Starbucks will launch its first-ever 3D-printed location in Brownsville The corporation has been teasing the opening of the store which is drive-thru-only and was created in partnership with PERI 3D Construction the official Starbucks Instagram account shared video of the store being built showing construction crews working alongside a robotic arm as it pours on layers of concrete to build the structure which includes the trademark Starbucks logo looks like your typical location for the popular coffee giant texas: our first 3d printed store in the u.s.,” the caption of the IG post reads "Will it melt in the summer?" one person asked in the comments I approve this," a second person said "Can’t wait to see this," a third person added "How fast did you get it up and going That’s cool!" a fourth person wrote "Why are we 3D printing buildings?" another wondered The iconic Starbucks logo is displayed on a high-rise building in Hell's Kitchen Getty Images In an interview with TODAY, Adeola Olubamiji, Ph.D., the CEO of Pathfinder Consulting and an expert in printing technologies, provided a quick breakdown of the science behind the structure. “You’re building from scratch, from nothing, layer by layer,” Olubamiji said. “You feed a material in, like powder, and then it makes it into a semi-solid.” “This technology combines the semi-solid with a polymer, so that each layer adheres to the next layer due to the polymer that connects them together, much like an adhesive," she added. Olubamiji also feels that 3D-printing could become more prominent based off Starbucks' willingness to incorporate it into their building.  Starbucks' first-ever 3D-printed store will open at 2491 Boca Chica Boulevard in Brownsville, Texas this Friday, May 2. By Andrew Holleran is a trending news writer on Men's Journal He's covered sports and pop culture for more than a decade The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V., its licensors, and contributors. All rights are reserved, including those for text and data mining, AI training, and similar technologies. For all open access content, the relevant licensing terms apply. The building is embedded between the axes of the adjacent volumes. Two almost equally sized cuboids with simple and clear form and contrast in materiality and appearance are shifted against each other.  © Hiepler BrunierThe simple material board for architecture and interior is extremely reduced in favor of clarity and calm: concrete The first three materials are conntected to the product world of PERI the screed instead reminds of an industrial hall flooring and the cladding and parquet on the first floor contrasts the cool concrete surfaces and creates a warm and comfortable atmosphere This is enhanced by the warm architectural and decorative lighting (PSLAB) Only a few colorful eyecatchers disturb the puristic harmony You'll now receive updates based on what you follow Personalize your stream and start following your favorite authors If you have done all of this and still can't find the email Metrics details An Author Correction to this article was published on 24 June 2020 This article has been updated Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical “corkscrew-like” membrane tubes Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis highlighting the mechanical stresses imposed by ESCRT-III Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes Here we investigate how ESCRT-III polymerization shapes membranes and how it influences their assembly on membranes we develop in vitro assays based on the essential core of purified human ESCRT-III proteins (CHMP4B We use C-terminally truncated versions of CHMP4B and CHMP2B to facilitate polymerization as well as full-length CHMP3 We design confocal microscopy experiments with membrane nanotubes of controlled geometries pulled from Giant Unilamellar Vesicles (GUVs) to study the effect of membrane mean curvature and topology on ESCRT-III protein recruitment and polymerization at the