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
Golgi
endosomes
Exosomes
(50-100nm)
lysosomes
late endosomes
(MVB)
Cells release various types of membrane vesicles in their
environment: extracellular vesiclesPlasma membrane-derived vesicles
(ectosomes, microvesicles…)
Extracellulardomains
Antigen PresentationMHC Class IMHC Class II
Adhesion moleculesTetraspanins (CD63, CD9…)
IntegrinsMFGE8, others
Membrane Transport/Fusion
(lipid-bound)Annexins, Flotillins
RAB, ARF
ESCRT components
Cytoskeletal proteinsActin, cofilin,
moesin, Tubulin
Signal transductionTrimeric G proteins
14-3-3, Syntenin
Histones
Other transmbproteins
LAMPsTfR
mRNAs miRNAs
Other non-coding RNAs
cytosol
PROTEINS
NUCLEIC ACIDS
LIPIDS
EnzymesElongation factors
General structure and composition of exosomes/EVs
recovered from tissue culture conditioned medium or biological fluids
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
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
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
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
RAB7SynteninSyndecan
ALIX
RAB11
RAB35
Wnt
PLP
Savina, J Cell Sci 2002
Hsu, J Cell Biol 2010
Abrami, Cell Reports 2013
Baietti, Nat Cell Biol 2012
Ceramide
Tetraspanins
ESCRT
Various machineries are described for formation of
intraluminal vesicles and exosome secretion
EarlyEndosome
PMGross, 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
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 degradationPS externalization (flippase-, floppase-scramblase+)
Myosin light Chain phosphorylation
Membrane fission
Ghossoub, Nature
Comm 2014
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