Equipe «Trafic, Signalisation et Ciblage Intracellulaires»

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1 Equipe «Trafic, Signalisation et Ciblage Intracellulaires» L. Johannes & C.Lamaze D. Koester, thèse : cavéoles et mécanique membranaire R. Ruez, thèse: cavéoles/signal IFN E. Girard, thèse: trafic cholestérol C. Viaris, post doc: tri endosomal et signal Jak/Stat C. Blouin, post doc: régulation de la voie Jak/Stat N. Zanin, M2: imagerie dynamique du signal IFN B. Sinha, post doc : tension membranaire K. Allesandri, thèse: confinement cellulaire 3D

2 Multiple endocytic pathways in mammalian cells? µm 120 nm 100 nm Macropinosome???? nm Recycling endosome? Sorting Endosome Caveosome Endoplasm ic reticulum Golgi Late Endosome Lysosomes AP-2 Clathrin Dynamin Eps15 caveolin Johannes and Lamaze, Traffic 3, (2002) Laboratoire «Trafic et Signalisation» UMR 144 CNRS

3 Endocytosis is required for a vast number of functions : - Provides for nutrients - Maintain composition of extracellular environment - Enhance communication between cells - Provide protective functions such as immunity - Remove dying cells - Modulate composition of plasma membrane

4 Endocytic mechanisms Phagocytosis Pinocytosis «Cell eating» «Cell drinking» Clathrin-dependent endocytosis Clathrin-independent endocytosis Macropinocytosis

5 Steps in Receptor -mediated endocytosis Clathrin, adaptors, sorting motifs

6 Formation of clathrin coated vesicles 1 Recruitment of coat components 2 Assembly of clathrin coats to form deeply invaginated pits 3 Fission of vesicles

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8 Clathrine hub chaînes légères chaîne lourde domaine terminal boîte clathrine L-L/I-D/E/N-L/F-D/E β-adaptines γ-adaptine β-arrestines AP180/CALM epsine amphiphysine HIP1R

9 Adaptors Adaptors recognize signals on proteins

10 Complexe AP-2 oreilles Eps15 epsine AP180/CALM amphiphysine dab2 HIP1R COOH α NH2 LL Yxxφ µ2 σ2 PIP2 synaptotagmine? β2 NH2 COOH Eps15 epsine AP180 clathrine β arrestine LL tête

11 AP1 AP2 AP3 AP4

12 Recruitment of receptors to clathrin-coated pits

13 Internalisation signals LDL receptor Tyrosine-based internalisation motifs

14 Sorting signals located in the cytoplasmic tail of membrane proteins are necessary to ensure their correct targeting YXXO LL NPXY LDL Receptor N P V Y Limp II D E R A P L I Transferrin Receptor Y T R F Invariant chain D D Q R D LL Mannose-6-phosphate Receptor Y S K V Tyrosinase D D Y H S L L

15 Steps in receptor mediated endocytosis Clathrin, AP2, sorting motifs Eps15, Dynamin, amphiphysin

16 Shibere mutant of drosophila : endocytosis is blocked

17 Dynamin is a GTPase : Forms oligomers rings in the neck of forming vesicles Dynamin domains «pinchase» or «popase» or «constrictase»

18 Streps in receptor mediated endocytosis Uncoating Clathrin, AP2, sorting motifs Dynamin, amphiphysin, EPS15 Hsc70, auxilin

19 Uncoating involves HSC70, Auxilin, Synaptojanin, endophilin

20 Uncoated vesicles fuse with endosomes Endosomes Late endosomes Lysosomes

21 After internalisation. ph 6.6 Endosomes Late endosomes ph 5.5 Lysosomes ph 4.5

22 Macropinocytosis Pinocytic pathways Clathrin-coated vesicle Non-coated vesicle µm dynamin Caveolae 120 nm 100 nm nm Endosome From Lamaze and Schmid (1995) Cur. Opin. Cell Biol.

23 Selective inhibition of clathrin-dependent endocytosis Eps15 dominant negative mutant assembly of coated vesicles Dynamin dominant negative mutant Inhibition of the fission process Clathrin sirna Inhibition of clathrin synthesis Laboratoire «Trafic et Signalisation» UMR 144 CNRS

24 α IL2 β γ Interleukin 2 receptors β and γ belong to the cytokine receptor superfamily β and γ are found in IL4, 7, 9 and 15 receptors lymphocytes growth and differentiation JAK/STAT signaling cascade

25 IL2 receptors are not localized in clathrin-coated structures at the plasma membrane of lymphocytes Lamaze and coll, 2001

26 Tf receptors distribution at the plasma membrane of lymphocytes In flat lattices (%) In coated pits (%) Others (%) IL2 receptors Tf receptors

27 Sphingomyelin Glycosphingolipids glycerolipids (PS, PE) cholesterol Ikonen and Simons, 2001

28 An increasing degree of complexity. IL2-R Lamaze et al., 2001 Mol Cell Gibert et al., 2010 Cell Microbiol STxB, CTxB, virus Römer et al., 2007 Nature Römer et al., 2010 Cell From Mayor and Pagano, Nature Rev Mol Biol Cell 2007 Laboratoire «Trafic, Signalisation et Ciblage Intracellulaires» UMR 144 CNRS

