Exosomes are derived from limiting membranes of MVBs (multivesicular bodies). They carry and transfer selected membrane and cytoplasmic proteins, mRNA and microRNA into target cells. It is due to this shipping of information that exosomes are considered to be the most promising therapeutic tool for multiple diseases. However, whereas knowledge on the composition of exosomes is rapidly increasing, the mode of selective recruitment into exosomes as well as target cell selection is poorly understood. We suggest that at least part of this task is taken over by tetraspanins. Tetraspanins, which are involved in morphogenesis, fission and fusion processes, are enriched in exosomes, and our previous work revealed that the recruitment of distinct tetraspanins into exosomes follows very selective routes, including a rearrangement of the tetraspanin web. Furthermore, only exosomes expressing a defined set of tetraspanins and associated molecules target endothelial cells, thereby contributing to angiogenesis and vasculogenesis. On the basis of these findings we hypothesize (i) that the protein assembly of exosomes and possibly the recruitment of microRNA will be regulated to a large extent by tetraspanins and (ii) that tetraspanins account for target cell selection and the tight interaction/uptake of exosomes by the target cell. Exosomes herald an unanticipated powerful path of cell–cell communication. An answer to how exosomes collect and transfer information will allow the use of Nature's concept to cope with malfunctions.
- multivesicular body (MVB)
- protein kinase C (PKC)
- target cell selection
Tetraspanins as a functionally important component of exosomes
Exosomes are most potent intercellular communicators that play a pivotal role in physiological and pathological processes [1,2]. Exosomes, limiting membranes of MVBs (multivesicular bodies), are delivered by many cells during ontogeny and in the adult organism and are found in all body fluids . Exosomes contain selected membrane and cytosolic proteins, mRNA and miRNA (microRNA) in a functionally active form . The selective enrichment of ‘marker’ proteins as well as of miRNA makes exosomes a very attractive means for non-invasive diagnosis, including longitudinal follow-up studies, which is coming in use, particularly, in oncology . Beyond this, exosomes are suggested to bind to, and be taken up by, selected target cells [6,7]. Thereby, exosomes can severely alter the fate of the target cell, which may become activated, differentiated or dedifferentiated according to the delivered proteins, mRNA or miRNA [8,9]. These findings advocate using exosomes therapeutically, which has in the last few years become appreciated in immunotherapy, based on the observation that exosomes derived from antigen-presenting cells are fully equipped to initiate activation of naive T-cells . With the more recent recovery of exosomal mRNA to be transcribed in target cells and of target cell RNA silencing by exosomal miRNA, hope has been created for most potent and safe exosome-based gene therapy . This, however, awaits more precise knowledge on the mode, whereby selected messages become incorporated into exosomes and, as far as ‘therapeutic’ exosomes are generated, on the exosomes’ selection of target cells.
Tetraspanins are a family of four-transmembrane proteins [12,13]. Tetraspanins form complexes by interacting between themselves as well as a large variety of transmembrane and cytosolic proteins . These tetraspanin complexes are located in microdomains, termed TEM domains (tetraspanin-enriched membrane domains) . TEM domains are different from rafts and clathrin-coated pits, but share with these structures a specific capacity to facilitate vesicular fusion and/or fission [15,16]. Accordingly, tetraspanins are highly enriched in exosomes . Tetraspanins act as ‘molecular facilitators’, which modulate, stabilize or prevent activities of associated molecules . They promote spreading, migration and cable formation by adjusting integrin compartmentalization, internalization, recycling and signalling . By regulating protein traffic, tetraspanins become involved in cell adhesion , and by modulating biosynthesis of associated molecules, such as MMPs (matrix metalloproteinases), they influence invasiveness . The main functions are cellular penetration, invasion and fusion . Thus sperm cannot fuse with CD9- and/or CD81-deficient eggs , and several viruses and parasites essentially depend on tetraspanins for cell entry and spread . Whether these activities are exclusively promoted by cell–cell contact or involve exosomal message transfer remains to be clarified . Nonetheless, there is evidence that exosomal message delivery may, indeed, be a major activity of tetraspanins .
