The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension.

Human WIPI ß-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan.

Wetting of phase-separated droplets on plant vacuole membranes leads to a competition between tonoplast budding and nanotube formation.

Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration of lytic vacuoles in embryos [M. Feeney et al., Plant Physiol. 177, 241-254 (2018)], PSVs undergo division during the later stages of seed maturation. Here, we study the biophysical mechanism of PSV morphogenesis in vivo, discovering that micrometer-sized liquid droplets containing storage proteins form within the vacuolar lumen through phase separation and wet the tonoplast (vacuolar membrane). We identify distinct tonoplast shapes that arise in response to membrane wetting by droplets and derive a simple theoretical model that conceptualizes these geometries. Conditions of low membrane spontaneous curvature and moderate contact angle (i.e., wettability) favor droplet-induced membrane budding, thereby likely serving to generate multiple, physically separated PSVs in seeds. In contrast, high membrane spontaneous curvature and strong wettability promote an intricate and previously unreported membrane nanotube network that forms at the droplet interface, allowing molecule exchange between droplets and the vacuolar interior. Furthermore, our model predicts that with decreasing wettability, this nanotube structure transitions to a regime with bud and nanotube coexistence, which we confirmed in vitro. As such, we identify intracellular wetting [J. Agudo-Canalejo et al., Nature 591, 142-146 (2021)] as the mechanism underlying PSV morphogenesis and provide evidence suggesting that interconvertible membrane wetting morphologies play a role in the organization of liquid phases in cells.

Intracellular wetting mediates contacts between liquid compartments and membrane-bound organelles.

Protein-rich droplets, such as stress granules, P-bodies, and the nucleolus, perform diverse and specialized cellular functions. Recent evidence has shown the droplets, which are also known as biomolecular condensates or membrane-less compartments, form by phase separation. Many droplets also contact membrane-bound organelles, thereby functioning in development, intracellular degradation, and organization. These underappreciated interactions have major implications for our fundamental understanding of cells. Starting with a brief introduction to wetting phenomena, we summarize recent progress in the emerging field of droplet-membrane contact. We describe the physical mechanism of droplet-membrane interactions, discuss how these interactions remodel droplets and membranes, and introduce "membrane scaffolding" by liquids as a novel reshaping mechanism, thereby demonstrating that droplet-membrane interactions are elastic wetting phenomena. "Membrane-less" and "membrane-bound" condensates likely represent distinct wetting states that together link phase separation with mechanosensitivity and explain key structures observed during embryogenesis, during autophagy, and at synapses. We therefore contend that droplet wetting on membranes provides a robust and intricate means of intracellular organization.

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.

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.

Unrestrained ESCRT-III drives micronuclear catastrophe and chromosome fragmentation.

The ESCRT-III membrane fission machinery maintains the integrity of the nuclear envelope. Although primary nuclei resealing takes minutes, micronuclear envelope ruptures seem to be irreversible. Instead, micronuclear ruptures result in catastrophic membrane collapse and are associated with chromosome fragmentation and chromothripsis, complex chromosome rearrangements thought to be a major driving force in cancer development. Here we use a combination of live microscopy and electron tomography, as well as computer simulations, to uncover the mechanism underlying micronuclear collapse. We show that, due to their small size, micronuclei inherently lack the capacity of primary nuclei to restrict the accumulation of CHMP7-LEMD2, a compartmentalization sensor that detects loss of nuclear integrity. This causes unrestrained ESCRT-III accumulation, which drives extensive membrane deformation, DNA damage and chromosome fragmentation. Thus, the nuclear-integrity surveillance machinery is a double-edged sword, as its sensitivity ensures rapid repair at primary nuclei while causing unrestrained activity at ruptured micronuclei, with catastrophic consequences for genome stability.

Controlled division of cell-sized vesicles by low densities of membrane-bound proteins.

