Exosomes: Mediators of Communication in Eukaryotes

In addition to the established mechanisms of intercellular signaling, a new way of communication has gained much attention in the last decade: communication mediated by exosomes. Exosomes are nanovesicles (with a diameter of 40-120 nm) secreted into the extracellular space by the multivesicular endosome after its outer membrane fuses with the plasma membrane. Once released, exosomes modulate the response of the recipient cells that recognize them. This indicates that exosomes operate in a specifi c manner and participate in the regulation of the target cell. Remarkably, exosomes occur from unicellular organisms to mammals, suggesting an evolutionarily conserved mechanism of communication. In this review we describe the cascade of exosome formation, intracellular traffi c, secretion, and internalization by recipient cells, and review their most relevant eff ects. We also highlight important steps that are still poorly understood.


INTRODUCTION
Cell-cell communication is imperative for life.There are diff erent pathways of intercellular communication, such as the expression of signalling molecules on plasma membranes or the secretion of soluble ligands (Grimmelikhuijzen and Hauser, 2012), gap junctions and tunneling nanotubes that allow electrical and metabolic coupling among cells (Abounit and Zurzolo, 2012;Orellana et al., 2012).In addition to these modes of communication, cells release membrane vesicles into the extracellular environment that aff ect target cells (Simons and Raposo, 2009).
In the 1980s, the groups of Stahl and Johnstone described the secretion of nano-sized vesicles during reticulocyte maturation (Harding et al., 1983;Pan et al., 1985).These vesicles were named exosomes and were thought to be necessary to remove unneeded proteins from cells (Johnstone et al., 1987).Later, Raposo et al. described that exosomes had antigen-presenting capacity (Raposo et al., 1996).Thereafter, the study of functional eff ects of exosomes has grown steadily (Simons and Raposo, 2009;Thery et al., 2009;Record et al., 2011;Pant et al., 2012).
As depicted in figure 1, four sequential steps have been described in the literature for exosome-mediated communication.To date, this cascade comprising exosome formation, intracellular traffi c, secretion and internalization by recipient cells, has been best characterized in immune cells (Thery et al., 2009;Bobrie et al., 2011) while the information in Biol Res 46: 5-11, 2013 other cell types is fragmented.Considering this, we propose that communication mediated by exosomes between cells must have been conserved throughout evolution, although how this pathway may have evolved is not yet known.In this review we aim to illustrate the current state of knowledge of this particular way of cellular communication.

Exosome biogenesis
Multivesicular endosomes (MVE, also called multivesicular bodies or MVB) give rise to exosomes as follows.The membrane of late endosomes invaginates and forms small vesicles that are pinched off into the endosomal space.These are the intralumenal vesicles (ILV) and the whole is the MVE (Fig. 1A).Notice that the internal face of an ILV membrane corresponds to the cytoplasmic face of the endosome limiting membrane, and the content of the ILV is originated from the cytosol prior to ILV formation.This anatomical fact has functional relevance for exosomes.A set of MVEs fuse their limiting membranes to the plasma membrane and the ILVs with their cargo into the extracellular space.These secreted vesicles are exosomes (Fig. 1B-C) (Simons and Raposo, 2009).
Formation of ILVs in the late endosome involves the endosomal sorting complex required for transport (ESCRT) proteins (Babst, 2011).ESCRT proteins are components of four ESCRT complexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III (Hanson et al., 2009).Each of these complexes is sequentially and transiently recruited to the forming MVE until a vesicle is fully shaped and released as an ILV into the endosomal space (Hurley and Emr, 2006).However, increasing evidence suggests that some lipids such as ceramide may play a key role in ILV formation, independently of ESCRT complexes (Trajkovic et al., 2008;Babst, 2011).
As mentioned above, a set of MVE fuse with the plasma membrane while other MVEs follow a degradative route and fuse with lysosomes.This pathway leads to the degradation of ILVs content.Which are the mechanisms that guide MVE fusion with the plasma membrane or with lysosomes?Evidence to date is incomplete but supports the existence of diff erent populations of MVEs (White et al., 2006) and/or diff erent subpopulations of ILVs within a pool of common MVEs.Accordingly, MVEs rich in Rab7 GTPase and ILVs containing phosphatidylinositol-3-phosphate and ubiquitinated proteins are sorted to lysosomes (Vanlandingham and Ceresa, 2009).Conversely, MVEs rich in Rab11 GTPase and ILVs with high amounts of ceramide are sorted for exosome secretion (Zerial and McBride, 2001;Savina et al., 2005).Rab GTPases coordinate intracellular traffi c, such as vesicle formation, transport and fusion with the target membranes (Zerial and McBride, 2001).In this way, the recruitment of specifi c Rabs and their eff ectors to MVEs membrane directs their fi nal destination towards degradation or exosome secretion.

