Am Chem Soc ; 125: (A) Caged phosphopeptide is unable to bind target protein (B) Irradiation of caged phosphopeptide releases free phosphoserine-containing peptide. (C) Released phosphoserine-peptide binds to the protein Nguyen A et al. Nat Biotechnol ; 22: 993 Caged phosphopeptides reveal a temporal role for in G1 arrest and S-phase checkpoint function.

28 Merci ! Merci !


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