Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation.

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

Experimental determination and mathematical modeling of standard shapes of forming autophagosomes.

The formation of autophagosomes involves dynamic morphological changes of a phagophore from a flat membrane cisterna into a cup-shaped intermediate and a spherical autophagosome. However, the physical mechanism behind these morphological changes remains elusive. Here, we determine the average shapes of phagophores by statistically investigating three-dimensional electron micrographs of more than 100 phagophores. The results show that the cup-shaped structures adopt a characteristic morphology; they are longitudinally elongated, and the rim is catenoidal with an outwardly recurved shape. To understand these characteristic shapes, we establish a theoretical model of the shape of entire phagophores. The model quantitatively reproduces the average morphology and reveals that the characteristic shape of phagophores is primarily determined by the relative size of the open rim to the total surface area. These results suggest that the seemingly complex morphological changes during autophagosome formation follow a stable path determined by elastic bending energy minimization.

Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2.

Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here, we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKCα and PKCδ were deemed to be involved in this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKCs in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.

Quantitative 3D correlative light and electron microscopy of organelle association during autophagy.

In macroautophagy, disk-shaped double-membrane structures called phagophores elongate to form cup-shaped structures, becoming autophagosomes upon closure. These autophagosomes then fuse with lysosomes to become autolysosomes and degrade engulfed material. Autophagosome formation is reported to involve other organelles, including the endoplasmic reticulum (ER) and mitochondria. Organelles are also taken up by autophagosomes as autophagy cargos. However, few studies have performed systematic spatiotemporal analysis of inter-organelle relationships during macroautophagy. Here, we investigated the organelles in contact with phagophores, autophagosomes, and autolysosomes by using three-dimensional correlative light and electron microscopy with array tomography in cells starved 30 min. As previously reported, all phagophores associate with the ER. The surface area of phagophores in contact with the ER decreases gradually as they mature into autophagosomes and autolysosomes. However, the ER still associates with 92% of autophagosomes and 79% of autolysosomes, suggesting that most autophagosomes remain on the ER after closure and even when they fuse with lysosomes. In addition, we found that phagophores form frequently near other autophagic structures, suggesting the presence of potential hot spots for autophagosome formation. We also analyzed the contents of phagophores and autophagosomes and found that the ER is the most frequently engulfed organelle (detected in 65% of total phagophores and autophagosomes). These quantitative three-dimensional ultrastructural data provide insights into autophagosome-organelle relationships during macroautophagy.Key words: 3D-CLEM, autophagosome, electron microscopy, endoplasmic reticulum, lysosome.

Transient visit of STX17 (syntaxin 17) to autophagosomes.

STX17 (syntaxin 17) mediates autophagosome-lysosome fusion, and the translocation of STX17 to autophagosomes is characteristic of this process. STX17 arrives at autophagosomes when they are closed, stays there for approximately 10 min to promote fusion with lysosomes, and leaves when the autolysosomes are mature. However, the mechanism of this transient visit remains largely unknown. Here, we summarize the current knowledge about this phenomenon, including a recently discovered retrieval mechanism, and discuss remaining questions.Abbreviations: MAM: mitochondria-associated membrane; SNX: sorting nexin; STX17: syntaxin 17.

Organelle degradation in the lens by PLAAT phospholipases.

The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.

Should I bend or should I grow: the mechanisms of droplet-mediated autophagosome formation.

Phase-separated droplets with liquid-like properties can be degraded by macroautophagy/autophagy, but the mechanism underlying this degradation is poorly understood. We have recently derived a physical model to investigate the interaction between autophagic membranes and such droplets, uncovering that intrinsic wetting interactions underlie droplet-membrane contacts. We found that the competition between droplet surface tension and the increasing tendency of growing membrane sheets to bend determines whether a droplet is completely engulfed or isolated in a piecemeal fashion, a process we term fluidophagy. Intriguingly, we found that another critical parameter of droplet-membrane interactions, the spontaneous curvature of the membrane, determines whether the droplet is degraded by autophagy or - counterintuitively - serves as a platform from which autophagic membranes expand into the cytosol. We also discovered that the interaction of membrane-associated LC3 with the LC3-interacting region (LIR) found in the autophagic cargo receptor protein SQSTM1/p62 and many other autophagy-related proteins influences the preferred bending directionality of forming autophagosomes in living cells. Our study provides a physical account of how droplet-membrane wetting underpins the structure and fate of forming autophagosomes.

Wetting regulates autophagy of phase-separated compartments and the cytosol.