Exosomal target cell selection
Exosomes can transfer metastatic capacity . We noted that exosomes derived from the highly metastatic rat pancreatic adenocarcinoma line, BSp73ASML, did not suffice by themselves to confer a fully metastatic phenotype, but exosomes together with a soluble tumour matrix did . These metastasis-promoting exosomes are enriched, besides other molecules, in α6β4, EpCAM (epithelial cell adhesion molecule), Claudin-7, c-Met, uPAR (urokinase-typeplasminogen activator receptor), MMPs and tetraspanins, including CD151 and Tspan8, the two metastasis-promoting tetraspanins . In BSp73ASML cells and exosomes, CD151 is preferentially associated with α6β4, and the association of α6β4 with Tspan8 becomes strengthened during exosome assembly. BSp73ASML-derived exosomes preferentially target lung and lymph node stroma cells, where they initiate up-regulation or de novo expression of several adhesion molecules, chemokines, growth factors, angiogenic factors and proteases, thus supporting pre-metastatic niche formation . The targeting profile of BSp73ASML-derived exosomes differs significantly from that of exosomes derived from a Tspan8-transfected non-metastatic variant of the BSp73ASML, BSp73AS-Tspan8, where exosomes derived from the latter line rather selectively target endothelial cells and endothelial cell progenitors . As BSp73ASML- and BSp73AS-Tspan8-derived exosomes do not differ in their tetraspanin profile, but BSp73AS-Tspan8-derived exosomes do not express α6β4, we speculated that the association of CD151 and/or Tspan8 with α6β4 in BSp73ASML-derived exosomes may be decisive for metastasis-promoting activity activities. In fact, BSp73AS-Tspan8 cells transfected with the cDNA of the β4 integrin chain gain in metastatic capacity , and exosomes derived from this line lose the capacity to bind endothelial cells. Instead, there is evidence that BSp73AS-Tspan8/β4-derived exosomes preferentially bind to, and become integrated into, stromal cells in vitro and become enriched in liver and lung after intravenous injection of exosomes (S. Rana and M. Zöller, unpublished work). As already mentioned, BSp73AS-Tspan8-derived exosomes, expressing CD151 and Tspan8, but not α6β4, most strongly initiate angiogenesis and contribute to vasculogenesis [28,30], instead BSp73AS-derived exosomes, which express neither Tspan8 nor α6β4, display no effect on metastasis formation or angiogenesis [27,30]. This finding implies that, at least in rats, exosome-initiated angiogenesis essentially requires Tspan8. Similar to metastasis-promoting exosomes, Tspan8 is required, but is not sufficient, for exosome binding and uptake by endothelial cells, which requires an association of Tspan8 with the α4 integrin chain . Exosomes derived from BSp73AS-β4 cells hardly become incorporated into endothelial cells. Thus the Tspan8-α4 complex is the crucial entity to allow exosome incorporation into endothelial cells and their progenitors . Notably, both metastasis- and angiogenesis-promoting exosomes indicate that a defined exosomal tetraspanin complex is decisive for target cell selection, where the tetraspanin and associated proteins are of equal importance.
Tetraspanins and exosome assembly
Tetraspanins, beyond clustering potential ligands for receptors on selective targets, might contribute to exosome assembly. This hypothesis is supported by indirect, but quite strong, evidence. Thus proteins and mRNA are differentially recruited in BSp73AS-mock- compared with BSp73AS-Tspan8-derived exosomes, the two cells differing only by transfection with an empty compared with a Tspan8 cDNA-containing vector . Several proteins with equal expression in both cells have been selectively enriched in AS-Tspan8-derived exosomes, among others galactoside-binding protein-3 and VCAM-1 (vascular cell adhesion molecule-1) . Differences at the mRNA level have been even more striking: 285 mRNAs were enriched by a factor of >3 in BSp73AS-Tspan8 exosomes, but another 37 mRNAs were enriched in BSp73AS-derived exosomes . Enrichment of proteins, mRNA and miRNA in exosomes is suggested to be directed by mono-ubiquitylation (proteins)  or a zip code in the 3′-UTR (3′-untranslated region; mRNA)  or by physical and functional coupling of RISCs (RNA-induced silencing complexes) to components of the sorting complex and thereby to MVB .