Loading of exosomes
In eukaryotes, most secreted proteins use the endoplasmic reticulum/Golgi-dependent secretory pathway (Keller and Simons, 1997).These proteins have a signal peptide which directs their fate towards the exocytic route (Nickel, 2005).However, proteins that follow unconventional secretory pathways, such as the exosome-mediated route, do not use this signal peptide.What commands the secretion of cellular components within vesicles and how are molecules sorted into exosomes?Ubiquitination is the best characterized signal that commands protein sorting into ILVs, for both cytosolic proteins and the cytosolic tail of membrane receptors (Baietti et al., 2012).Ubiquitinated-proteins interact sequentially with the ESCRT complex and with specifi c domains of late endosomes.This leads to the invagination of the late endosome membrane together with the tagged-protein and the fi nal formation of an ILV (Babst, 2011;MacDonald et al., 2012).However, some non-ubiquitinated proteins such as the transferrin receptor interact with the ESCRT complex and are sorted into exosomes, suggesting that diff erent protein interactions can provide access to ESCRT processing (Marsh and van Meer, 2008).In fact, an alternative mechanism to ubiquitin-tagging has been recently described for a plasma membrane receptor (Baietti et al., 2012).Syndecans -transmembrane proteins that off er heparin sulphate to the cell surface-recruit the fi broblast growth factor receptor into MVEs.To achieve this, the cytosolic domain of syndecan interacts with the adaptor protein syntenin which in turn connects syndecans to Alix, an auxiliary component of the ESCRT machinery.This syndecan-syntenin-Alix pathway leads to the exosome secretion of fi broblast growth factor receptor (Baietti et al., 2012).It remains to be explored whether this new pathway is suffi cient to recruit other plasma receptors, such as the transferrin receptor, into exosomes.vesicular traffi c (Levy and Shoham, 2005).Although this fi eld is quite speculative to date, exosomes from all cell types are enriched in tetraspanins, thus they are plausible candidates to mediate selective cargo recruitment into these vesicles.
The sorting process of protein complexes into ILVs is a highly regulated mechanism.Most proteins secreted by exosomes derive from parent cell membranes, the cytosol or the Golgi, but rarely the endoplasmic reticulum or mitochondria.Some of the proteins sorted into ILV participate in antigen presentation (MHC-I, MHC-II), cell adhesion (integrins), cell structure and motility (actins, tubulin, myosin, etc.), stress regulators (heat shock proteins 70 and 90), metabolic enzymes (b-enolase, peroxidases, pyruvate kinase) proteins of ESCRT machinery, proteins of the signalling cascade (kinases), tetraspanins (CD9, CD63, CD81, CD82), proteins involved in transcription and protein synthesis (histones, ribosomal proteins, ubiquitin), and proteins involved in traffi cking and membrane fusion (Rabs, annexins) (Lakkaraju and Rodriguez-Boulan, 2008).Additionally, cytosolic proteins remain in the lumen of exosomes and those derived from the plasma membrane remain in the vesicle membrane, maintaining the same topology they had in the cell, with potential roles in sequestering soluble ligands (Pan et al., 1985;Thery et al., 2009).
Although exosomes were originally described in 1980's, interest in these vesicles was renewed in recent years when exosomes were reported to contain functional mRNA and microRNA (Valadi et al., 2007).The functional consequences of transfer of genetic material include the induction, amplifi cation and/or modulation of immune responses, as well as acquisition of new functional properties in other recipient cells.For example, glioblastoma cells carrying EGFRvIII mRNA have been shown to stimulate tubule formation in endothelial cells and proliferation of glioblastoma cells, thereby promoting acquisition of the angiogenic phenotype and metastatic abilities (Skog et al., 2008).Exosomes have been shown to be highly enriched in defi ned mRNA species compared to their distributions in the donor cell (Skog et al., 2008).This fact supports the existence of a sorting mechanism, but it has not been characterized so far.
Exosomes contain not only proteins and nucleic acids but also a specific lipid composition; they are rich in cholesterol, sphingomyelin and ceramide; and they also exhibit phosphatidylserine (Thery et al., 2009).Interestingly, bioactive lipids such as prostaglandins are also sorted into exosomes (Subra et al., 2010).ExoCarta -a free to use web-database of proteins, RNA and lipids identifi ed in exosomes-has been compiled and reveals molecules that are more often found in exosomes and those that are specifi c to certain cell types (http://www.exocarta.org)(Mathivanan and Simpson, 2009).
In conclusion, the eff ort to characterize the composition of exosomes has been largely successful but the data on a mechanism to recruit the cargo have been meager.This shows a clear need to characterize the interactions between proteins, lipids and RNAs that allow specific cargo packaging into exosomes.

