Vps68 cooperates with ESCRT-III in intraluminal vesicle formation

The endosomal sorting complex required for transport (ESCRT)-III mediates budding and abscission of intraluminal vesicles (ILVs) into multivesicular endosomes. To further define the role of the ESCRT-III associated protein Mos10/Vps60 in ILV formation, we screened for new interaction partners by SILAC/MS. Here, we focused on the newly identified interaction partner Vps68. Our data suggest that Vps68 cooperates with ESCRT-III in ILV formation. The deletion of VPS68 caused a sorting defect similar to the SNF7 deletion, when the cargo load was high. The composition of ESCRT-III was altered, the level of core components was higher and the level of associated proteins was lower in the deletion strain. This suggests that a shift occurs from an active complex to a disassembly competent complex and that this shift is blocked in the Δvps68 strain. We present evidence that during this shift Snf7 is replaced by Mos10. Vps68 has an unusual membrane topology. Two of its potential membrane helices are amphipathic helices localized to the luminal side of the endosomal membrane. Based on this membrane topology we propose that Vps68 and ESCRT-III cooperate in the abscission step by weakening the luminal and cytosolic leaflets of the bilayer at the abscission site.

Applications of cell resealing to reconstitute microRNA loading to extracellular vesicles

MicroRNAs (miRNAs) are cargo carried by extracellular vesicles (EVs) and are associated with cell–cell interactions. The response to the cellular environment, such as disease states, genetic/metabolic changes, or differences in cell type, highly regulates cargo sorting to EVs. However, morphological features during EV formation and secretion involving miRNA loading are unknown. This study developed a new method of EV loading using cell resealing and reconstituted the elementary miRNA-loading processes. Morphology, secretory response, and cellular uptake ability of EVs obtained from intact and resealed HeLa cells were comparable. Exogenously added soluble factors were introduced into multivesicular endosomes (MVEs) and their subsequent secretion to the extracellular region occurred in resealed HeLa cells. In addition, miRNA transport to MVEs and miRNA encapsulation to EVs followed a distinct pathway regulated by RNA-binding proteins, such as Argonaute and Y-box binding protein 1, depending on miRNA types. Our cell-resealing system can analyze disease-specific EVs derived from disease model cells, where pathological cytosol is introduced into cells. Thus, EV formation in resealed cells can be used not only to create a reconstitution system to give mechanistic insight into EV encapsulation but also for applications such as loading various molecules into EVs and identifying disease-specific EV markers.

ROR1 sustains multivesicular endosomes by interacting with HRS and STAM1

The authors have requested that this preprint be withdrawn due to erroneous posting.

RAB31 marks and controls an ESCRT-independent exosome pathway

Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.

ROR1 sustains multivesicular endosomes by interacting with HRS and STAM1

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) regulates caveolae formation and caveolae-dependent endocytosis by interacting with caveolae components, which in turn sustains pro-survival signaling toward AKT from multiple RTKs, including EGFR, and MET. We report here a novel function of ROR1 as a scaffold for HRS and STAM1, two essential components of ESCRT-0. The present results show that ROR1 facilitates interactions of HRS and STAM1, thereby preventing the lysosomal degradation of HRS. Furthermore, interaction of ROR1 with STAM1 was found to be required to sustain binding of ROR1 to HRS as well as HRS subcellular localization. Additionally, ROR1 localized in both the limiting membrane and intraluminal vesicles (ILVs) of Rab5-induced multivesicular endosomes (MVEs) containing HRS, CD63, and EEA1 was found to regulate the formation of Rab5-induced MVEs by an association with the GTP-bound form of Rab5 in cancer cells. Notably, ROR1 depletion inhibits CD63-positive MVEs formation and reduces exosomes release. Our findings provide the first evidence that the onco-embryonic antigen receptor ROR1 regulates exosome biogenesis via MVE formation in cancer cells.

Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

The canonical Wnt signaling pathway serves as a hub connecting diverse cellular physiological processes, such as β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many components of the ESCRT (Endocytic Sorting Components Required for Transport) machinery interacted with Lrp6 within 5 minutes of Wnt3a treatment. This supports the proposal of a central role of multivesicular endosomes in canonical Wnt signaling. Interestingly, proteomic analyses identified the Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, as being strongly enriched in the proximity of Lrp6. We provide evidence that TFG specifically co-localized with MVBs after Wnt stimulation. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.SignificanceWnt/β-catenin signaling is a conserved pathway involved in cell differentiation and in the regulation of many other processes, including cell growth and proliferation, macropinocytosis, and cell metabolism. Endocytosis is required to regulate Wnt signaling, but the precise factors at play are still elusive. Here, we describe a biotin-dependent proximity labeling approach using ascorbate peroxidase-tagged Lrp6, a Wnt co-receptor. Proteomic analysis of biotinylated-enriched targets identified numerous multivesicular endosome proteins that were recruited to the receptor shortly after addition of Wnt protein. Additionally, we identified the protein TFG as one of the strongest interactors with Lrp6. TFG co-localized with Wnt-induced multivesicular endosomes. Xenopus embryo assays revealed that TFG is required in vivo for canonical Wnt signaling.

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.

Roles for ER:endosome membrane contact sites in ligand-stimulated intraluminal vesicle formation

Multivesicular endosomes/bodies (MVBs) sort membrane proteins between recycling and degradative pathways. Segregation of membrane proteins onto intraluminal vesicles (ILVs) of MVBs removes them from the recycling pathway and facilitates their degradation following fusion of MVBs with lysosomes. Sorting of many cargos onto ILVs depends on the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, although ESCRT-independent mechanisms also exist. In mammalian cells, efficient sorting of ligand-stimulated epidermal growth factor receptors onto ILVs also depends on the tyrosine phosphatase, PTP1B, an ER-localised enzyme that interacts with endosomal targets at membrane contacts between MVBs and the ER. This review focuses on the potential roles played by ER:MVB membrane contact sites in regulating ESCRT-dependent ILV formation.

To be or not to be... secreted as exosomes, a balance finely tuned by the mechanisms of biogenesis

The release of extracellular vesicles such as exosomes provides an attractive intercellular communication pathway. Exosomes are 30- to 150-nm membrane vesicles that are generated in endosomal compartment and act as intercellular mediators in both physiological and pathological context. Despite the growing interest in exosome functions, the mechanisms responsible for their biogenesis and secretion are still not completely understood. Knowledge about these mechanisms is important because they control the composition, and hence the function and secretion, of exosomes. Exosomes are produced as intraluminal vesicles in extremely dynamic endosomal organelles, which undergo various maturation processes in order to form multivesicular endosomes. Notably, the function of multivesicular endosomes is balanced between exosome secretion and lysosomal degradation. In the present review, we present and discuss each intracellular trafficking pathway that has been reported or proposed as regulating exosome biogenesis, with a particular focus on the importance of endosomal dynamics in sorting out cargo proteins to exosomes and to the secretion of multivesicular endosomes. An overall picture reveals several key mechanisms, which mainly act at the crossroads of endosomal pathways as regulatory checkpoints of exosome biogenesis.