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Semelhante a ISEV2014 - Introduction to EV biogenesis and secretion (C. Thery)
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ISEV2014 - Introduction to EV biogenesis and secretion (C. Thery)
- 1. Clotilde Théry, PhD, team « Exosomes and tumor growth » Institut Curie, INSERM U932, « Immunity and Cancer », Paris Molecular mechanisms of extracellular vesicle / exosome biogenesis and secretion
- 2. Golgi endosomes Exosomes (50-100nm) lysosomes late endosomes (MVB) Cells release various types of membrane vesicles in their environment: extracellular vesicles Plasma membrane-derived vesicles (ectosomes, microvesicles…)
- 3. Extracellular domains Antigen Presentation MHC Class I MHC Class II Adhesion molecules Tetraspanins (CD63, CD9…) Integrins MFGE8, others Membrane Transport/Fusion (lipid-bound) Annexins, Flotillins RAB, ARF ESCRT components Cytoskeletal proteins Actin, cofilin, moesin, Tubulin Signal transduction Trimeric G proteins 14-3-3, Syntenin Histones Other transmb proteins LAMPs TfR mRNAs miRNAs Other non- coding RNAs cytosol PROTEINS NUCLEIC ACIDS LIPIDS Enzymes Elongation factors General structure and composition of exosomes/EVs recovered from tissue culture conditioned medium or biological fluids
- 4. EVs can interact in various ways with surrounding cells Théry et al, Nature Rev Immunol 2009
- 5. TGN Recycling endosomes exosomes MVB CD63 tubules 1) transfer towards periphery 2) retention at periphery (actin cortical skeleton) 3) docking to plasma membrane 4) fusion with plasma membrane RAB27B RAB27A and RAB27B OVA RAB27B RAB27A and RAB27B control different steps of the exosome secretion pathway based on Ostrowski et al, Nature Cell Biol 2010 MHC class II
- 6. signaling membrane trafficking Targeting / adhesion MVB formation chaperones cytoskeleton metabolism Flottilin-1 Alix, clathrin, Tsg101, Ubiquitin Hsc70, Hsp90 cyclophilin A Actin, Tubulin Ezrin, Moesin, Radixin, Cofilin 1 myosin Annexins (1, 2, 4, 5, 6, 11) Rab (2, 7, 10, 11B), Rab GDI, Rap1B MHC II MHC I Enzymes: GAPDH, Pyruvate kinase, Enolase, Phosphoglycerate kinase 1, Peroxiredoxin 1 Protein synthesis : Ef11, eEF2, ADP ribosylation factor 14-3-3, G, Syntenin Transmembrane molecules T cell stimulating molecules CD26/Dipeptidylpeptidase CD13/Aminopeptidase ATPase Na+/K+ transporting CD98 Histone 2/4, Complement factor, MVP, ferritin, atypically secreted proteins others protein composition of a « canonical » exosome MFGE8 IntegrinsTetraspanins (CD63, CD81, CD9) compilation of proteomic studies 1999-2010: http://microvesicles.org generally purified by differential ultracentrifugation (final: 100,000g) Chaput and Théry, Semin Immunopathol. 2011
- 7. Early endosomes Late endosomes Extracellular Other intracellular compartments Exosomes bearing late endosome markers (CD63, Tsg101, Alix, Hsc70) (+CD9, Mfge8) RAB27 ? or Vesicles co-purified with late endosome-derived exosomes (CD9, Mfge8) Diverse subpopulations of vesicles secreted by different intracellular mechanisms are co-purified by the currently used protocols different functions ? Intracellular based on Bobrie, Colombo et al, JEV 2012 Bobrie and Théry, Biochem Soc Trans 2013
- 8. Various RABs are required for exosome secretion PM MVB Golgi RAB27A (RAB27B) CD63 TSG101 Ostrowski, Nat Cell Biol 2010 Bobrie, Cancer Res 2012 Peinado, Nat Med 2012 Hoshino, Cell Rep 2013 RAB7 Syntenin Syndecan ALIX RAB11 RAB35 Wnt PLP Savina, J Cell Sci 2002 Hsu, J Cell Biol 2010 Abrami, Cell Reports 2013 Baietti, Nat Cell Biol 2012
- 9. Ceramide Tetraspanins ESCRT Various machineries are described for formation of intraluminal vesicles and exosome secretion Early Endosome PM Gross, Nat Cell Biol 2012 Baietti, Nat Cell Biol 2012 Abrami, Cell reports 2013 Colombo, J Cell Sci 2013 Trajkovic, Science 2008 Kosaka, J Biol Chem 2010 Dreux, Cell Host Microb 2012 Abrami, Cell reports 2013 Chairungdua, J Cell Biol 2010 Van Niel, Dev Cell 2011 Perez-Fernandez, JBC 2013
- 10. Mechanisms of formation of plasma membrane-derived EVs Hugel et al, Physiology 2005 PM Muralidharan-Chari, Curr Biol 2009 TSG101(+) ALIX(-) Booth, J cell Biol 2006 Nabhan PNAS 2012 Romancino, FEBS Lett 2013 Ca2+-dpdt actin cytoskeleton degradation PS externalization (flippase-, floppase-scramblase+) Myosin light Chain phosphorylation Membrane fission Ghossoub, Nature Comm 2014
- 11. Cells secrete different types of membrane-enclosed vesicles, collectively called Extracellular Vesicles (EVs) The small vesicles purified by high-speed ultracentrifugation are not as homogeneous as initially thought: they contain vesicles with different biogenesis and secretion mechanisms The term “exosomes” could apply to vesicles originating from different intracellular MVBs Challenging questions Do all these EV and/or subtypes of exosomes display the same functions? In biological fluids, do some, or all (or none!) of these EVs have prognostic, diagnostic or therapeutic value Conclusion Need to establish new separation protocols and new characterization criteria of EVs