The proliferation of life on earth is based on the ability of single cells to divide into two daughter cells. During cell division, the plasma membrane undergoes a series of morphological transformations which ultimately lead to membrane fission. Here, we show that analogous remodeling processes can be induced by low densities of proteins bound to the membranes of cell-sized lipid vesicles. Using His-tagged fluorescent proteins, we are able to precisely control the spontaneous curvature of the vesicle membranes. By fine-tuning this curvature, we obtain dumbbell-shaped vesicles with closed membrane necks as well as neck fission and complete vesicle division. Our results demonstrate that the spontaneous curvature generates constriction forces around the membrane necks and that these forces can easily cover the force range found in vivo. Our approach involves only one species of membrane-bound proteins at low densities, thereby providing a simple and extendible module for bottom-up synthetic biology.

Phase separation organizes the site of autophagosome formation.

Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS.

Formation of Autophagosomes Coincides with Relaxation of Membrane Curvature.

Autophagy is an intracellular degradation process that employs complex membrane dynamics to isolate and break down cellular components. However, many unanswered questions remain concerning remodeling of autophagic membranes. Here, we focus on the advantages of theoretical modeling to study the formation of autophagosomes and to understand the origin of autophagosomal membranes. Starting from the well-defined geometry of final autophagosomes, we ask the question of how these organelles can be formed by combining various pre-autophagosomal membranes such as vesicles, membrane tubules, or sheets. We analyze the geometric constraints of autophagosome formation by taking the area of the precursor membranes and their internal volume into account. Our results suggest that vesicle fusion contributes little to the formation of autophagosomes. In the second part, we quantify the curvature of the precursors and report that the formation of autophagosomes is associated with a strong relaxation of membrane curvature energy. This effect we find for a wide range of membrane asymmetries. It is especially strong for small distances between both autophagosomal membranes, as observed in vivo. We quantify the membrane bending energies of all precursors by considering membrane asymmetries. We propose that the generation and supply of pre-autophagosomal membranes is one limiting step for autophagosome formation.

Interactive Autophagy: Monitoring a Novel Form of Selective Autophagy by Macroscopic Observations.

Traditional lectures and cookbook laboratory exercises are today's standard tools in scientific teaching and learning. However, these conventional methods are suboptimal. Combining active learning techniques with physical experiences can improve educational success significantly. Still, hands-on material which supports active and physical teaching concepts is rare. Here, we introduce an interactive, performance-based method.As an example, we studied autophagosome formation. We observed assembly of the phagophore by membrane fusion, cargo isolation by bending the phagophore and membrane scission. We extracted characteristic time scales of autophagosome formation. Moreover, we observed capturing the autophagic cargo within a single membrane for the first time. In this chapter, we provide an easy tool to engage participants in the process of scientific perception. We are convinced that "hands-on" experiments and interactive analyses will encourage students to participate more actively in classes and thus, will improve learning. Moreover, we anticipate that the approach enhances translation of scientific concepts between different fields by providing scientists with a fresh view on, e.g., membrane-bound processes and can improve communication of science to the public.

Charged giant unilamellar vesicles prepared by electroformation exhibit nanotubes and transbilayer lipid asymmetry.

Giant unilamellar vesicles (GUVs) are increasingly used as a versatile research tool to investigate membrane structure, morphology and phase state. In these studies, GUV preparation is typically enhanced by an externally applied electric field, a process called electroformation. We find that upon osmotic deflation, GUVs electroformed from charged and neutral lipids exhibit inward pointing lipid nanotubes, suggesting negative spontaneous curvature of the membrane. By quenching a fluorescent analog of the charged lipid, zeta potential measurements and experiments with the lipid marker annexin A5, we show that electroformed GUVs exhibit an asymmetric lipid distribution across the bilayer leaflets. The asymmetry is lost either after storing electroformed GUVs at room temperature for one day or by applying higher voltages and temperatures during electroformation. GUVs having the same lipid composition but grown via gel-assisted swelling do not show asymmetric lipid distribution. We discuss possible mechanisms for the generation and relaxation of lipid asymmetry, as well as implications for studies using electroformed vesicles. The observed effects allow to control the molecular assembly of lipid bilayer leaflets. Vesicle tubulation as reported here is an example of protein-free reshaping of membranes and is caused by compositional lipid asymmetry between leaflets.