macroscopic scale by using high-speed AFM (HS-AFM) and cryo-electron microscopy (cryoEM) we obtain nanometer resolution images showing the preferential membrane shape induced upon ESCRT-III assembly on small liposomes and preformed tubes and the corresponding organization of the protein filaments at their surface a CHMP4B-ΔC spirals observed by HS-AFM on a lipid bilayer b Cryo-EM image of CHMP4B-ΔC spiral on deformable LUVs c Top view (top) and side view (bottom) of a cryo-EM tomogram (Supplementary Movie 2) showing CHMP4B-ΔC spirals (red: CHMP4 filaments polymerized on lipids; blue: filaments polymerized in bulk; yellow: lipids) d The different geometries used to study ESCRT-III proteins/membrane interactions The protein location is indicated by a green shadow (ii) Proteins outside a nanotube pulled from a GUV: on the tube (iii) Proteins inside a nanotube pulled from a GUV: on the tube (iv) Spontaneously formed tubule inside a GUV in geometry (iii): on the internal tube e Confocal images corresponding to a GUV fusion experiment in which CHMP4B-ΔC is binding in geometry (iii) f Sorting ratio for 17 nanotubes from 17 GUVs in 8 independent GUV preparations and variable diameters (e) N measurements were made: <20 nm: N = 19; 20–40 nm: N = 28; 40–60 nm: N = 11; 60–80 nm: N = 6; >80 nm: N = 8 g Confocal images corresponding to a GUV fusion experiment where CHMP4B-ΔC binds in geometry (ii) h Sorting ratio for 24 nanotubes from 24 GUVs in 10 independent GUV preparations and of variable diameters (g) N measurements were performed: <20 nm: N = 11; 20–40 nm: N = 8; 40–60 nm: N = 5; 60–80 nm: N = 4; >80 nm: N = 11 i Cryo-EM image of CHMP4B polymerized outside deformable membrane nanotubes j Cryo-EM image of CHMP4B-ΔC filaments polymerized onto non-deformable GlaCer tubes h Source data are provided as a Source Data file these results do not support previous models of a stiff CHMP4B spiral acting as a loaded spring that could induce membrane bending in the absence of the other ESCRT-III proteins flattens membranes or assembles along the main axis of tubes where the mean curvature is null a HS-AFM image of CHMP2B-ΔC rings on a flat The quantification of ring diameters is shown b Confocal images corresponding to a GUV fusion experiment in which CHMP2B-ΔC is exposed to a geometry (iv) induced by the I-BAR domain of IRSp53 (non-fluorescent) tubulating the membrane when present on the exterior of the GUV c Confocal images corresponding to a GUV fusion experiment in which CHMP2A-ΔC + CHMP3 are binding in geometry (iii) d Confocal images corresponding to a GUV fusion experiment in which CHMP2A-ΔC + CHMP3 are binding in geometry (iv) showing the affinity of the assembly for internal positively curved tubes e Left: Confocal images corresponding to a GUV fusion experiment in which CHMP2A-ΔC + CHMP3 are binding in geometry (ii) Right: Quantification of the sorting ratio for 24 nanotubes of variable diameters from 25 GUVs in 9 independent GUV preparations N measurements have been performed: <20 nm: N = 20; 20–40 nm: N = 74; 40–60 nm: N = 41; 60–80 nm: N = 6; >80 nm: N = 4 The red dashed line corresponds to a sorting ratio equal to 1 f Cryo-EM image of CHMP2A-ΔC/CHMP3 filaments polymerized outside deformable membrane nanotubes g Cryo-EM image of CHMP2A-ΔC/CHMP3 filaments polymerized outside non-deformable GlaCer tubes e Source data are provided as a Source Data file However, in the presence of spontaneously formed internal tubules in the GUVs with a positive mean curvature (geometry (iv) Fig. 2d) we noticed a strong enrichment of the proteins on these structures we observed with cryo-EM that these proteins generate positive membrane curvature since the fraction of tubular structures is increased as compared to the control (31 ± 5%) when CHMP2A (0.5 µM) and CHMP3 (3 µM) are added since the sorting ratio increases with tube curvature (the inverse of the radius) up to about 5 for tube diameters smaller than 20 nm it demonstrates that the CHMP2A/CHMP3 complex can