Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodeling superfamily.

Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.

TMEM41B Is a Pan-flavivirus Host Factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission.

The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity.

Mechanisms of Autophagy Initiation.

Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and noncatalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9 (autophagy-related 9), COPII (coat protein complex II) vesicles, and possibly other sources. In the wake of reconstitution and super-resolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes.

The endosomal sorting complexes required for transport (ESCRT) pathway mediates cellular membrane remodeling and fission reactions. The pathway comprises five core complexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and Vps4. These soluble complexes are typically recruited to target membranes by site-specific adaptors that bind one or both of the early-acting ESCRT factors: ALIX and ESCRT-I/ESCRT-II. These factors, in turn, nucleate assembly of ESCRT-III subunits into membrane-bound filaments that recruit the AAA ATPase Vps4. Together, ESCRT-III filaments and Vps4 remodel and sever membranes. Here, we review recent advances in our understanding of the structures, activities, and mechanisms of the ESCRT-III and Vps4 machinery, including the first high-resolution structures of ESCRT-III filaments, the assembled Vps4 enzyme in complex with an ESCRT-III substrate, the discovery that ESCRT-III/Vps4 complexes can promote both inside-out and outside-in membrane fission reactions, and emerging mechanistic models for ESCRT-mediated membrane fission.

Visualizing intermediate stages of viral membrane fusion by cryo-electron tomography.

Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion.

Conserved structures of ESCRT-III superfamily members across domains of life.

Structural and evolutionary studies of cyanobacterial phage shock protein A (PspA) and inner membrane-associated protein of 30 kDa (IM30) have revealed that these proteins belong to the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, which is conserved across all three domains of life. PspA and IM30 share secondary and tertiary structures with eukaryotic ESCRT-III proteins, whilst also oligomerizing via conserved interactions. Here, we examine the structures of bacterial ESCRT-III-like proteins and compare the monomeric and oligomerized forms with their eukaryotic counterparts. We discuss conserved interactions used for self-assembly and highlight key hinge regions that mediate oligomer ultrastructure versatility. Finally, we address the differences in nomenclature assigned to equivalent structural motifs in both the bacterial and eukaryotic fields and suggest a common nomenclature applicable across the ESCRT-III superfamily.

A single particle analysis method for detecting membrane remodelling and curvature sensing.

In biology, shape and function are related. Therefore, it is important to understand how membrane shape is generated, stabilised and sensed by proteins and how this relates to organelle function. Here we present an assay that can detect curvature preference and membrane remodelling using free-floating liposomes using protein concentrations in a physiologically relevant ranges. The assay reproduced known curvature preferences of BAR domains and allowed the discovery of high curvature preference for the PH domain of AKT and the FYVE domain of HRS. In addition, our method reproduced the membrane vesiculation activity of the ENTH domain of Epsin1 and showed similar activity for the ANTH domains of PiCALM and Hip1R. Finally, we found that the curvature sensitivity of the N-BAR domain of Endophilin inversely correlates to membrane charge and that deletion of its N-terminal amphipathic helix increased its curvature specificity. Thus, our method is a generally applicable qualitative method for assessing membrane curvature sensing and remodelling by proteins.

Tetraspanin proteins in membrane remodeling processes.

Membrane remodeling is a fundamental cellular process that is crucial for physiological functions such as signaling, membrane fusion and cell migration. Tetraspanins (TSPANs) are transmembrane proteins of central importance to membrane remodeling events. During these events, TSPANs are known to interact with themselves and other proteins and lipids; however, their mechanism of action in controlling membrane dynamics is not fully understood. Since these proteins span the membrane, membrane properties such as rigidity, curvature and tension can influence their behavior. In this Review, we summarize recent studies that explore the roles of TSPANs in membrane remodeling processes and highlight the unique structural features of TSPANs that mediate their interactions and localization. Further, we emphasize the influence of membrane curvature on TSPAN distribution and membrane domain formation and describe how these behaviors affect cellular functions. This Review provides a comprehensive perspective on the multifaceted function of TSPANs in membrane remodeling processes and can help readers to understand the intricate molecular mechanisms that govern cellular membrane dynamics.

Hsp90 Mediates Membrane Deformation and Exosome Release.

Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now find that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and membrane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deforming function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release.

Molecular mechanism of GTP binding- and dimerization-induced enhancement of Sar1-mediated membrane remodeling.