Secretion of exosomes
The secretion of exosomes has been described both in vivo and in vitro as a non-conventional secretory mechanism (Nickel, 2005).As mentioned above, once the MVEs fuse with the plasma membrane, ILVs are secreted as exosomes (Fig. 1C).Electron microscopy analysis of exosomes show a cup-shaped morphology and a size between 40-120 nm (Simons and Raposo, 2009).Exosomes have been isolated in vitro but also in vivo in bodily fl uids such as blood, urine, breast milk, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid and synovial fl uid (Record et al., 2011).
Exosomes are released both constitutively and in a regulated manner.For example, B cells secrete detectable levels of exosomes only following the activation of a cell surface receptor (Saunderson et al., 2008) while most tumor cells constitutively secrete exosomes (Record et al., 2011).Importantly, distinct subpopulations of exosomes are secreted from polarized cells; apical and basolateral exosomes (Tauro et al., 2012).This indicates that exosome secretion can be even more complex, since a diff erent mechanism may coexist in the cell to target exosome release to the appropriate membrane.
Intracellular calcium is a physiological second messenger in the secretion of exosomes (Savina et al., 2003;Faure et al., 2006).In murine macrophages, dendritic and neuroblastoma cells, activation of P2X 7 receptors (purinergic receptor P2X) by ATP increases intracellular calcium and also secretion of exosomes (Qu et al., 2009;Emmanouilidou et al., 2010).Pharmacological elevation of intracellular calcium also induces exosome secretion in cortical neurons, oligodendrocytes and erythroleukemia cells (Savina et al., 2003;Faure et al., 2006;Fruhbeis et al., 2012), which confi rms the role of calcium as second order messenger.Together with a cytosolic calcium increase, the expression of Rab11 GTPase is needed for exosome secretion in erythroleukemia cells in vitro (Savina et al., 2005).In fact, calcium dependent-exosome secretion is enhanced when cells with low endogenous levels of the Rab11 are manipulated to overexpress this GTPase (Savina et al., 2005).Although the direct link between calcium and Rabs is missing, two other Rab proteins have been implicated in the fusion of MVE membrane to the plasma membrane of mammalian cells.For example, Rab35 is involved in the secretion of myelin protein-enriched exosomes in oligodendroglial cells and Rab27a/b but not Rab11 mediates MVE transport and docking to the plasma membrane in HeLa cells in vitro (Hsu et al., 2010;Ostrowski et al., 2010).Finally, in the motor nerve endings of the fl y D. melanogaster exosome secretion is regulated by Rab11 but not by Rab27 or Rab35, while secretion of vesicles loaded with neurotransmitter required Rab3, which suggests a specifi c pathway for exosomes (Koles et al., 2012).These results refl ect the variety of secretory pathways during exosome formation within a cell and in diverse cell types (Koles et al., 2012).Collectively, calcium and specifi c Rab GTPases participate in the fusion of MVEs to the plasma membrane as a step in the secretion of exosomes.Taken together, this evidence indicates that exosome secretion, besides being constitutive, is also regulated.
Eukaryotic cells have developed elaborate mechanisms of quality control within their classical ER/Golgi-dependent secretory pathway.It is likely that a quality control is also operating in the exosome pathway.However, the molecular machinery involved in the transport of MVEs to the plasma membrane, their docking and fusion to release exosomes is poorly understood.