polymerize on membrane in a positive curvature-dependent matter In vivo, ESCRT-III complexes function on membranes with a negative Gaussian curvature. We therefore co-encapsulated CHMP4B and CHMP2B (both fluorescent) as well as CHMP4B and CHMP2A/CHMP3 (with fluorescent CHMP4B and CHMP2A) at low micromolar concentrations in EPC GUVs and fused them with GUVs containing PI(4,5)P2 from which a tube was pulled (Fig. 1d Bottom: Fourier-Transform (FT) with the distances corresponding to the Bragg peaks Right: The red line represents the direction of the tube axis and the blue line to the perpendicular direction along the tube section m Source data are provided as a Source Data file we have found that in the absence of other ESCRT partners these minimal complexes can be recruited to the neck of membrane tube structures exhibiting a negative Gaussian curvature they have some affinity for membranes with a positive mean curvature showing that CHMP4B with CHMP2B can mechanically deform SUVs CHMP4B has to assemble first on liposomes to nucleate the helical membrane tube deformation by either CHMP2B or CHMP2A/CHMP3 Populations of ESCRT filaments bound to tubular membranes resulting from sub-tomogram averaging Upper line: orthoslices viewed from the cross sections of tubes Second line: orthoslices viewed from the top of tubes Third line: 3D reconstructions viewed from the cross sections of tubes Fourth line: reconstructions viewed from the top of tubes Bottom line: schematic representations of the CHMP4B-ΔC/CHMP2B-ΔC-decorated pipes a Single ESCRT individual filaments bound to lipid tubes b Paired ESCRT filaments bound to lipid tubes c High density of filaments bound to lipid tubes Arrows point to structures perpendicular to the tube axis our analyses demonstrate that the observed macroscopic tubulation into a corkscrew-like architecture is driven by distinct nanometer ultra-structures of ESCRT filaments it establishes that not only CHMP1B interacts with positive curved membranes the latter have been implicated in vivo in membrane remodeling with an opposite membrane geometry Common reagents were purchased from VWR reagents 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS L-α-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (PE-Biotin 870282P) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE Gold nanorods Streptavidin-conjugated gold nanorods (C12-10-850-TS-DIH-50) were purchased from Nanopartz™ Streptavidin-coated polystyrene beads (diameter 3.2 μm) for the tube pulling experiments were purchased from Spherotech CHMP3 (full length) was expressed in Escherichia coli BL21 cells (New England BioLabs, # C2530H) for 3 h at 37 °C11 cells were harvested by centrifugation (4000g for 20 min at 4 °C) and the bacterial pellet was resuspended in 50 ml of binding buffer A (20 mM Bicine pH 9.3 The bacteria were lysed by sonication and CHMP3-FL was purified by Ni2+ chromatography A final gel filtration chromatography step was performed in buffer B (20 mM Hepes pH 7.6 CHMP2A-ΔC containing residues 9–161 was expressed as MBP-fusion protein in Escherichia coli BL21 cells61 for 1 h at 37 °C Cells were harvested by centrifugation (4000g for 20 min at 4 °C) and the bacterial pellet was resuspended in 50 ml of binding buffer C (20 mM Hepes pH 7.6 and CHMP2A-ΔC was purified on an amylose column CHMP2A-ΔC was labeled overnight at 4 °C with Alexa Fluor 405 NHS Ester (Thermo Fisher Scientific) using a molar ratio (Alexa Fluor:protein) of 2:1 A final gel filtration chromatography step was performed in a buffer B CHMP3-FL and CHMP2A-ΔC were concentrated to 20 μM and immediately frozen in liquid nitrogen with 0.1% of methyl cellulose (Sigma-Aldrich) as cryo-protectant All aliquots were kept at −80 °C prior to experiments was expressed in Escherichia coli BL21 cells for 4 h at 37 °C Cells were lysed by sonication in buffer D (50 mM Tris-HCl pH 7.4 10 mM DTT and protease inhibitor (Complete EDTA free Roche) at the concentration indicated by the manufacturer) and the