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

EVs can interact in various ways with surrounding cells

Théry et al, Nature Rev Immunol 2009

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