Author:

Keywords:

exosome, syndecan, syntenin

Abstract:

Syndecans, heparan sulfate proteoglycans, are essential for fine-regulating signalling events between cells. The extracellular heparan sulfate chains of these membrane proteins attract several signalling molecules, such as morphogens, to cell surfaces, but the mechanisms of their regulatory functions remain largely unknown. We have found that, through the PDZ protein syntenin, a well-known syndecan cytoplasmic adaptor, syndecans are connected to ALIX, an auxiliary component of the ESCRT machinery, implicated in vesicular budding, and marker of small signalling vesicles released by cells known as ‘exosomes’. Here, we explore whether the syndecan-syntenin-ALIX connection might regulate the biogenesis of exosomes, impacting on the cellular release of morphogen signals.Syndecan 1 is released in exosomes and co-fractionates, together with syntenin and ALIX, with exosomal marker proteins such as CD63. While syntenin over-expression results, depending on ALIX, in an increased release of exosomes, the depletion of syntenin (or ALIX) by RNAi treatment blocks the exosomal accumulation of syndecan and marker proteins. Strikingly, syndecan RNAi markedly suppresses exosome production. Syntenin has an effect on a subset of exosomes: while CD63 and syntenin largely reside in the same exosome population, as shown by immuno-fractionation and by fluorescence cross-correlation spectroscopy, flotillin-1 co-purifies mainly with CD63-negative vesicles and its release is not affected by syntenin. Syntenin-exosomes are of endosomal origin and depend on endosomal trafficking, as shown by RNAi targeting RAB7, a small GTPase regulating late endocytic membrane traffic. Electron microscopy analysis of syntenin-depleted cells shows that MVBs are largely devoid of intraluminal vesicles, indicating that syntenin supports the intraluminal budding and then production of exosomes. The molecular machinery responsible for the biogenesis of syntenin-exosomes is ESCRT-dependent, as depletion by RNAi of several components of the ESCRT complexes affects the formation of these exosomes. The presence of heparan sulfate chains on syndecans is necessary for producing exosomes, and the release of exosomes, impaired in HS-depleted cells, can be rescued by anti-syndecan antibody, suggesting that ligand-initiated syndecan oligomerization might be the driving force for the production of syntenin-exosomes. Syndecan-syntenin exosomes contain HS-dependent signalling cargo, such as p-FGFR, and can transfer FGF-responsiveness to recipient cells, hinting at the functional relevance of these exosomes.The results obtained show that interfering with syndecan-syntenin-ALIX reduces exosome production by affecting the biogenesis of MVBs and that syndecan-syntenin-ALIX complexes control the composition of a specific population of exosomes, potentially transferring specific information between cells. The molecular connection of syndecan with exosomes might thus support a novel role for proteoglycans in vesicular trafficking and trans-cellular signalling, supporting the release of growth factors and signalling components into the extracellular space. The establishment of a direct link between syndecans and exosomes, as a novel mechanism of morphogen spreading, may help explain the role of HSPG in the diffusion of morphogenetic signals, in particular how lipid-modified morphogens travel long-range. Syndecan-syntenin exosomes might also participate in the biology of HS-associated pathological processes, including neurodegenerative and oncogenic diseases.