soluble fraction was discarded after centrifugation (50,000g The pellet was washed three times with buffer E (50 mM Tris-HCl pH 7.4 2% Triton X-100 and 2 mM β-mercaptoethanol) The last wash was performed in absence of urea and Triton X-100 The extraction of CHMP2B was performed in 50 mM Tris-HCl pH 7.4 CHMP2B was purified by Ni2+-chromatography in buffer F (50 mM Tris-HCl pH 7.4 The protein was eluted in 50 mM Tris-HCl pH 7.4 Refolding was performed by rapid dilution of CHMP2B into buffer G (50 mM Tris-HCl pH 7.4 50 mM l-arginine) and a final concentration of 2 μM CHMP2B was concentrated by passing it over a Ni2+ column in buffer H (50 mM Tris-HCl pH 7.4 200 mM NaCl) and eluted in buffer I (50 mM Tris-HCl pH 7.4 CHMP2B was labeled overnight at 4 °C with Alexa Fluor 488 C5 Maleimide (Thermo Scientific) with a molar ratio (Alexa Fluor:protein) of 2:1 A final gel filtration chromatography step was performed on a superdex75 column in buffer J (50 mM Tris-HCl pH 7.4 and immediately frozen in liquid nitrogen with 0.1% of methyl cellulose (Sigma-Aldrich) as a cryo-protectant Cells were harvested by centrifugation (4000g for 20 min at 4 °C) and the bacterial pellet was resuspended in 50 ml of binding buffer K (50 mM Hepes pH 7.6 The CHMP4B protein was purified on an amylose column CHMP4B was labeled overnight at 4 °C with Alexa 555 succimidyl ester or 633 succimidyl ester (Thermo Fisher Scientific) using a molar ratio (Alexa Fluor:protein) of 2:1 A final gel filtration chromatography step was performed in the buffer KJ CHMP4B were concentrated to 15 μM and immediately frozen in liquid nitrogen with 0.1% of methyl cellulose (Sigma Aldrich) as cryo-protectant A 300 kV FEG (Field Emission Gun) POLARA microscope (FEI Netherlands) equipped with an energy filter and a direct detector (K2 camera the imaging was performed at a magnification of 81,000 with a pixel size of 1.21 Å using a movie mode collecting 40 successive frames for a total dose of 50 electrons per Å2 The different frames were subsequently aligned Back projection was performed using IMOD and SIRT reconstruction was carried out using Tomo3d The segmentation was performed manually using IMOD Tube radius was determined upon aligning and averaging particles cropped from the tube axis as the center of sub-volumes of 88 pixels (46.6 nm) using as alignment mask a cylinder of 22 nm radius the center of the box was displaced to the tube’s membrane surface and the selected oversampling geometry was 16 cropping points per radius separated by 6 pixels along the tube axis Reference free sub-tomogram averaging was performed on sub-volumes of 343 nm in Dynamo Lipid stock solutions were mixed at a total concentration of 1 mg/ml in chloroform with following molar ratio: 54.7% EPC; 10% DOPS; 10% DOPE; 15% cholesterol; 10% PI(4,5)P2; 0.2% DSPE-PEG2000-Biotin; 0.1% PE–Rhodamine for the charged GUVs and 98.8% EPC 0.2% DSPE-PEG2000-Biotin for the non-charged GUVs (containing encapsulated proteins) All proteins have been incubated with GUVs at a concentration of 500 nM in a buffer containing 100 mM glucose 25 mM Tris pH 7.4 and 50 mM NaCl for 30′ together with 10 nM TEV which was sufficient to cleave at least 90% of the MBP tags in 15′ at room temperature (not shown) CHMP4B stock solution being at 300 mM NaCl + 300 mM KCl the encapsulation mixture has a salt concentration of ~100 mM after fusion with a PI(4,5)P2 vesicle of equal size After addition of 80 µl of CHMP2A and 50 µl of CHMP3 the encapsulation mixture has a NaCl concentration of ~90 mM After fusion with a PI(4,5)P2 vesicle of equal size the NaCl concentration drops to about 45 mM the encapsulation mixture has a NaCl concentration of about 100 mM the encapsulation mixture has a NaCl concentration of ~100 mM After fusion with a PI(4,5)P2-containing vesicle of equal size The final protein concentrations in the GUVs after fusion are listed in Supplementary Table 6 two types of GUVs extracted from each PVA slide were mixed with the relative external buffer