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19/10/041 Etude d interactions protéines-protéines in vivo (in cellulo) : Exemple des récepteurs de la famille erbB/HER Pierre Hubert (LISM - IBSM)

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Presentation on theme: "19/10/041 Etude d interactions protéines-protéines in vivo (in cellulo) : Exemple des récepteurs de la famille erbB/HER Pierre Hubert (LISM - IBSM)"— Presentation transcript:

1 19/10/041 Etude d interactions protéines-protéines in vivo (in cellulo) : Exemple des récepteurs de la famille erbB/HER Pierre Hubert (LISM - IBSM)

2 19/10/042 Douglas Hanahan & Robert A. Weinberg The Hallmarks of Cancer Cell, 100, 57–70, 2000

3 19/10/043 Récepteurs à activité tyrosine-kinase Human ~ 70 C. elegans ~ 40 Yeast ~ 0 (dual)

4 19/10/044 Organisation en domaines du récepteur de l EGF, et mécanisme d activation par trans-autophosphorylation

5 19/10/045 Dimérisation

6 6 Signalisation > 10 4

7 19/10/047 Src kinase InteractionsProtéines-protéines

8 19/10/048 PPPVPPRR EYINQ

9 19/10/049 EGFR ErbB2FGFR + egf+ anticorps+fgf et héparine D Grb2 PH-PTB SH2 Bennasroune et al., Crit. Rev. Oncol. Hematol. 2004, 50, 23-38

10 19/10/0410 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 1- Dimérisation de récepteurs de l EGF par une méthode « double hybride », dans des myoblastes de souris (C2C12)

11 19/10/0411 Méthode « double-hybride » classique, rappel

12 19/10/0412 Complémentation de mutants de la ß-galactosidase FACS, FDG = fluorescein digalactoside

13 19/10/0413 Complémentation de mutants de la ß-galactosidase ? Dimérisation - EGF

14 19/10/0414 Cinétiques, courbes effet-dose

15 19/10/0415 Effets de anticorps

16 19/10/0416 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 2- FRET I : Rapporteurs fluorescents de phosphorylation ( cellules HeLa, NIH 3T3 & B82 )

17 19/10/0417 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

18 19/10/0418 Rappel bis :

19 19/10/0419

20 19/10/0420 Copyright ©2001 by the National Academy of Sciences Fig. 1. General design of ratiometric indicators for monitoring tyrosine kinase activities In vitro

21 19/10/0421 Principe de la microscopie confocale

22 19/10/0422 F. Gaits, K. Hahn, Shedding light on cell signaling: Interpretation of FRET biosensors. Sci. STKE 2003, pe3 (2003)

23 19/10/0423 Fig. 2. 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)Srcreporter Localisation IP + WB Réversibilité Egf Egf AG AG - - +

24 19/10/0424

25 19/10/0425 Fig. 3. Cellular response of the Crk-based reporter WB Localisation

26 19/10/0426

27 19/10/0427 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 3- FRET II (FLIM) : Imagerie dynamique de la phosphorylation du récepteur de l EGF (cellules MCF7 et COS7)

28 19/10/0428 Overview presenting the described constructs FLAME = Sensors (détecteurs) ratiométriques

29 19/10/0429 Translocation and binding of soluble PTB domain to EGFR-ECFP in MCF7 cells EGF-++pb

30 19/10/0430 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 4- Fluorescence et transduction du signal : Récepteurs-GFPs et EGF-QD (quantum dots) (cellules CHO et A431)

31 19/10/0431 Comparative sizes of common fluorophores and protein carriers used in FRET imaging. Elizabeth A Jares-Erijman1 & Thomas M Jovin Nature Biotech. 2003; 21,

32 19/10/0432InternalisationPhosphorylation = endocytose (transferrine) 200 pM egf-QDs

33 19/10/0433 Cinétiques

34 19/10/0434

35 19/10/0435

36 19/10/0436 Transport rétrograde des egf-QDs

37 19/10/0437

38 19/10/0438 Fusion d endosomes

39 19/10/0439 Analyse quantitative BindingerbB1 / erbB3 Internalisation masques

40 19/10/0440 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 5- FRET III : Localisation dynamique de complexes protéiques (cellules A431, NIH 3T3, endothéliales B11)

41 19/10/0441 Ea = efficacité du transfert d énergie Interaction Grb2 / shc

42 19/10/0442 Etc…


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