Micron-sized domains in quasi single-component giant vesicles.

Giant unilamellar vesicles (GUVs), are a convenient tool to study membrane-bound processes using optical microscopy. An increasing number of studies highlights the potential of these model membranes when addressing questions in membrane biophysics and cell-biology. Among them, phase transitions and domain formation, dynamics and stability in raft-like mixtures are probably some of the most intensively investigated. In doing so, many research teams rely on standard protocols for GUV preparation and handling involving the use of sugar solutions. Here, we demonstrate that following such a standard approach can lead to the abnormal formation of micron-sized domains in GUVs grown from only a single phospholipid. The membrane heterogeneity is visualized by means of a small fraction (0.1 mol%) of a fluorescent lipid dye. For dipalmitoylphosphatidylcholine GUVs, different types of membrane heterogeneities were detected. First, the unexpected formation of micron-sized dye-depleted domains was observed upon cooling. These domains nucleated about 10 K above the lipid main phase transition temperature, TM. In addition, upon further cooling of the GUVs down to the immediate vicinity of TM, stripe-like dye-enriched structures around the domains are detected. The micron-sized domains in quasi single-component GUVs were observed also when using two other lipids. Whereas the stripe structures are related to the phase transition of the lipid, the dye-excluding domains seem to be caused by traces of impurities present in the glucose. Supplementing glucose solutions with nm-sized liposomes at millimolar lipid concentration suppresses the formation of the micron-sized domains, presumably by providing competitive binding of the impurities to the liposome membrane in excess. It is likely that such traces of impurities can significantly alter lipid phase diagrams and cause differences among reported ones.

The Conserved ESCRT-III Machinery Participates in the Phagocytosis of Entamoeba histolytica.

The endosomal sorting complex required for transport (ESCRT) orchestrates cell membrane-remodeling mechanisms in eukaryotes, including endocytosis. However, ESCRT functions in phagocytosis (ingestion of ≥250 nm particles), has been poorly studied. In macrophages and amoebae, phagocytosis is required for cell nutrition and attack to other microorganisms and cells. In Entamoeba histolytica, the voracious protozoan responsible for human amoebiasis, phagocytosis is a land mark of virulence. Here, we have investigated the role of ESCRT-III in the phagocytosis of E. histolytica, using mutant trophozoites, recombinant proteins (rEhVps20, rEhVps32, rEhVps24, and rEhVps2) and giant unilamellar vesicles (GUVs). Confocal images displayed the four proteins located around the ingested erythrocytes, in erythrocytes-containing phagosomes and in multivesicular bodies. EhVps32 and EhVps2 proteins co-localized at the phagocytic cups. Protein association increased during phagocytosis. Immunoprecipitation and flow cytometry assays substantiated these associations. GUVs revealed that the protein assembly sequence is essential to form intraluminal vesicles (ILVs). First, the active rEhVps20 bound to membranes and recruited rEhVps32, promoting membrane invaginations. rEhVps24 allowed the detachment of nascent vesicles, forming ILVs; and rEhVps2 modulated their size. The knock down of Ehvps20 and Ehvps24genes diminished the rate of erythrophagocytosis demonstrating the importance of ESCRT-III in this event. In conclusion, we present here evidence of the ESCRT-III participation in phagocytosis and delimitate the putative function of proteins, according to the in vitro reconstruction of their assembling.

Fusion and scission of membranes: Ubiquitous topological transformations in cells.

The 2016 Nobel Prizes were awarded to Yoshinori Ohsumi for autophagy and to David Thouless, Duncan Haldane and Michael Kosterlitz for topological transitions. Both of these phenomena are intrinsically related when it comes to membranes. Here, we give a brief account on topological transformations of lipid membranes, commonly known as membrane fusion and membrane scission, and introduce the underlying topological invariant, the genus. The genus of a shape is a useful concept to distinguish unambiguously the processes of membrane fusion/scission and offers a simple method to describe complex, cellular membrane structures, such as fenestrated cristae. We distinguish and highlight the connection between topological transformations of lipid membranes and the recent awards, and point out the extraordinarily large number of topological changes during autophagy.