Compartmentalization of cellular material in droplet-like structures is a hallmark of liquid-liquid phase separation1,2, but the mechanisms of droplet removal are poorly understood. Evidence suggests that droplets can be degraded by autophagy3,4, a highly conserved degradation system in which membrane sheets bend to isolate portions of the cytoplasm within double-membrane autophagosomes5-7. Here we examine how autophagosomes sequester droplets that contain the protein p62 (also known as SQSTM1) in living cells, and demonstrate that double-membrane, autophagosome-like vesicles form at the surface of protein-free droplets in vitro through partial wetting. A minimal physical model shows that droplet surface tension supports the formation of membrane sheets. The model also predicts that bending sheets either divide droplets for piecemeal sequestration or sequester entire droplets. We find that autophagosomal sequestration is robust to variations in the droplet-sheet adhesion strength. However, the two sides of partially wetted sheets are exposed to different environments, which can determine the bending direction of autophagosomal sheets. Our discovery of this interplay between the material properties of droplets and membrane sheets enables us to elucidate the mechanisms that underpin droplet autophagy, or 'fluidophagy'. Furthermore, we uncover a switching mechanism that allows droplets to act as liquid assembly platforms for cytosol-degrading autophagosomes8 or as specific autophagy substrates9-11. We propose that droplet-mediated autophagy represents a previously undescribed class of processes that are driven by elastocapillarity, highlighting the importance of wetting in cytosolic organization.

High-speed single-molecule imaging reveals signal transduction by induced transbilayer raft phases.

Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59 cluster rafts and GM1 cluster rafts were stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK and continually recruited Lyn and H-Ras right beneath them in the inner leaflet with dwell lifetimes <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the nonraftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Because GM1 cluster rafts recruited Lyn and H-Ras as efficiently as CD59 cluster rafts, and because the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft-lipid interactions and thus serve as a key signal transduction platform.

Modeling Membrane Morphological Change during Autophagosome Formation.

Autophagy is an intracellular degradation process that is mediated by de novo formation of autophagosomes. Autophagosome formation involves dynamic morphological changes; a disk-shaped membrane cisterna grows, bends to become a cup-shaped structure, and finally develops into a spherical autophagosome. We have constructed a theoretical model that integrates the membrane morphological change and entropic partitioning of putative curvature generators, which we have used to investigate the autophagosome formation process quantitatively. We show that the membrane curvature and the distribution of the curvature generators stabilize disk- and cup-shaped intermediate structures during autophagosome formation, which is quantitatively consistent with in vivo observations. These results suggest that various autophagy proteins with membrane curvature-sensing properties control morphological change by stabilizing these intermediate structures. Our model provides a framework for understanding autophagosome formation.

Development of a Series of Practical Fluorescent Chemical Tools To Measure pH Values in Living Samples.

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate p Ka for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) SiRpH5 has suitable p Ka and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.

Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets.

ATG2 is one of the autophagy-related (ATG) proteins essential for autophagosome formation and localizes to isolation membranes and lipid droplets in mammalian cells. Here, we investigated the requirement of regions in ATG2A for its organellar localization and function. The N-terminal amino acids 1-198 and the C-terminal amino acids 1830-1938 are required for the localization to isolation membranes and lipid droplets, respectively. The C-terminal region is not required for the localization to isolation membranes and for autophagy. We also identified an amphipathic helix in ATG2A that is required for both its localization to organelles and autophagosome formation. These data suggest that the dual localization of ATG2A is regulated by different regions.

ATG conjugation-dependent degradation of the inner autophagosomal membrane is a key step for autophagosome maturation.

Although the autophagy-related (ATG) conjugation systems are thought to be important for a late step of autophagosome formation, their precise function has been poorly understood because they are also required for localization of the most important autophagosomal marker LC3. In our recent study we found that, using the autophagosomal SNARE STX17 (syntaxin 17) as an alternative marker, autophagosome-like structures were generated in ATG conjugation system-deficient cells. Those structures could fuse with lysosomes but the degradation of the inner autophagosomal membrane was significantly delayed. We suggest that the ATG conjugation-dependent closure of autophagosomes causes the inner autophagosomal membrane to become sensitive to lysosomal degradation.

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy.

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.

The ATG conjugation systems are important for degradation of the inner autophagosomal membrane.

In macroautophagy, cytoplasmic contents are sequestered into the double-membrane autophagosome, which fuses with the lysosome to become the autolysosome. It has been thought that the autophagy-related (ATG) conjugation systems are required for autophagosome formation. Here, we found that autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 17-positive autophagosome-like structures could be generated even in the absence of the ATG conjugation systems, although at a reduced rate. These syntaxin 17-positive structures could further fuse with lysosomes, but degradation of the inner autophagosomal membrane was significantly delayed. Accordingly, autophagic activity in ATG conjugation-deficient cells was strongly suppressed. We suggest that the ATG conjugation systems, which are likely required for the closure (i.e., fission) of the autophagosomal edge, are not absolutely essential for autolysosome formation but are important for efficient degradation of the inner autophagosomal membrane.

Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells.

Autophagy is mediated by a unique organelle, the autophagosome. Autophagosome formation involves a number of autophagy-related (ATG) proteins and complicated membrane dynamics. Although the hierarchical relationships of ATG proteins have been investigated, how individual ATG proteins or their complexes contribute to the organization of the autophagic membrane remains largely unknown. Here, systematic ultrastructural analysis of mouse embryonic fibroblasts (MEFs) and HeLa cells deficient in various ATG proteins reveals that the emergence of the isolation membrane (phagophore) requires FIP200 (also known as RB1CC1), ATG9A and phosphatidylinositol (PtdIns) 3-kinase activity. By contrast, small premature isolation-membrane-like and autophagosome-like structures were generated in cells lacking VMP1 and both ATG2A and ATG2B, respectively. The isolation membranes could elongate in cells lacking ATG5, but did not mature into autophagosomes. We also found that ferritin clusters accumulated at the autophagosome formation site together with p62 (also known as SQSTM1) in autophagy-deficient cells. These results reveal the specific functions of these representative ATG proteins in autophagic membrane organization and ATG-independent recruitment of ferritin to the site of autophagosome formation.

Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site.

Autophagosome formation is governed by sequential functions of autophagy-related (ATG) proteins. Although their genetic hierarchy in terms of localization to the autophagosome formation site has been determined, their temporal relationships remain largely unknown. In this study, we comprehensively analyzed the recruitment of mammalian ATG proteins to the autophagosome formation site by live-cell imaging, and determined their temporal relationships. Although ULK1 and ATG5 are separated in the genetic hierarchy, they synchronously accumulate at pre-existing VMP1-positive punctate structures, followed by recruitment of ATG14, ZFYVE1, and WIPI1. Only a small number of ATG9 vesicles appear to be associated with these structures. Finally, LC3 and SQSTM1/p62 accumulate synchronously, while the other ATG proteins dissociate from the autophagic structures. These results suggest that autophagosome formation takes place on the VMP1-containing domain of the endoplasmic reticulum or a closely related structure, where ULK1 and ATG5 complexes are synchronously recruited.

Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy.

Mitochondria can be degraded by autophagy in a process termed mitophagy. The Parkinson-disease-associated ubiquitin ligase Parkin can trigger mitophagy of depolarized mitochondria. However, it remains to be determined how the autophagy machinery is involved in this specific type of autophagy. It has been speculated that adaptor proteins such as p62 might mediate the interaction between the autophagosomal LC3 family of proteins and ubiquitylated proteins on mitochondria. Here, we describe our systematic analysis of the recruitment of Atg proteins in Parkin-dependent mitophagy. Structures containing upstream Atg proteins, including ULK1, Atg14, DFCP1, WIPI-1 and Atg16L1, can associate with depolarized mitochondria even in the absence of membrane-bound LC3. Atg9A structures are also recruited to these damaged mitochondria as well as to the autophagosome formation site during starvation-induced canonical autophagy. In the initial steps of Parkin-mediated mitophagy, the structures containing the ULK1 complex and Atg9A are independently recruited to depolarized mitochondria and both are required for further recruitment of downstream Atg proteins except LC3. Autophagosomal LC3 is important for efficient incorporation of damaged mitochondria into the autophagosome at a later stage. These findings suggest a process whereby the isolation membrane is generated de novo on damaged mitochondria as opposed to one where a preformed isolation membrane recognizes mitochondria.

Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging.

Receptor dimerization is important for many signaling pathways. However, the monomer-dimer equilibrium has never been fully characterized for any receptor with a 2D equilibrium constant as well as association/dissociation rate constants (termed super-quantification). Here, we determined the dynamic equilibrium for the N-formyl peptide receptor (FPR), a chemoattractant G protein-coupled receptor (GPCR), in live cells at 37°C by developing a single fluorescent-molecule imaging method. Both before and after liganding, the dimer-monomer 2D equilibrium is unchanged, giving an equilibrium constant of 3.6 copies/µm(2), with a dissociation and 2D association rate constant of 11.0 s(-1) and 3.1 copies/µm(2)s(-1), respectively. At physiological expression levels of ∼2.1 receptor copies/µm(2) (∼6,000 copies/cell), monomers continually convert into dimers every 150 ms, dimers dissociate into monomers in 91 ms, and at any moment, 2,500 and 3,500 receptor molecules participate in transient dimers and monomers, respectively. Not only do FPR dimers fall apart rapidly, but FPR monomers also convert into dimers very quickly.

Hierarchical organization of the plasma membrane: investigations by single-molecule tracking vs. fluorescence correlation spectroscopy.

Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.