In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17–Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17–Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

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The spatiotemporal regulation of light-gated ion channels is a powerful tool to study physiological pathways and develop personalized theranostic modalities. So far, most existing light-gated channels are limited by their action spectra in the ultraviolet (UV) or visible region. Simple and innovative strategies for the specific attachment of photoswitches on the cell surface without modifying or genetically encoding channel structures, and more importantly, that enable the remote activation of ion-channel functions within near-infrared (NIR) spectral window in living systems, remain a challenging concern. Herein, metabolic glycan biosynthesis is used to achieve site-specific covalent attachment of near-infrared-light-mediated lanthanide-doped upconversion nanocrystals (UCNs) to the cell surface through copper-free click cyclization. Upon irradiation with 808 nm light, the converted emission at 480 nm could activate a light-gated ion channel, channelrhodopsins-2 (ChR2), and thus remotely control the cation influx. This unique strategy provides valuable insights on the specific regulation membrane-associated activities in vivo.

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure.

Prompt reperfusion after cerebral ischemia is critical for neuronal survival. Any strategies that extend the limited reperfusion window will be of great importance. Acidic postconditioning (APC) is a mild acidosis treatment that involves inhaling CO2 during reperfusion following ischemia. APC attenuates ischemic brain injury although the underlying mechanisms have not been elucidated. Here we report that APC reinforces ischemia-reperfusion-induced mitophagy in middle cortical artery occlusion (MCAO)-treated mice, and in oxygen-glucose deprivation (OGD)-treated brain slices and neurons. Inhibition of mitophagy compromises neuroprotection conferred by APC. Furthermore, mitophagy and neuroprotection are abolished in Park2 knockout mice, indicating that APC-induced mitophagy is facilitated by the recruitment of PARK2 to mitochondria. Importantly, in MCAO mice, APC treatment extended the effective reperfusion window from 2 to 4 h, and this window was further extended to 6 h by exogenously expressing PARK2. Taken together, we found that PARK2-dependent APC-induced mitophagy renders the brain resistant to ischemic injury. APC treatment could be a favorable strategy to extend the thrombolytic time window for stroke therapy.