The Sar1 GTPase initiates coat protein II (COPII)-mediated protein transport by generating membrane curvature at subdomains on the endoplasmic reticulum, where it is activated by the guanine nucleotide exchange factor (GEF) Sec12. Crystal structures of GDP- and GTP-bound forms of Sar1 suggest that it undergoes a conformational switch in which GTP binding enhances the exposure of an amino-terminal amphipathic helix necessary for efficient membrane penetration. However, key residues in the amino terminus were not resolved in crystal structures, and experimental studies have suggested that the amino terminus of Sar1 is solvent-exposed in the absence of a membrane, even in the GDP-bound state. Therefore, the molecular mechanism by which GTP binding activates the membrane-remodeling activity of Sar1 remains unclear. Using atomistic molecular dynamics simulations, we compare the membrane-binding and curvature generation activities of Sar1 in its GDP- and GTP-bound states. We show that in the GTP-bound state, Sar1 inserts into the membrane with its complete (residues 1 to 23) amphipathic amino-terminal helix, while Sar1-GDP binds to the membrane only through its first 12 residues. Such differential membrane-binding modes translate into significant differences in the protein volume inserted into the membrane. As a result, Sar1-GTP generates positive membrane curvature 10 to 20 times higher than Sar1-GDP. Dimerization of the GTP-bound form of Sar1 further amplifies curvature generation. Taken together, our results present a detailed molecular mechanism for how the nucleotide-bound state of Sar1 regulates its membrane-binding and remodeling activities in a concentration-dependent manner, paving the way toward a better understanding COPII-mediated membrane transport.

Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE: Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.

BIN1 modulation in vivo rescues dynamin-related myopathy.

The mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2RW/+ and Dnm2RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2RW/+ mice and rescued the perinatal lethality and survival of Dnm2RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations.

In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair.

The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.

Structure and dynamics of ESCRT-III membrane remodeling proteins by high-speed atomic force microscopy.

Endosomal sorting complex required for transport (ESCRT) proteins assemble on the cytoplasmic leaflet of membranes and remodel them. ESCRT is involved in biological processes where membranes are bent away from the cytosol, constricted, and finally severed, such as in multivesicular body formation (in the endosomal pathway for protein sorting) or abscission during cell division. The ESCRT system is hijacked by enveloped viruses to allow buds of nascent virions to be constricted, severed, and released. ESCRT-III proteins, the most downstream components of the ESCRT system, are monomeric and cytosolic in their autoinhibited conformation. They share a common architecture, a four-helix bundle with a fifth helix that interacts with this bundle to prevent polymerizing. Upon binding to negatively charged membranes, the ESCRT-III components adopt an activated state that allows them to polymerize into filaments and spirals and to interact with the AAA-ATPase Vps4 for polymer remodeling. ESCRT-III has been studied with electron microscopy and fluorescence microscopy; these methods provided invaluable information about ESCRT assembly structures or their dynamics, respectively, but neither approach provides detailed insights into both aspects simultaneously. High-speed atomic force microscopy (HS-AFM) has overcome this shortcoming, providing movies at high spatiotemporal resolution of biomolecular processes, significantly increasing our understanding of ESCRT-III structure and dynamics. Here, we review the contributions of HS-AFM in the analysis of ESCRT-III, focusing on recent developments of nonplanar and deformable HS-AFM supports. We divide the HS-AFM observations into four sequential steps in the ESCRT-III lifecycle: (1) polymerization, (2) morphology, (3) dynamics, and (4) depolymerization.

Lipid Monolayer on Cell Surface Protein Templates Functional Extracellular Lipid Assembly.

When the ancestors of men moved from aquatic habitats to the drylands, their evolutionary strategy to restrict water loss is to seal the skin surface with lipids. It is unknown how these rigid ceramide-dominated lipids with densely packed chains squeeze through narrow extracellular spaces and how they assemble into their complex multilamellar architecture. Here it is shown that the human corneocyte lipid envelope, a monolayer of ultralong covalently bound lipids on the cell surface protein, templates the functional barrier assembly by partly fluidizing and rearranging the free extracellular lipids in its vicinity during the sculpting of a functional skin lipid barrier. The lipid envelope also maintains the fluidity of the extracellular lipids during mechanical stress. This local lipid fluidization does not compromise the permeability barrier. The results provide new testable hypotheses about epidermal homeostasis and the pathophysiology underlying diseases with impaired lipid binding to corneocytes, such as congenital ichthyosis. In a broader sense, this lipoprotein-mediated fluidization of rigid (sphingo)lipid patches may also be relevant to lipid rafts and cellular signaling events and inspire new functional materials.

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin-mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.

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.