1. Etude d ’interactions protéines-protéines in vivo (in cellulo) :
Exemple des récepteurs de la famille erbB/HER
Pierre Hubert
(LISM - IBSM)
19/10/04

2. 19/10/04 Douglas Hanahan & Robert A. Weinberg The Hallmarks of Cancer
Cell, 100, 57–70, 2000
19/10/04

3. Récepteurs à activité tyrosine-kinase
Human ~ 70
C. elegans ~ 40
Yeast ~ 0 (dual)
19/10/04

4. Organisation en domaines du récepteur de l ’EGF,
et mécanisme d ’activation
par trans-autophosphorylation
19/10/04

5. Dimérisation
19/10/04

6. > 10 4
Signalisation
19/10/04

7. Src
kinase
Interactions
Protéines-protéines
19/10/04

8. PPPVPPRR
EYINQ
19/10/04

9. D Grb2 PH-PTB SH2 EGFR ErbB2 FGFR + egf + anticorps +fgf et héparine
Bennasroune et al., Crit. Rev. Oncol. Hematol. 2004, 50, 23-38
19/10/04

10. dans des myoblastes de souris (C2C12)
1- Dimérisation de récepteurs de l ’EGF par une méthode « double hybride »,
dans des myoblastes de souris (C2C12)
Nature Biotech. 2000, 18,
Epidermal growth factor receptor dimerization monitored in live cells
Bruce T. Blakely, Fabio M. V. Rossi, Bonnie Tillotson, Michelle Palmer, Angeles Estelles & Helen M. Blau
19/10/04

11. Méthode « double-hybride » classique, rappel
19/10/04

12. Complémentation de mutants de la ß-galactosidase
FACS, FDG = fluorescein digalactoside
19/10/04

13. ? Complémentation de mutants de la ß-galactosidase Dimérisation - EGF
19/10/04

14. Cinétiques, courbes effet-dose
19/10/04

15. Effets de ≠ anticorps
19/10/04

16. Rapporteurs fluorescents de phosphorylation
2- FRET I :
Rapporteurs fluorescents de phosphorylation
(cellules HeLa, NIH 3T3 & B82)
PNAS | December 18, 2001 | vol. 98 | no. 26 |
Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells
Alice Y. Ting, Kristin H. Kaindagger, Richard L. Klemkedagger, and Roger Y. Tsien
19/10/04

17. Rappel :
Interactions between two proteins can be imaged by detecting fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores. FRET is a photophysical phenomenon where energy is transferred non-radiatively from a donor fluorophore to an acceptor fluorophore with an efficiency defined by:
where r is the distance between the two fluorophores, and R0 is the distance at which 50% energy transfer takes place (typically 2–6 nm). There are a number of ways to measure FRET in a microscope that can be broadly divided into intensity-based methods and fluorescence decay-kinetics-based methods
19/10/04

18. Rappel bis :
19/10/04

19. 19/10/04

20. Copyright ©2001 by the National Academy of Sciences
Fig General design of ratiometric indicators for monitoring tyrosine kinase activities
In vitro
19/10/04

21. Principe de la microscopie confocale
19/10/04

22. F. Gaits, K. Hahn, Shedding light on cell signaling: Interpretation of FRET biosensors.
Sci. STKE 2003, pe3 (2003)
19/10/04

23. Localisation IP + WB Réversibilité
Fig B82 cells transfected with the EGFR indicator responded to EGF stimulation with an [approx] 30% increase in yellow to cyan emission ratio (transition from blue to red pseudocolor)
Src
reporter
Localisation IP + WB Réversibilité
Egf AG
19/10/04

24. 19/10/04

25. Fig. 3. Cellular response of the Crk-based reporter
WB Localisation
19/10/04

26. 19/10/04

27. Imagerie dynamique de la phosphorylation du récepteur de l ’EGF
3- FRET II (FLIM) :
Imagerie dynamique de la phosphorylation du récepteur de l ’EGF
(cellules MCF7 et COS7)
J. Biol. Chem., 279, , 2004
Imaging Phosphorylation Dynamics of the Epidermal Growth Factor Receptor
Martin Offterdinger, Virginie Georget, Andreas Girod, and Philippe I. H. Bastiaens
19/10/04

28. Overview presenting the described constructs
FLAME = Sensors (détecteurs) ratiométriques
19/10/04

29. Translocation and binding of soluble PTB domain to EGFR-ECFP in MCF7 cells+
+pb
19/10/04

30. 4- Fluorescence et transduction du signal :
Récepteurs-GFPs et EGF-QD (quantum dots)
(cellules CHO et A431)
Nature Biotechnology 22, (2004)
Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction
Diane S Lidke, Peter Nagy, Rainer Heintzmann, Donna J Arndt-Jovin, Janine N Post, Hernan E Grecco, Elizabeth A Jares-Erijman & Thomas M Jovin
19/10/04

31. Comparative sizes of common fluorophores and protein carriers used in FRET imaging.
Elizabeth A Jares-Erijman1 & Thomas M Jovin
Nature Biotech. 2003; 21,
19/10/04

32. Internalisation Phosphorylation = endocytose (transferrine)
200 pM egf-QDs
19/10/04

33. Cinétiques
19/10/04

34. 19/10/04

35. 19/10/04

36. Transport rétrograde des egf-QDs
19/10/04

37. 19/10/04

38. Fusion d ’endosomes
19/10/04

39. Analyse quantitative Binding erbB1 / erbB3 Internalisation masques
19/10/04

40. Localisation dynamique de complexes protéiques
5- FRET III :
Localisation dynamique de complexes protéiques
(cellules A431, NIH 3T3, endothéliales B11)
Molecular Biology of the Cell, 2002, 13,
Coordinated Traffic of Grb2 and Ras during Epidermal Growth Factor Receptor Endocytosis Visualized in Living Cells
Xuejun Jiang, and Alexander Sorkin
19/10/04

41. Interaction Grb2 / shc Ea = efficacité du transfert d ’énergie
19/10/04

42. Etc…
19/10/04