Shedding light on the cell biology of extracellular vesicles.

TL;DR: Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material.

Abstract: Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively They are present in biological fluids and are involved in multiple physiological and pathological processes Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles

Summary

(Fig 1a-c).

• There is now evidence that each cell type tunes extracellular vesicle biogenesis depending on its physiological state, and to release extracellular vesicles with particular lipid, protein and nucleic acid compositions 5 (Fig. 1d ).
• Because most published reports of extracellular vesicles have focused on their potential functions rather their origins, it is still unclear which sub-species of vesicles is responsible for any given effect.
• The current available protocols to recover extracellular vesicles from cell culture supernatants or liquid biopsies result in a heterogeneous population of vesicles of unknown origin 23 .
• Moreover, to validate respective roles of exosomes and microvesicles, efforts are being made to uncover mechanisms underlying the targeting of the different cargoes that these vesicles transport to the site of extracellular vesicle biogenesis, the generation and secretion of vesicles, and their fate in target cells.
• Here, the authors review current knowledge and delineate unknown aspects of the essential cellular processes that govern the biology of mammalian extracellular vesicles, including their potential physiological roles, as well as their relevance to disease and to clinical applications.

[H1] Biogenesis of extracellular vesicles

• Exosomes and microvesicles have different modes of biogenesis (but both involve membrane trafficking processes): exosomes are generated within the endosomal system as ILVs and secreted during fusion of MVEs with the cell surface, whereas microvesicles originate by an outward budding at the plasma membrane 10 .
• T cells generate primarily extracellular vesicles from the cell surface with characteristics of exosomes, likely exploiting at the plasma membrane molecular components and mechanisms that are usually associated with the endosomal biogenesis of ILVs 28 .
• In many cases these shared mechanisms hinder the possibility to distinguish among them 5 .
• Mechanistic details of extracellular vesicle biogenesis have just started to be uncovered as discussed below.
• First, cargoes scheduled for secretion within extracellular vesicles must be targeted to the site of production, either at the plasma membrane (for microvesicles) or at the limiting membrane of MVE (for exosomes).

[H3] Cargoes and their targeting to the site of extracellular vesicle generation.

• The nature and abundance of extracellular vesicle cargoes 30 (Fig 1d ) is cell type specific and is often influenced by the physiological or pathological state of the donor cell, the stimuli that modulate their production and release, and the molecular mechanisms that lead to their biogenesis 31 .
• Cargoes are the first regulators of extracellular vesicle formation.
• Exosomal membrane cargoes reach endosomes from the Golgi apparatus or are internalized from the plasma membrane before being sorted to ILVs during endosome maturation 34 (Fig 1 ).
• Hence cargoes that are preferentially recycled to the plasma membrane are likely not enriched in exosomes unless their recycling is impaired, as is the case for the transferrin receptor in reticulocytes 35 .
• Syntenin [G] protein, by acting both in the recycling 36 and in the sorting of syndecan [G] in MVEs 37 for exosome biogenesis, appears as a potential regulator of the crossroad between endocytic recycling and endosomal targeting of potential exosomal cargoes.

Modulation of endocytosis or recycling of cargoes to the plasma membrane

• Would also impinge on their targeting at the site of microvesicle biogenesis.
• The small GTPase ADP-ribosylation factor 6 (ARF6) was identified as a regulator of selective recruitment of proteins, including β1 integrin receptors, MHC class I molecules, membrane type 1-matrix metalloproteinase 1 (MT1-MMP) and the vesicular soluble N-ethylmaleimidesensitive factor attachment protein receptor (v-SNARE) VAMP3 into tumourderived microvesicles 38, 39 .
• In addition to ARF6-regulated endosomal trafficking, VAMP3 mediates the trafficking and incorporation of MT1-MMP into tumor-derived microvesicles in a CD9-dependent manner.
• This suggests that VAMP3-and ARF6-positive recycling endosomes are a site of MT1-MMP recycling to the cell surface and trafficking to microvesicles.
• Such crosstalk between recycling and microvesicle biogenesis is also illustrated by studies reporting that the small GTPase Rab22a co-localizes with budding microvesicles and mediates packaging and loading of cargo proteins in hypoxic breast cancer cells 40 .

[H3] Machineries involved in the biogenesis of exosomes.

