Experimental determination and mathematical modeling of standard shapes of forming autophagosomes

オートファゴソーム標準形態の実験的決定と数理モデル --オートファジーを司る膜構造体の形の特徴を実験と理論で解明--. 京都大学プレスリリース. 2024-01-12.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

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.Comment: 33 pages, 8 figure

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

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

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

A single-molecule tracking technique coupled with mathematical modeling was developed for fully determining the dynamic monomer–dimer equilibrium of molecules in or on the plasma membrane, which will provide a framework for understanding signal transduction pathways initiated and regulated by dynamic dimers of membrane-localized receptors

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, lysosom

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

AbstractSingle-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 (2–200nm) 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

ABSTRACT 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. By further assessing the precision of the position determination, and the signal/noise and signal/ background ratios, we found that two single molecules in dual color can be colocalized to within 64–100 nm (68–90% detectability) in the membrane of cells for GFP and Alexa633. The detectability of true colocalization at the molecular level and the erroneous inclusion of incidental approaches of two molecules as colocalization have to be compromised at different levels in each experiment, depending on its purpose. This technique was successfully demonstrated in living cells in culture, monitoring colocalization of single molecules of E-cadherin fused with GFP diffusing in the plasma membrane with single molecules of Alexa633 conjugated to anti-E-cadherin Fab externally added to the culture medium. This work established a benchmark for monitoring the colocalization of two single molecules, which can be applied to wide ranges of studies for molecular interactions, both at the levels of single molecules and collections of molecules

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. 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