16: Exosomes Biogenesis and Markers

Exosomes

• Extracellular vesicles (EVs) are not cells, but contain cellular materials within a phospholipid bilayer
• Exosomes form a specific class of EVs
• Exosomes range in size from ~40-100 nm
• Unique protein and lipid composition defines exosomes

Exosome Biogenesis

• Exosomes originate as intraluminal vesicles (ILVs) of endosomal multi-vesicular bodies (MVBs)
• These MVBs fuse with the cell surface through an exocytic process
• Exosomes get released with the exocytic fusion of MVBs and the plasma membrane

Exosomal Markers

• Exosomes transport cell-specific and cell-independent biomarkers, depending on origin cell
• Exosomes contain proteins involved in membrane transport, fusion, and MVB biogenesis
• Proteins found in exosomes include:
• Rab GTPases
• Annexins
• Flotillin
• Alix
• TSG101
• Exosomses are involved in heat shock protein processes
• Contain intergrins and tetraspanins
• CD63
• CD9
• CD81
• CD82
• Exosomal marker proteins include Alix, flotillin, TSG101, and CD63
• Enrichment in cholesterol, sphingolipids, ceramide, and glycerophospholipids with long and saturated fatty-acyl chains is another exosome feature, uniquely defining their lipid bilayer

Vesicle transport - Nobel Prize

• The 2013 Nobel Prize in Physiology or Medicine was awarded for discoveries of machinery regulating vesicle trafficking.
• James E. Rothman, Randy W. Schekman and Thomas C. Südhof were the recipients

Endosomal Sorting Complexes (ESCRT)

• The ESCRT machinery is a set of cytosolic protein complexes (ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III)
• ESCRT complexes facilitate membrane remodeling, leading to MVB formation and budding
• ESCRT components have been studied in organisms like yeast and humans
• The ESCRT machinery is involved in MVB biogenesis, cellular abscission, and viral budding
• MVB biogenesis sees ubiquitin-tagged proteins enter endosomes via vesicle formation
• Viral budding often requires ESCRT machinery, as is the case with HIV

More on Exosome Biogenesis

• MVBs appear along the endocytic pathway
• They are characterized by the presence of vesicles (ILVs) in their lumen, formed by inward budding from the limiting Membrane
• The RAB family of small GTPase proteins regulates intracellular vesicular trafficking
• These steps include:
  • Vesicle budding
  • Vesicle and organelle mobility via cytoskeleton interaction
    • Vesicle docking to target, membrane fusion

SNAREs

• Soluble NSF-attachment protein receptor (SNARE) complexes facilitates lipid bilayer fusion between the compart- ments
• VAMP7 is essential for the release of acetylcholinesterase-containing exosomes
• R-SNARE protein YKT6 is needed for the secretion of WNT3A containing exosomes

Exosome Binding and Uptake

• Topology of exosomes is similar to cells, with external receptors/ligands and internal cytoplasmic proteins/RNAs
• Exosomes interact with cells through EV release, surface binding, membrane fusion, and endocytosis

Wnt Signaling

• Wnt proteins regulate interactions during embryogenesis
• Aberrant Wnt signaling is associated with cancers
• Wnt proteins bind Frizzled family receptors (GPCRs)
• This includes GSK3β differential phosphorylation and destruction complex-mediated control of beta-catenin localization
• Release of beta-catenin from a destruction complex, followed by entry into the nucleus and transcription activation, forms a complex with TCF to activate transcription of Wnt target genes

Exosomes and Wnt Interactions

• Differential regulation controls Wnt molecules and affects morphogen spreading and signaling
• Different forms of Wnts might appear to be not be mutually exclusive
• Vesicle-bound Wnt removal only lowers Wnt activity, so other secretion routes exist, maybe through direct plasma membrane release, and that has different extracellular trafficking properties

Wnt Secretion

• Exosome-dependent Wnt secretion uses exosome sorting machinery

Exosomes and Wnt Activity Assays

• Adding Wnt3A-conditioned medium induces TCF/Wnt reporter activity in HEK293 cells
• Compared to Wnt, purified exosomes have a significant capability to induce Wnt signalling
• 40% of Wnt3A is secreted on exosomes from L cells

Wnt Cargo Receptor Evi

• Wnt cargo receptor Evi is needed to secrete both forms of Wnts into the supernatant
• Decreasing protein Evi by short hairpin RNA (shRNA)-mediated silencing decreases Wnt released
• Evi is required for Wnt protein release on exosomes

Other Regulators

• Under normal conditions, Wnt3A localizes in puncta inside enlarged endosomes, similar to its MVB colocalization with CD81 and TSG101
• Dysregulation with bafilomycin causes disassembly of endosomes, Bafilomycin A1 prevents separation of Evi Wnt complexes
• Bafilomycin A1 leads to mixed secretory lysosomal organelles, and inhibits acidification blocking proper sorting into MVBs
• GW4869 is an exosome biogenesis inhibitor, but the Wnt3A-positive puncta stay inside the enlarged endosomes, with the later maturation and trafficking of MVB impairs
• NH4Cl, but not GW4869, inhibits exosomal Wnt secretion implying that the inhibitor affects general membrane segregation at MVBs and may switch the equilibria from exosomal to other form of Wnt secretion

Retromer Complex

• This complex sorts membrane proteins and recycles them trans-Golgi from endosomes
• SNX3 sorting route may determine if unloaded Evi cycles to the Golgi or the Evi-Wnt complexes are packaged on exosomes, since depletion of retromer leads to lysosomal sorting
• Retromer works upstream of MVB sorting of Evi-Wnt complexes, evident through the effect of ESCRT and Ykt6 RNAi on Wnt/Evi and the exosomal marker CD81

Shh morphogen

• The paper’s findings support Shh morphogen transport using vesicle transport, secretion along cytonemes/filopodia leading to spatial restricted signaling