• Exosomes are generated as ILVs within the lumen of endosomes during their maturation into MVEs, a process that involves particular sorting machineries.
• Clustering of several cone-shaped tetraspanins could then induce inward budding of the microdomain in which they are enriched 55 (Fig 2).
• Therefore several mechanisms could concomitantly or sequentially act on forming MVEs, thereby allowing the sorting of diverse cargoes at different stages of maturation of the MVE 78 ; alternatively or concomitantly, distinct subpopulations of MVEs may exist and may be targeted by different machineries 5,49 (Fig 3 ).
• Overall, this data support a model, whereby the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of cargoes likely generating heterogeneous populations of ILVs and exosomes within common or distinct subpopulations of MVE 5,6 .
• Overall, as major regulators of the composition of exosomes, endosomal sorting machineries appear as main determinants of their functional properties.

[H3] Machineries involved in the biogenesis of microvesicles.

• Whereas blebbing from the plasma during apoptosis has long been known to produce microvesicles in the form of apoptotic bodies 79 , the release of microvesicles from the plasma membrane of healthy cells and the mechanisms involved in this secretion have only started to emerge recently.
• These observations suggest that other lipids, and the domains they form, contribute to microvesicle biogenesis.
• The activity of Rho family of small GTPases [G] and of the Rho-associated protein kinase (ROCK), which are important regulators of actin dynamics, induce microvesicle biogenesis in different populations of tumor cells 84 .
• Cytosolic components fated for secretion into microvesicles require their binding to the inner leaflet of the plasma membrane.
• It is still unclear how nucleic acids, which are generally found in microvesicles, are targeted to cell surface.

12 [H1] The release of extracellular vesicles

• Once formed, microvesicles pinch off from the plasma membrane whereas exosome secretion requires the transport and apposition of MVEs to the plasma membrane to fuse with and release ILVs (as exosomes) into the extracellular milieu.
• The different intracellular events leading to their secretion are likely to impose a time difference between generation and release of both types of extracellular vesicles.
• Release of microvesicles would be likely faster as cargoes only need to remain at the plasma membrane to be targeted to microvesicles and their subsequent release would directly follow their generation and fission.
• On the contrary, release of exosomes requires additional steps to sort cargoes to MVEs, then to ILVs and extrasteps to target MVEs to the plasma membrane and to prime them for secretion.

[H3] Avoiding MVE degradation.

• MVEs are primarily destined to fuse with lysosomes for degradation.
• Mechanisms preventing their degradation and allowing MVE secretion exist, thereby enabling exosome secretion (Fig 3 and 4 ).
• Some insights into how the balance between targeting MVEs for secretion and degradation is established have recently emerged.
• A similar balance exists between exosome secretion and macroautophagythe process that drives degradation of superfluous or damaged cellular components in the lysosome to maintain cellular homeostasis and that promotes energy conservation under stress.
• This suggests that the capacity of MVEs to secrete exosomes is counter-balanced by their fusion with the autophagosome.

[H3] Transport of MVEs.

• As discussed above, MVEs fuse either with lysosomes for degradation of their content or with the plasma membrane.
• In both cases a two-step process involving their transport and fusion is required, but the effectors involved in targeting MVEs to the lysosomes or to the plasma membrane are certainly distinct.
• Exosome secretion is provided by the oriented secretion of these vesicles towards the immunological synapse between antigen-presenting cells and T cells during antigen presentation 52, 104 .
• The molecular motors involved in this process remain to be determined but certainly counterbalance those that regulate transport of MVEs towards lysosomes.
• Rab27 also controls secretion of secretory lysosomes so called lysosome related organelles 109 , which suggests that MVEs capable of exosome secretion may be considered as a specialized compartment rather than a simple MVE subtype.

[H3] Fusion of

• Additional SNARE proteins involved in exosome secretion such as YkT6 121 in Drosophila, SYX-5 in C. elegans 122 and syntaxin 1a 123 in mammals reflect again the diversity of regulators that could be involved in exosome secretion, most likely depending on the organism, the cell type or the MVE subtypes.
• It should be noted that most of the studies on the intracellular regulators of exosome release came from analysis of exosomal pellets isolated from supernatants from cell cultures treated with inhibitors or interfering RNAs against potential targets, ignoring the complexity of intracellular pathways that might be affected in the producing cells by these perturbations.
• Moreover, the quantity of extracellular vesicles recovered in the supernatant does not take into account the fraction of vesicles that remains tethered (not fully released) at the plasma membrane of the producing cells 95 or the fraction of extracellular vesicles that can be recaptured by the same cell 124 .
• A better understanding of this step certainly requires the development of new tools and techniques to follow docking and fusion of MVEs with the plasma membrane.