The mode of tetraspanin internalization could, in addition, contribute to the selectivity of exosome assembly. Tetraspanins are suggested to become internalized via a tyrosine-based internalization motif, YXXΦ  that allows attachment of the AP2 (adaptor protein 2) complex, which plays an essential role in cargo transport . However, the motif is not present in all tetraspanins (e.g. CD9) or can be located too close to the plasma membrane (e.g. Tspan8) . Alternatively, tetraspanins may become internalized via associated molecules with an YXXΦ (where Φ is a hydrophobic residue) motif [35,36]. CD151 and CD49d both contain a tyrosine-based internalization motif [34,37,38]. Thus CD9 and/or Tspan8 could become internalized via their association with either of these molecules. However, because only BSp73AS-Tspan8-, but not BSp73AS-derived exosomes interact with endothelial cells [28,30], a common path of Tspan8, CD9, CD151 and CD49d internalization became unlikely. Furthermore, the Tspan8 web in exosomes differs from the Tspan8 web in BSp73ASML or BSp73AS-Tspan8 cells, where in both cells Tspan8 preferentially associates with CD9 and α3.
To answer the question of how Tspan8 becomes recruited into exosomes, we evaluated which molecules associate with Tspan8 during internalization. To define the region(s) of Tspan8 that are involved in these mostly weak associations, the N- and/or the C-terminal regions of Tspan8 were exchanged by the corresponding regions of CD9 and CD151 or the large extracellular loop of CD9 and CD151 was exchanged with that of Tspan8. We found that, during internalization, which is facilitated by an acid pH  (as in tumours and promoting angiogenesis) or PKC (protein kinase C) activation , where PKC is associated with many tetraspanins , Tspan8 rebuilds a new web that does not contain CD9 and CD151 or α3 as in resting cells, but – according to availability – α4 or α6β4 and, importantly, INS2 (intersection-2), a multimodular protein involved in clathrin-mediated endocytosis , and dynamin (S. Rana and M. Zöller, unpublished work). Intersections contain Eps15 homology domains, binding of Eps15 promoting endocytosis  and are known to interact with dynamin and synaptojanin , where dynamin binding may regulate the fission process [45,46]. None of these molecules, INS2, dynamin, α4 or β4, associates with Tspan8 during biosynthesis, which excludes a major contribution of Tspan8 exosomes derived from the synthetic route of MVB generation. Instead, there is evidence that, under stress conditions, Tspan8, but also α4 and β4, become recruited into distinct membrane microdomains (S. Rana and M. Zöller, unpublished work) that rather resemble classical rafts  than cholesterol- and glycolipid-enriched membrane microdomains mostly formed by tetraspanins [13,48]. The molecular pathways that account for this membrane microdomain organization remain to be explored. Nonetheless, the stress-induced membrane microdomain reorganization obviously recruits α4 into close proximity to Tspan8, where rapid recycling of α4 [49,50] could well support the pronounced release of α4-associated Tspan8 into exosomes.
Conclusion and outlook
Taken together, these findings suggest that individual tetraspanins separately integrate into exosomes making use of distinct fission and fusion machineries and organizing a web/recruiting molecules distinct from that in the resting cell and different for individual tetraspanins. Although not yet fully understood, this tetraspanin-mediated fine tuning of exosome assembly is well compatible with a major contribution of tetraspanins in exosomal message transfer under physiological and pathological conditions. Because of the potential therapeutic power of exosomes, it will be worth defining for individual tetraspanins/tetraspanin-associated molecule complexes the potential target cell/target cell receptor. This will allow generating tailored exosomes to be used as a very selective therapeutics.
This work was supported by the German Research Foundation [grant number Zo40/12–1], the National Cancer Institute, Heidelberg, Germany and the Karlsruhe Institute of Technology, Karlsruhe, Germany.
4th European Conference on Tetraspanins: An Independent Meeting held at the University of Birmingham, Birmingham, U.K., 8–10 September 2010. Organized by Fedor Berditchevski (Birmingham, U.K.), Jane McKeating (Birmingham, U.K.), Peter Monk (Sheffield, U.K.), Lynda Partridge (Sheffield, U.K.), Mike Tomlinson (Birmingham, U.K.) and Annemiek van Spriel (University Medical Centre, Nijmegen, The Netherlands). Edited by Mike Tomlinson (Birmingham, U.K.).
Abbreviations: INS2, intersection-2; miRNA, microRNA; MMP, matrix metalloproteinase; MVB, multivesicular body; PKC, protein kinase C; TEM domain, tetraspanin-enriched membrane domain
- © The Authors Journal compilation © 2011 Biochemical Society