29 Question: do they exist as individual pathways or is it a molecular regulation? -problem: degree of plasticity of the plasma membrane switch from a topological approach to a functional approach: role in signaling? Signaling receptors: the IFNR system

30 Endocytosis and Signaling : A recent but tight partnership dyn wt dyn K44A Cell proliferation PLCγ Ca 2+ Endocytosis controls EGF-R signaling Vieira AV, Lamaze C, Schmid SL., Science 1996 EGF-R ERK1, ERK2 PI3-kinase Signaling molecules controls EGF-R endocytosis Lamaze et al., Nature 1996 Grb2/mSOS/ras MAP kinase PP Y-kinase SH2/SH3 proteins Rho/Rac PLCγ PIs PKC PI-3 kinase PIP2 transcription Cell proliferation EGF-R degradation Laboratoire «Trafic et Signalisation» UMR 144 CNRS

31 IFNs roles: antiviral, antibacterial, antitumoral Pharmacological uses: multiple sclerosis, hepatitis B and C, leukemia, lymphoma 3 types of IFN-R (IFNAR, IFNGR, type III) Several ligands (IFNa, IFNb) 2 chains Binding of the ligand activates the associated JAK JAKs Phosphorylate the receptor Recruitment and activation of STATs Nuclear translocation of STATs and transcription IFNα/β: shared effects + specificities Question: How is the specificity achieved? Nature Reviews Immunology 5, (2005) JAK = Janus kinase STAT = Signal Transducer and Activator of transcription IFN = interferon

32 IFN-Rs and the Jak/Stat pathway IFNAR1 IFNα IFNAR2 IFNγ IFNGR1 IFNGR2 Clathrin-dept endocytosis Caveolae Clathrin and caveolae- Indept endocytosis TYK2 JAK1 JAK1 JAK2 Conner & Schmid, 2003 P STAT1/ STAT2 P STAT1 STAT2 IRF9 P P STAT1 P STAT1 STAT1 P pstat1 + - Stat1 P P I R F 9 ISRE P Transcription Transcription P GAS Nucleus Antiviral Antitumoral immunomodulatory + IFN - IFN Laboratoire «Trafic, Signalisation et Ciblage Intracellulaires» UMR 144 CNRS

33 What is the portal of entry of IFNγRs and IFNαRs? Clathrin-dependent pathway Clathrin-independent pathway Tf-R IFN-Rs IL2-R? (Lamaze et al; 2001) RNAi clathrin Hub clathrin Eps15 DN RhoA GTP Dynamin RhoA GDP Rho GDI Laboratoire «Trafic et Signalisation» UMR 144 CNRS

34 Model IFNγ IFNα Rafts Lipid rafts Stat1 signaling and IFNγ activity IFNGR endocytosis IFNAR endocytosis Stat1 signaling IFNα activity Marchetti, M., M.N. Monier, A. Fradagrada..and C. Lamaze Stat-mediated Signaling Induced by Type I and Type II Interferons (IFNs) Is Differentially Controlled through Lipid Microdomain Association and Clathrin-dependent Endocytosis of IFN Receptors. I. Kerr( FEBS lett, 2003) Stat signaling and IFN-R trafficking are independent events Mol Biol Cell. 17: H.Johnson ( J. Immunol., 2002) Lipid rafts, exclusive sites of endocytosis of IFNGRs

35 A distinct role associated to each pathway

36 Human mutations in IFNGRs IFNGR2-T168N mutant: adds an extra N-glycosylation site on the extracellular IFNGR2: defect in biological activity of IFN-γ (Vogt et al., 2005 Nat. Genet. J Exp Med 2008) IFNGR2 IFNGR1 IFNGR2 IFNGR1 T168N Jak2 Jak1 Jak2 Jak2 Jak1 Stat1 Stat1 Jak2 Jak1 Jak1 Nucleus Stat1 Stat1 GAS

37 Endocytic properties of IFNGR IFNGR2 IFNGR1 T168N Jak2 Jak1 WT R2-/ WT R1-/- Compet WT T168N % internalisation (disp.) Time % internalisation (disp.) % internalisation (disp.) Time Time Jak2 Jak1

38 30 min 15 min 0 min Wild type pstat1 Time of IFNg stimulation T168N R1-/- WT T168N WT T168N WT T168N WT T168N 0 R2-/- Gain of Glycosylation Mutant Impairs STAT1 Activation and Nuclear Translocation

39 min R1R2 R1R2 + PNG T168N T168N + PNG PNGase F Can Restore Wildtype Phenotype Signaling WT T168N +PNG PStat1 20min

40 In T168N cells, IFNGR complex raft association is impaired (IFNγ*) WT T168N T168N+PNG Marchetti et al. Mol Biol Cell 2006 Surface DRM IFNGR (% of Total Bound)