[H3] Release of microvesicles.

• The release of microvesicles requires their fission from the plasma membrane, a mechanism that is dependent on the interaction of actin and myosin with a subsequent ATP-dependent contraction 85, 125 .
• As such, the activation of small GTP binding proteins including ARF6 and ARF1 leads to the phosphorylation of the myosin light chain (MLC) and actomyosin contraction, which allows the vesicles to bud off from the membranes of cancer cells 39 126 127 .
• In HeLa cells another regulator of actin dynamics, Cdc42 has been shown to be involved, but the underlying mechanism is still not known 84 .
• Interestingly, TSG101 and VPS4-ATPase, mostly involved in exosomes generation as part of the ESCRT machinery, were reported to participate in the scission and release of ARMMs (subtype of microvesicles containing ARRDC1) 25 .
• This scenario mirrors the exposure of phosphatidylserine by lipid translocation, which as discussed above, can promote membrane bending and microvesicle budding.

[H1] Targeting to recipient cells

• Once released into the extracellular space extracellular vesicles can reach recipient cells and deliver their content to elicit functional responses and promote phenotypical changes that will impact on their physiological or pathological status.
• Extracellular vesicle-mediated intercellular communication requires docking at the plasma membrane, followed by the activation of surface receptors and signalling, vesicle internalization or their fusion with target cells (Fig 5 ).
• The mode of vesicle interaction with the cell surface and the mechanisms that mediate the transfer of extracellular vesicle cargoes are not fully unravelled.
• These processes are complex and depend on the origin of extracellular vesicles and on the identity and origin of the recipient cells, as well as seem to be linked to the downstream effects and processes instigated by these vesicles 134 .
• Current studies have been mostly focused on investigating membrane interaction and intercellular fate of pools of exosomes, but despite different content and size, the principles of uptake and general intercellular trafficking of different sub-populations of extracellular vesicle are likely to be shared.

[H3] Uptake and intracellular fate of extracellular vesicles.

• Once they have bound to recipient cells extracellular vesicles may remain at the plasma membrane 135 ; 52 or may be internalized by clathrin-mediated or clathrin-independent endocytosis, such as macropinocytosis [G] and phagocytosis [151] [152] [153] as well as through endocytosis via caveolae and lipid rafts [157] [158] [159] .
• Specific composition of extracellular vesicles will influence their fate.
• The presence of Amyloid precursor protein on one exosome subtype from neuroblastoma cells will specifically target them to neurons contrary to a CD63 enriched exosome subtype that binds both neurons and glial cells 154 .
• As an illustrative example, it has been shown that microvesicles derived from microglia [G] show largely different dynamics of interaction with membranes of microglia and astrocytes [G] 157 .
• Following interaction with the plasma membrane of recipients cells 157 and after uptake by different mechanisms, extracellular vesicles follow the endocytic pathway and reach MVEs, which in most cases, are targeted to the lysosome 160, 161 .

Once docked at the plasma membrane, extracellular vesicles can elicit functional responses by binding to and activating receptors expressed on the recipient cells (Fig 5). First examples were B cells and dendritic cells derived

• Exosomes that were able to present antigen to T cells and induce specific antigenic response 15, 16 .
• Tumour derived microvesicles were shown to carry fibronectin, which when bound to integrin on non-transformed fibroblasts was able to promote their anchorage independent growth (one of the hallmarks of tumorigenesis), contributing to the acquisition of transformed phenotype by healthy cells 163 .
• Cargo delivered by extracellular vesicles can also activate various responses and processes in the recipient cell after internalization.
• Extracellular vesicles can transport various lipid species including eicosanoids, fatty acids, and cholesterol as well as lipid translocases, thereby contributing to the regulation of bioactive lipid species 167 .
• Their transfer to recipient cells, requiring back fusion, has been proposed to favour transcellular spreading of amyloids 168 .

