Apical dehydration impairs the cystic fibrosis airway epithelium barrier via a ß1-integrin/YAP1 pathway.

Defective hydration of airway surface mucosa is associated with lung infection in cystic fibrosis (CF), partly caused by disruption of the epithelial barrier integrity. Although rehydration of the CF airway surface liquid (ASL) alleviates epithelium vulnerability to infection by junctional protein expression, the mechanisms linking ASL to barrier integrity are unknown. We show here the strong degradation of YAP1 and TAZ proteins in well-polarized CF human airway epithelial cells (HAECs), a process that was prevented by ASL rehydration. Conditional silencing of YAP1 in rehydrated CF HAECs indicated that YAP1 expression was necessary for the maintenance of junctional complexes. A higher plasma membrane tension in CF HAECs reduced endocytosis, concurrent with the maintenance of active ß1-integrin ectopically located at the apical membrane. Pharmacological inhibition of ß1-integrin accumulation restored YAP1 expression in CF HAECs. These results indicate that dehydration of the CF ASL affects epithelial plasma membrane tension, resulting in ectopic activation of a ß1-integrin/YAP1 signaling pathway associated with degradation of junctional proteins.

Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth.

Lysosomes must maintain the integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but it is unknown whether dedicated programmes for maintaining the integrity of the lysosome membrane facilitate pancreatic cancer growth. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor Myoferlin as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosomal localization of Myoferlin is necessary and sufficient for the maintenance of lysosome health and provides an early acting protective system against membrane damage that is independent of the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances the pro-oncogenic activities of the lysosome.

An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission.

The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity.

ALIX- and ESCRT-III-dependent sorting of tetraspanins to exosomes.

The intraluminal vesicles (ILVs) of endosomes mediate the delivery of activated signaling receptors and other proteins to lysosomes for degradation, but they also modulate intercellular communication when secreted as exosomes. The formation of ILVs requires four complexes, ESCRT-0, -I, -II, and -III, with ESCRT-0, -I, and -II presumably involved in cargo sorting and ESCRT-III in membrane deformation and fission. Here, we report that an active form of the ESCRT-associated protein ALIX efficiently recruits ESCRT-III proteins to endosomes. This recruitment occurs independently of other ESCRTs but requires lysobisphosphatidic acid (LBPA) in vivo, and can be reconstituted on supported bilayers in vitro. Our data indicate that this ALIX- and ESCRT-III-dependent pathway promotes the sorting and delivery of tetraspanins to exosomes. We conclude that ALIX provides an additional pathway of ILV formation, secondary to the canonical pathway, and that this pathway controls the targeting of exosomal proteins.

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells.

In specialized cell types, lysosome-related organelles support regulated secretory pathways, whereas in nonspecialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol-storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in nonspecialized cells and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel mucolipin-1, as well as FYCO1, the AP1 adaptor, and its partner Gadkin. We conclude that endo-lysosomes in nonspecialized cells can acquire secretory functions elicited by cyclodextrin and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling.

The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE.