Context-specific regulation of extracellular vesicle biogenesis and cargo selection

Abstract

To coordinate, adapt and respond to biological signals, cells convey specific messages to other cells. An important aspect of cell-cell communication involves secretion of molecules into the extracellular space. How these molecules are selected for secretion has been a fundamental question in the membrane trafficking field for decades. Recently, extracellular vesicles (EVs) have been recognized as key players in intercellular communication, carrying not only membrane proteins and lipids but also RNAs, cytosolic proteins and other signalling molecules to recipient cells. To communicate the right message, it is essential to sort cargoes into EVs in a regulated and context-specific manner. In recent years, a wealth of lipidomic, proteomic and RNA sequencing studies have revealed that EV cargo composition differs depending upon the donor cell type, metabolic cues and disease states. Analyses of distinct cargo 'fingerprints' have uncovered mechanistic linkages between the activation of specific molecular pathways and cargo sorting. In addition, cell biology studies are beginning to reveal novel biogenesis mechanisms regulated by cellular context. Here, we review context-specific mechanisms of EV biogenesis and cargo sorting, focusing on how cell signalling and cell state influence which cellular components are ultimately targeted to EVs.

Figure 1:. A road map of EV biogenesis.

a | After endocytosis, early endosomes undergo maturation into multivesicular bodies (MVBs) containing intraluminal vesicles formed through inward membrane budding into the endosome lumen. Intraluminal vesicles can be directed towards lysosomal degradation by MVB-lysosome fusion or towards secretion into the extracellular space through MVB-plasma membrane fusion. The immunoelectron micrograph (i) shows gold-labelled transferrin receptor on the surface of exosomes. b | A variety of biogenesis pathways give rise to ectosomes, but in all cases stem from surface blebs or protrusions that are cut off from the plasma membrane. Bladder cancer cells release large oncosomes (ii), shown by staining with cholera toxin B and visualization by confocal microscopy. Smaller “classical” ectosomes immunostained for annexin A1 are visible by structured illumination microscopy budding off of the plasma membrane of colorectal cancer cells (iii). Much smaller ectosomes can also bud off of the plasma membrane through a pathway depending on arrestin domain-containing protein 1 (ARRDC) (iv). These ARMMs (ARRDC1-mediated microvesicles), are detected on the surface of cells expressing ARRDC1-mCherry by immunoelectron microscopy with gold-labelled anti-mCherry. Small ectosomes may also bud off of the tips and sides of surface protrusions (v). Inset (v) exemplifies ectosome budding off the tips of microvilli in the brush border of an ATP-treated rat small intestine. c | Other types of extracellular vesicles include migrasomes, EVs containing internal vesicles that arise from retraction fibers (transmission electron micrograph (vi)), and vesicles originating from amphisomes that are formed when when the outer autophagosome membrane fuses with a late endosome. d | Apoptotic bodies arise from orderly fragmentation of apoptotic cells. Caspase-3 substrates include key players in apoptotic body formation, including ROCK1 and other regulators such as Pannexin-1 and Plexin-B2^,,. ROCK1 activates actomyosin contractility, resulting in blebbing from the plasma membrane itself or from the tips of surface protrusions termed apoptopodia^,. Apoptopodia can undergo beading along their length, visualized by DIC microscopy of apoptotic THP-1 monocytes in (vii).

Figure 2:. Mechanisms of EV biogenesis and cargo sorting.

a | In the ESCRT pathway, ESCRT-0 (Hrs:STAM dimers) and other ESCRT components underly flat clathrin domains on the endosome and cluster ubiquitinated cargoes for incorporation into ILVs. Current models suggest that ESCRT-I and -II mediate membrane bending, and ESCRT-III filaments promote membrane scission in concert with VPS4. ESCRT also recruits deubiquitinating enzymes (DUbs) that remove ubiquitin from cargoes. b | An ALIX-syntenin pathway of ILV formation bypasses early ESCRT machinery. Acting as a scaffolding protein through its Bro1 domain and PRR^,,, ALIX interacts with LBPA, phosphatidic acid (PA), other phospholipids (PLs), CHMP4B (ESCRT-III) and TSG101 (ESCRT-I). The V-domain of ALIX also engages short LYPX(n)L motifs on syntenin, which serves as a cargo adaptor for exosome cargoes like CD63 and syndecan. Syntenin captures cargo through its tandem PDZ domains, which can engage syndecan. c | Lipids and membrane proteins may promote ILV generation by acting as cone-shaped wedges that bend the endosome membrane. nSMase2 removes the head group of sphingomyelin (SM) to produce ceramide (Cer), a cone-shaped lipid sufficient for in vitro ILV formation. nSMase2 is activated by factor associated with nSMase (FAN) and is pharmacologically inhibited by the small molecule GW4869. Cargoes sorted through this pathway localize to lipid rafts enriched in flotillin and cholesterol. Tetraspanins are also cone-shaped and can promote membrane bending. d | Large ectosomes originate as plasma membrane blebs that decouple from the cortical actin cytoskeleton (1). Myosin-II sliding promotes drawstring-like closing of the bleb (2). The bud is cut off from the plasma membrane (3), releasing an ectosome into the extracellular space (4).

Figure 3:. Examples of context-specific regulation of cargo sorting.

a | Src directly phosphorylates syndecan and syntenin to regulate integrin cargo sorting through ARF6-PLD2^,. b | Oncogenic mutant KRAS signaling in colon cancer cells inhibits Ago2 entry into small EVs by promoting MEKI/II-dependent phosphorylation of Ago2. Phosphorylation of Ago2 inhibits its incorporation into ILVs and increases its association with processing bodies (PBs), resulting in altered RNA and protein content in small EVs compared to small EVs secreted from isogenic colon cancer cells with wild-type KRAS. Nonetheless, certain miRNAs are secreted at enhanced levels from mutant KRAS-expressing cells, and are presumably bound by other yet to be identified RNA-binding proteins. c | Lipidated LC3 assists in nonselective engulfment of autophagy cargoes (denoted as stars) by the phagophore, a double-membrane organelle that fuses to itself to create an autophagosome. In an independent process termed LC3-dependent EV loading and secretion (LDELS), lipidated LC3 captures RNA-binding proteins (via LC3 interaction motifs) and associated RNAs onto the endosome membrane to promote nSMase2-dependent exosome biogenesis. Secretion of LC3-induced exosomes may occur via normal fusion of MVBs with the plasma membrane or after fusion of MVBs and autophagosomes to form amphisomes. Under conditions of lysosomal dysfunction, such as with inhibition of acidification by Bafilomycin A1 (BafA1), there is enhanced secretion of autophagic proteins (blue and pink stars), both inside and outside of EVs, presumably due to fusion of both amphisomes and autolysosomes with the plasma membrane. Arrows represent membrane fusion events. d | Hypoxia activates HIF-mediated transcriptional upregulation of targeted circRNAs, miRNAs, and mRNAs, which enter EVs along with the protein products of the affected mRNAs. e | Oxidative stress from reactive oxygen species leads to caveolin-1 (Cav-1) phosphorylation, stabilizing the RNA-binding protein hnRNPA2B1 and promoting its O-GlcNAcylation. This post-translational modification enhances hnRNPA2B1 binding to microRNAs that are trafficked to large EVs. f | Leptin signaling activates transcription of Hsp90, which stabilizes Tsg101 to increase the number of endosomes and EVs. However, Hsp90 also antagonizes this process through its interaction with AMPKα1.