[H1] Conclusions and perspectives

• Much progress has been made in recent years in understanding the basic biology of extracellular vesicles, but further investigations are required to fully resolve the functional capabilities of these vesicles.
• Thus, extracellular vesicles hold a great potential for clinical application.
• Their use in clinical research have already demonstrated that extracellular vesicles secreted by immune cells (dendritic cells) stimulate the immune system and can therefore be exploited as antitumor vaccines 176, 177 .
• Such strategy could be considered for fungi, bacteria, parasitic protozoa and helminths 172 .
• Other undergoing assays are based on in vitro manipulation of extracellular vesicles with the loading of a particular cargo (for example interfering RNAs; suicide mRNA/protein [G] , miRNAs, drugs) to then deliver it to the target cell as a drug or for bioengineering purposes 184, 185 .

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Shedding light on the cell biology of extracellular
vesicles.
Guillaume van Niel, Gisela d’Angelo, Graca Raposo
To cite this version:
Guillaume van Niel, Gisela d’Angelo, Graca Raposo. Shedding light on the cell biology of extracellular
vesicles.. Nature Reviews Molecular Cell Biology, Nature Publishing Group, 2018, 19 (4), pp.213-228.
�10.1038/nrm.2017.125�. �hal-02359760�

1
Shedding light on the cell biology of extracellular vesicles 1
2
Guillaume van Niel
1
, Gisela D’Angelo
2
and Graça Raposo
2
3
4
1. France Center of Psychiatry and Neurosciences, INSERM U895, Paris 5
75014, France 6
7
2. Institut Curie, PSL Research University, CNRS UMR144, Structure and 8
Membrane Compartments, Paris F-75005 9
10
Correspondence should be addressed to: G.R graca.raposo@curie.fr 11
12
13
Abstract : 14
15
Extracellular vesicles are a heterogeneous group of cell-derived membranous 16
structures that comprises exosomes and microvesicles, which originate from 17
the endosomal system or are shed from the plasma membrane, respectively. 18
They are present in biological fluids and are involved in multiple physiological 19
and pathological processes. Extracellular vesicles are now considered as an 20
additional mechanism for intercellular communication allowing cells to 21
exchange proteins, lipids and the genetic material. Knowledge of the cellular 22
processes that govern extracellular vesicle biology is essential to shed light on 23
physiological and pathological functions of these vesicles as well as on clinical 24
applications involving their use and/or analysis. Yet, many unknowns still 25
remain in this expanding field related to their origin, biogenesis, secretion, 26
targeting and fate. 27
28
29
30
31
32
33
[H1] Introduction 34

2
35
Apart from the release of secretory vesicles by specialized cells, which carry, 36
for example, hormones or neurotransmitters, all cells are capable of secreting 37
different types of membrane vesicles, known as extracellular vesicles, and 38
this process is conserved throughout evolution from bacteria to humans and 39
plants
1
2,3
. Secretion of extracellular vesicles has been initially described as 40
means of eliminating obsolete compounds
4
from the cell. However, now we 41
know that extracellular vesicles are more than waste carriers, and the main 42
interest in the field is now focused on their capacity to exchange components 43
between cells — varying from nucleic acids to lipids and proteins — and to act 44
as signalling vehicles in normal cell homeostatic processes or as a 45
consequence of pathological developments
5,6,7
. 46
47
Even though one generic term — extracellular vesicles – is currently in use to 48
refer to all these secreted membrane vesicles, they are in fact highly 49
heterogeneous (Fig. 1), which has largely hampered characterization and 50
manipulation of their properties and functions. Insights into the biogenesis of 51
secreted vesicles was provided by transmission and immuno-electron 52
microscopy, and by biochemical means
8-10
. Based on the current knowledge 53
of their biogenesis, extracellular vesicles can be broadly divided into two main 54
categories: exosomes and microvesicles (Fig 1a). 55
56
The term exosome (which should not be confused with the exosome complex, 57
which is involved in RNA degradation
11
) was initially used to name vesicles of 58
an unknown origin released from a variety of cultured cells and carrying 5’-59
nucleotidase activity
12
. Subsequently, the term exosomes was adopted to 60
refer to membrane vesicles (30-100 nm in diameter) released by reticulocytes 61
[G] during differentiation
4
. In essence, exosomes are intraluminal vesicles 62
(ILVs) formed by the inward budding of endosomal membrane during 63
maturation of multivesicular endosomes (MVEs) — which are intermediates 64
within the endosomal system — and secreted upon fusion of MVEs with the 65
cell surface
13,14
(Fig 1a-c). In the mid 1990’s exosomes were reported to be 66
secreted by B lymphocytes
15
and dendritic cells
16
with potential functions 67
related to immune regulation, and considered for use as vehicles in anti-68