Copper ATPase CopA from Escherichia coli: Quantitative Correlation between ATPase Activity and Vectorial Copper Transport.

Cu-ATPases are membrane copper transporters present in all kingdoms of life. They play a central role in Cu homeostasis by pumping Cu ions across cell membranes with energy derived from ATP hydrolysis. In this work, the Cu-ATPase CopA from Escherichia coli was expressed and purified in fully functional form and demonstrated to bind Cu(I) with subfemtomolar affinity. It was incorporated into the lipid membrane of giant unilamellar vesicles (GUVs) whose dimensions match those of eukaryotic cells. An 1H NMR approach provided a quantitative ATPase activity assay for the enzyme either dissolved in detergent or embedded in GUV membranes. The activity varied with the Cu(I) availability in an optimized assay solution for either environment, demonstrating a direct correlation between ATPase activity and Cu(I) transport. Quantitative analysis of the Cu content trapped by the GUVs is consistent with a Cu:ATP turnover ratio of 1.

Posing for a picture: vesicle immobilization in agarose gel.

Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biological preparations for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 µm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small molecules, proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solutions, and complex biochemical reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resolution 3D images of GUVs.

Autophagosome closure requires membrane scission.

During the intracellular process of macroautophagy (hereafter autophagy), a membrane-bound organelle, the autophagosome, is generated de novo. The remodeling of the autophagic membrane during the life cycle of the organelle is a complex multistep process and involves several changes in the topology of the autophagic membrane. Here, we focus on the final step of autophagosome formation, the closure of the phagophore, during which the inner and outer autophagic membranes become separate entities. We argue that this topological membrane transformation is a membrane scission event. Surprisingly, not a single recent review describes this substep as membrane scission (or membrane fission). In contrast, a number of publications imply that membrane fusion is involved. We discuss the potential sources for misinterpretation and recommend to consistent use of the unambiguous term "membrane scission."

Membrane morphology is actively transformed by covalent binding of the protein Atg8 to PE-lipids.

Autophagy is a cellular degradation pathway involving the shape transformation of lipid bilayers. During the onset of autophagy, the water-soluble protein Atg8 binds covalently to phosphatdylethanolamines (PEs) in the membrane in an ubiquitin-like reaction coupled to ATP hydrolysis. We reconstituted the Atg8 conjugation system in giant and nm-sized vesicles with a minimal set of enzymes and observed that formation of Atg8-PE on giant vesicles can cause substantial tubulation of membranes even in the absence of Atg12-Atg5-Atg16. Our findings show that ubiquitin-like processes can actively change properties of lipid membranes and that membrane crowding by proteins can be dynamically regulated in cells. Furthermore we provide evidence for curvature sorting of Atg8-PE. Curvature generation and sorting are directly linked to organelle shapes and, thus, to biological function. Our results suggest that a positive feedback exists between the ubiquitin-like reaction and the membrane curvature, which is important for dynamic shape changes of cell membranes, such as those involved in the formation of autophagosomes.

Curvature of double-membrane organelles generated by changes in membrane size and composition.

Transient double-membrane organelles are key players in cellular processes such as autophagy, reproduction, and viral infection. These organelles are formed by the bending and closure of flat, double-membrane sheets. Proteins are believed to be important in these morphological transitions but the underlying mechanism of curvature generation is poorly understood. Here, we describe a novel mechanism for this curvature generation which depends primarily on three membrane properties: the lateral size of the double-membrane sheets, the molecular composition of their highly curved rims, and a possible asymmetry between the two flat faces of the sheets. This mechanism is evolutionary advantageous since it does not require active processes and is readily available even when resources within the cell are restricted as during starvation, which can induce autophagy and sporulation. We identify pathways for protein-assisted regulation of curvature generation, organelle size, direction of bending, and morphology. Our theory also provides a mechanism for the stabilization of large double-membrane sheet-like structures found in the endoplasmic reticulum and in the Golgi cisternae.