A sustained calcium response mediated by IP3 receptor anchoring to the desmosome is essential for apoptotic cell elimination.

Efficient elimination of apoptotic cells within epithelial cell sheets is crucial for preserving epithelial barrier integrity. It is well established that immediate neighbors of an apoptotic cell actively participate in its removal by enclosing it within a wall of actomyosin, pushing it out in a purse-string manner in a process called apical extrusion. Here, we found that sustained elevation of calcium ions in neighboring epithelial cells is necessary to generate the contractility required for apoptotic cell elimination.

Organelle landscape analysis using a multiparametric particle-based method.

Organelles have unique structures and molecular compositions for their functions and have been classified accordingly. However, many organelles are heterogeneous and in the process of maturation and differentiation. Because traditional methods have a limited number of parameters and spatial resolution, they struggle to capture the heterogeneous landscapes of organelles.

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.

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.

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.

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.

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.

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 zone. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy. 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 mice-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes.

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.

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 separation, but the mechanisms of droplet removal are poorly understood. Evidence suggests that droplets can be degraded by autophagy, a highly conserved degradation system in which membrane sheets bend to isolate portions of the cytoplasm within double-membrane autophagosomes. 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.

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.

Modeling Membrane Morphological Change during Autophagosome Formation.

Autophagy is an intracellular degradation process that is mediated by 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.

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 K 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.

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.

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.

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.

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.

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.

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.

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.

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.

Fluorescence imaging for monitoring the colocalization of two single molecules in living cells.

The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single fluorescent molecule colocalization imaging, to quantitatively detect the colocalization of two species of individual molecules. We first established a method for spatially correcting the two full images synchronously obtained in two different colors, and then for overlaying them with an accuracy of 13 nm.

Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts.

We have evaluated the sizes and lifetimes of rafts in the plasma membrane from the existing literature, with a special attention paid to their intrinsically broad distributions and the limited time and space scales that are covered by the observation methods used for these studies. Distinguishing the rafts in the steady state (reserve rafts) from those after stimulation or unintentional crosslinking of raft molecules (stabilized receptor-cluster rafts) is critically important. In resting cells, the rafts appear small and unstable, and the consensus now is that their sizes are smaller than the optical diffraction limit (250 nm).