3
tumoral immune responses. Exosome secretion is now largely extended to 69
many different cell types and their implications in intercellular communication 70
in normal and pathological states are now well documented
5
. 71
72
Microvesicles, formerly called “platelet dust”, were described as subcellular 73
material originating from platelets in normal plasma and serum
17
. Later, 74
ectocytosis, a process allowing the release of plasma membrane vesicles, 75
was described in stimulated neutrophils
18
. Although microvesicles were mainly 76
studied for their role in blood coagulation
19,20
, more recently they were 77
reported to have a role in cell–cell communication in different cell types, 78
including cancer cells
21
where they are generally called oncosomes. 79
Microvesicles range in size from 50-1000 nm in diameter, but can be even 80
larger (up to 10µm) in the case of oncosomes. They are generated by the 81
outward budding and fission of the plasma membrane and the subsequent 82
release of vesicles into the extracellular space
22
(Fig 1a-c). 83
84
There is now evidence that each cell type tunes extracellular vesicle 85
biogenesis depending on its physiological state, and to release extracellular 86
vesicles with particular lipid, protein and nucleic acid compositions
5
(Fig. 1d). 87
Because most published reports of extracellular vesicles have focused on 88
their potential functions rather their origins, it is still unclear which sub-species 89
of vesicles is responsible for any given effect. The current available protocols 90
to recover extracellular vesicles from cell culture supernatants or liquid 91
biopsies result in a heterogeneous population of vesicles of unknown origin
23
. 92
Moreover, the diversity of isolated extracellular vesicle populations is further 93
expanded by the inclusion of additional structures into the pool of extracellular 94
vesicles, such as the apoptotic bodies, migrasomes, which transport 95
multivesicular cytoplasmic contents during cell migration
24
or arrestin domain-96
containing protein 1-mediated microvesicles (ARMMS)
25
, which are largely 97
uniform, ~50 nm in diameter, microvesicles that have been shown to bud 98
directly from the plasma membrane in a manner resembling the budding of 99
viruses and dependent on arrestin domain-containing protein 1 (ARRDC1) 100
and on endosomal sorting complex required for transport (ESCRT) proteins 101
(similarly to a sub-population of exosomes; see also below). 102

4
103
104
The overlapping range of size, similar morphology and variable composition 105
challenge current attempts to devise a more precise nomenclature of 106
extracellular vesicles
26
27
. Nevertheless, novel isolation and characterization 107
methods are being developed to allow a more thorough description of 108
respective functions of the different types of extracellular vesicles and to 109
establish a suitable classification and terminology. Moreover, to validate 110
respective roles of exosomes and microvesicles, efforts are being made to 111
uncover mechanisms underlying the targeting of the different cargoes that 112
these vesicles transport to the site of extracellular vesicle biogenesis, the 113
generation and secretion of vesicles, and their fate in target cells. Here, we 114
review current knowledge and delineate unknown aspects of the essential 115
cellular processes that govern the biology of mammalian extracellular 116
vesicles, including their potential physiological roles, as well as their relevance 117
to disease and to clinical applications. 118
119
120
[H1] Biogenesis of extracellular vesicles 121
122
Exosomes and microvesicles have different modes of biogenesis (but both 123
involve membrane trafficking processes): exosomes are generated within the 124
endosomal system as ILVs and secreted during fusion of MVEs with the cell 125
surface, whereas microvesicles originate by an outward budding at the 126
plasma membrane
10
. This nomenclature is still questionable as extracellular 127
vesicle biogenesis pathways may differ according to the producing cell type. 128
For example, T cells generate primarily extracellular vesicles from the cell 129
surface with characteristics of exosomes, likely exploiting at the plasma 130
membrane molecular components and mechanisms that are usually 131
associated with the endosomal biogenesis of ILVs
28
. This peculiar biogenesis 132
of exosomes from the plasma membrane might be specific to T cells, which 133
also use the endosomal machinery for HIV budding at the plasma 134
membrane
29
. 135
136

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