2020) ESCRT-mediated phagophore sealing during mitophagy, Autophagy, 16:5, 826-841, ABSTRACT Inactivation of the endosomal sorting complex required for transport (ESCRT) machinery has been reported to cause autophagic defects, but the exact functions of ESCRT proteins in macroautophagy/ autophagy remain incompletely understood. Using live-cell fluorescence microscopy we found that the filament-forming ESCRT-III subunit CHMP4B was recruited transiently to nascent autophagosomes during starvation-induced autophagy and mitophagy, with residence times of about 1 and 2 min, respectively. Correlative light microscopy and electron tomography revealed CHMP4B recruitment at a late step in mitophagosome formation. The autophagosomal dwell time of CHMP4B was strongly increased by depletion of the regulatory ESCRT-III subunit CHMP2A. Using a novel optogenetic closure assay we observed that depletion of CHMP2A inhibited phagophore sealing during mitophagy. Consistent with this, depletion of CHMP2A and other ESCRT-III subunits inhibited both PRKN/PARKINdependent and -independent mitophagy. We conclude that the ESCRT machinery mediates phagophore closure, and that this is essential for mitophagic flux.

Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis plays important roles in infection, immunity and cancer development. A key question is how large macropinosomes are able to squeeze through the dense actin network underlying the plasma membrane in order to move towards the cell centre for maturation. Here we show that, immediately after macropinosomes have been sealed off from the plasma membrane, the PH-and FYVE domain-containing protein Phafin2 is recruited by a mechanism that involves binding to phosphatidylinositol 3-phosphate (PtdIns3P) generated in a non-canonical manner. Phafin2 in turn regulates the actin cross-linking protein Filamin A to promote entry of macropinosomes through the subcortical actin matrix and subsequent maturation. Depletion of Phafin2 inhibits macropinocytic internalization and maturation. We conclude that PtdIns3P and its effector Phafin2 are key components of a system that allows nascent macropinosomes to navigate through the dense subcortical actin network.

Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network.