Itinerant exosomes: emerging roles in cell and tissue polarity

Abstract

Cells use secreted signals (e.g. chemokines and growth factors) and sophisticated vehicles such as argosomes, cytonemes, tunneling nanotubes and exosomes to relay important information to other cells, often over large distances. Exosomes, 30-100-nm intraluminal vesicles of multivesicular bodies (MVB) released upon exocytic fusion of the MVB with the plasma membrane, are increasingly recognized as a novel mode of cell-independent communication. Exosomes have been shown to function in antigen presentation and tumor metastasis, and in transmitting infectious agents. However, little is known about the biogenesis and function of exosomes in polarized cells. In this review, we discuss new evidence suggesting that exosomes participate in the transport of morphogens and RNA, and thus influence cell polarity and developmental patterning of tissues.

Figure 1

The endosomal network and the multivesicular bodies (MVBs) pathway. Non-polarized cells (a) have a simpler endosomal system than polarized cells (b). (a) Newly synthesized proteins are transported from the trans-Golgi (TGN) network to the plasma membrane (green and blue arrows). Clustering of proteins into lipid rafts (blue) or interaction of specific motifs in the proteins with clathrin adaptors (green) act as sorting signals. Recycling of endocytosed membrane proteins occurs from early endosomes [also known as sorting endosomes, (EE/SE)] and recycling endosomes (RE, orange arrows). Proteins destined for degradation are sorted into membrane invaginations of the EE/SE. These intralumenal vesicle-containing regions of the EE/SE eventually mature into late endosomes, also known as multivesicular bodies (LE/MVBs). Ubiquitylation of proteins and the ESCRT machinery helps to sort cargo into LE/MVBs and might also participate in their biogenesis [15,85]. Not all ILVs are destined for lysosomal degradation. Fusion of ILVs with the late endosomal limiting membrane, a process called ‘back-fusion’, is essential for the cytoplasmic delivery of certain proteins and viruses [10]. In many cells, the limiting membrane of LE/MVBs fuses with the plasma membrane, releasing ILVs into the extracellular space; these vesicles are now referred to as exosomes. (b). Epithelial cells have distinct apical and basolateral membrane domains separated by tight junctions (brown), and a complex endosomal system that is essential for the establishment and maintenance of polarity [1]. In the biosynthetic route, at the TGN, specific signals help to sort proteins into carrier vesicles that are transported to either the apical or the basolateral membrane. GPI-anchors or association with lipid rafts transport proteins to the apical domain (blue) and proteins containing motifs such as NPXY, YXXΦ, LL and L which interact with clathrin adaptors are transported to the basolateral domain (green) [3]. In the endocytic route, apical and basolateral proteins are first internalized into early apical or basolateral endosomes [also known as sorting endosomes, (EAE/ASE and EBE/BSE)]. Recycling cargo is transported to common recycling endosomes (CREs) and sorted into separate apical and basolateral recycling routes. In epithelial cells, the apical recycling route also includes the Rab11+ apical recycling endosome (ARE) [3]. Little is known about sorting of ubiquitylated cargo, the ESCRT machinery or the biogenesis of LE/MVBs in polarized cells, and phenomena such as back-fusion have yet to be demonstrated. Epithelial cells originating from different organs do release exosomes at both the apical and the basolateral surfaces [11,23,61-64]. Apical and basolateral endocytic pathways have been shown to intersect at the level of late endosomes in epithelial cells [31]; however, because exosomes released apically have a different protein composition from those released at the basolateral surface [11,64], it is also possible that different populations of MVBs generate exosomes destined for apical or basolateral release (see text and Figure 2 for a discussion on polarized sorting into exosomes).

Figure 2

Sorting and trafficking of exosomes. (a) In non-polarized cells, GPI-anchoring, lipid raft-association or antibody-induced oligomerization sorts proteins into the exosomal pathway [26,27]. Degradation of the clathrin adaptor AP2 reroutes the transferrin receptor away from recycling (orange) and towards the MVB pathway (red) [19]. This is dependent upon binding of the YXXΦ motif of the transferrin receptor to the class E protein Alix. Many studies have shown that increasing intracellular calcium[25,35-39], decreasing membrane cholesterol [36,45,46] or inducing cellular stress [45,49] stimulates the release of exosomes. Once released, exosomes interact with cells by binding to specific receptors such as Tim-4 [57], an endogenous phosphatidylserine receptor [58], or other cell surface molecules [56], or by directly fusing with target cells [55]. (b) Epithelial cells release exosomes at both the apical (intestine, kidney) and basolateral (intestine) surfaces [11,23,61]. Exosomes released apically by intestinal epithelial cells have a different composition than those released basolaterally [11], raising the possibility of differential sorting into apical and basolateral exosomes. However, how this might occur is still largely speculative. Here, we depict two potential sorting mechanisms (red dashed arrows) that might function in polarized exosomal sorting; on the basis of mechanisms operating in non-polarized cells, we hypothesize that GPI-anchoring and association with lipid rafts (which are both apical targeting signals in the biosynthetic route in polarized cells [3]) direct proteins into MVBs destined for fusion with the apical membrane. Sorting into MVBs destined for basolateral release might use interactions between the ESCRT machinery and canonical basolateral sorting signals. In a manner analogous to that seen in non-polarized cells, binding of Alix to the YXXΦ motif of the transferrin receptor might direct it into exosomes destined for basolateral release. It is currently unknown whether apical and basolateral exosomes arise from two separate LE/MVBs or from a common LE/MVB pool [31]. The V0 subunit of the vacuolar ATPase has been implicated in the apical secretion of exosomes [77]; it remains to be investigated if Rab11, which is involved in exosome release in non-polarized cells [50], and syntaxin-3, which is involved in secretory granule exocytosis [52], also function in apical release of exosomes.

Figure 3

Potential role of exosomes in cell and tissue polarity. Exosomes released at either the apical or basolateral surfaces of epithelial cells can travel over several cell diameters [74]. Exosomes internalized by dendritic cells traffic through LAMP-1-positive late endsosomes or lysosomes [56]; whether this is true in epithelial cells is currently unknown. Studies have shown that exosomes carry morphogens, mRNA and miRNA, molecules that play important roles in cell and tissue morphogenesis[37,71,77]. Mechanisms involved in sorting morphogens and nucleic acids into exosomes have yet to be elucidated but might involve association with lipid rafts [76] or binding to proteins involved in endosomal sorting [84]. Upon internalization by target cells, exogenous mRNA delivered by exosomes is translated into protein [37], endogenous protein synthesis can be downregulated by exosomal miRNA, and morphogens can influence gene expression. Thus, by modulating the protein repertoire of target cells, it is likely that exosomes could participate in the generation of apicobasal polarity in epithelial cells, planar cell polarity of the epithelial sheet, or the developmental patterning of tissues such as the Drosophila wing and the vertebrate neural tube. Given that tumor-derived exosomes promote tumor invasion, exosomes might also contribute to the epithelial–mesenchymal transition. However, a direct role for exosomes in any of these processes remains to be demonstrated.