Exosome uptake by recipient cells
Once in the extracellular space, exosomes may be eliminated (Fig. 1D).This fate is illustrated by their presence in urine (Miranda et al., 2010).The biological significance of this elimination, if any, has not been explored.By contrast, exosomes have important effects on target cells (Fig. 1E).Interaction of exosomes with target cells follows two alternatives; endocytosis of the whole vesicle or and/or fusion with the plasma membrane.Membrane vesicles of all cellular origins express adhesion molecules on their surface, which could favor their endocytosis by recipient cells (Record et al., 2011).For example, surface molecules such as integrins, tetraspanins and phosphatidylserine in exosomes form complexes with cell surface molecules and participate in the attachment of exosomes, as studied in dendritic cells (Thery et al., 2009).In these cells, the whole exosome is internalized and sorted into recycling endosomes and then through late endosomes/lysosomes (Morelli et al., 2004).Co-incubation of exosomes and dendritic cells with specifi c antibodies to block various adhesion molecules reduces exosome internalization by these cells (Morelli et al., 2004).By this mechanism, dendritic cells process and then present to T lymphocytes peptides derived from the internalized exosomes.Alternatively, exosomes can be selectively internalized by macropinocytosis, a mechanism where macromolecules are taken by actinmembrane ruffles, and that does not necessarily depend on receptor-ligand interaction (Fitzner et al., 2011).On the other hand, fusion of exosomes has been shown to be a lipiddependent process, in which a high content of sphingomyelin/ ganglioside GM3 in exosomes is responsible for the increased fusion effi ciency in tumor cells (Parolini et al., 2009), while Isolation and characterization of exosomes from seed and leaved fl uids.(An et al. 2007;Regente et al. 2009)
In any case, an important unanswered question in the fi eld is how exosomes deliver their intralumenal content into the cytosol of the recipient cells.

Eff ect of exosome cargo on recipient cells
Since the discovery that exosomes participate in the cascade of antigen presentation (Raposo et al., 1996), this novel mechanism of intercellular communication has been implicated in various essential processes such as development (Korkut et al., 2009), immune responses (Thery et al., 2009), cancer and tumor metastasis (Peinado et al., 2012), and in the transmission of infectious agents like prions and viruses (Fevrier et al., 2004;Lenassi et al., 2010).Table I provides an overview of the most relevant fi ndings of exosome functional eff ects in eukaryotes to date.It is worth mentioning that studies on exosomes other than mammalian cells are scant but still support the role of exosomes as a conserved mechanism of communication throughout evolution.
Finally, exosomes are promising tools to target drugs or biological material to specifi c cells across diff erent biological barriers (O'Loughlin et al., 2012;Pant et al., 2012).For example, Alvarez-Erviti et al. (2011) obtained neuronal-targeted exosomes from genetically modified dendritic cells in vitro and loaded them with specific siRNA.After intravenous injection, these exosomes loaded with siRNA knocked down their target gene in brain neurons (Alvarez-Erviti et al., 2011).Thus, the possibility to deliver agents in a specifi c manner to defi ned target cells opens a wide avenue of research, even to overcome important challenges such as biological barriers, or tolerance to vesicles by using those derived from patient cells.

CONCLUSION
In this review we illustrate the sequential steps for exosomemediated communication and compile the evidence supporting this role in eukaryotes.Although exosomes have been described in many cells from a wide variety of organisms, many important gaps remain concerning their formation cascade and their physiological relevance.Addressing these questions implies important challenges, such as the development of sensitive techniques to purify these nanovesicles from small volumes of fluids and to determine precisely how and with what types of cells exosomes interact in vivo.The disclosure and growth of this research area will help to address these issues and lead to major advances in understanding exosomal functions as means for cell-to-cell communication.
Figure 1.Sequential steps necessary for exosome-mediated communication.Intralumenal vesicles (ILV) are formed after the invagination of the multivesicular endosome membrane.These ILVs are loaded with specifi c cargo originated from the plasma membrane and/ or the cytoplasm (A).Notice that the internal face of an ILV membrane corresponds to the cytoplasmic face of the endosome limiting membrane, and the ILV is loaded with cytosolic components.Upon fusion of the MVE with the plasma membrane (B), ILV are secreted as exosomes (C).Exosomes can be lost (D) or taken up by target cells (E) by fusion, or internalization mediated by surface molecules or by macropinocytosis.Additional abbreviation: ESCRT, endosomal sorting complex required for transport.