matching the osmolarity and centrifuged for 10 min at 1000g GUVs taken from the bottom of the Eppendorf were incubated with gold nanorods 20 min at room temperature and then added to the imaging chamber Gold nanorods Streptavidin-conjugated gold nanorods have a peak of absorption at λ = 834 nm with a tail spanning the wavelength of the infrared laser of the optical tweezers (λ = 1064 nm) The stock solution (typical concentration 1750 ppm) was diluted 1:100 upon incubation with GUVs and again diluted 1:40 when GUVs were transferred to the observation chamber Fusion of GUV pairs coated with the gold nanorods is achieved by bringing the GUVs hold by two micropipettes into close contact with micromanipulation and by locally heating the nanorods by focusing the infrared laser on the contact through the objective for experiments probing the affinity of the proteins for positive curvature the tube was formed by bringing briefly the GUV coated with proteins in contact with a streptavidin-coated bead trapped with the optical tweezer and moved away For experiments involving encapsulation and fusion the tube was pulled from the PI(4,5)P2-containing GUV prior to fusion using a streptavidin-coated bead hold by a third micromanipulator Fusion was then performed between the GUV pair The values of tube diameter (in nm) were deduced from the lipid fluorescence intensities the tube in comparison with the fluorescence in the GUV where \(I_{{\mathrm{tube}}}^{{\mathrm{lipid}}}\) and \(I_{{\mathrm{GUV}}}^{{\mathrm{lipid}}}\)represent the fluorescence intensities of the lipids in the tube and in the GUV The sorting ratio S (protein enrichment in the tube) was calculated using where \(I_{{\mathrm{tube}}}^{{\mathrm{protein}}}\) and \(I_{{\mathrm{GUV}}}^{{\mathrm{protein}}}\)represent the fluorescence intensities of the proteins in the tube and in the GUV All HS-AFM data were taken in amplitude modulation mode using a sample scanning HS-AFM [Research Institute of Biomolecule Metrology (RIBM) Switzerland) with spring constant of 0.15 N/m and a quality factor of ∼2 in buffer were used The cantilever-free amplitude is 1 nm (3 nm for imaging liposomes) and the set-point amplitude for the cantilever oscillation was set around 0.8 nm (2.7 nm for liposomes) all the HS-AFM recordings were performed in buffer containing 25 mM Tris pH 7.4 and 50 mM NaCl LUVs were thawed at room temperature and diluted to a concentration of 0.2 mg/ml in buffer (25 mM Tris Then the LUVs were incubated onto the freshly cleaved mica for 5–10 min and rinsed with the same buffer afterwards the surface was imaged without addition of protein the proteins were added to the AFM liquid chamber to reach a final concentration of 2 µM for CHMP4B The formation of CHMP4B spirals on SLBs occurred within 10 minutes after incubation To capture the effect of CHMP2B on CHMP4B spiral CHMP2B was only added after the formation of CHMP4B spirals was confirmed by HS-AFM imaging The HS-AFM experiments for dynamic membrane deformation were performed using liposomes (SUVs) composed of 50.7% EPC; 10% DOPS; 10% DOPE; 15% cholesterol; 10% PI(4,5)P2; 0.2% DSPE-PEG2000-Biotin; 0.1% PE–Rhodamine The SUVs were obtained by sonicating a LUV mixture for 30 s The SUVs were incubated for 5 min on freshly cleaved mica CHMP4B was added to reach a final concentration of 2 µM in the chamber CHMP2B (at a final concentration of 1 µM) was added but only after confirmed spiral formation (typically after 10 min of CHMP4B addition) on randomly formed membrane patches on mica surface All the HS-AFM images were processed with Igor Pro with a built-in script from RIBM (Japan) all reported values are presented as mean ± SD Further information on research design is available in the Nature Research Reporting Summary linked to this article One example tomogram as well as our sub-tomogram averages have been deposited in the EMBD An amendment to this paper has been published and can be accessed via a link at the top of the paper Molecular mechanisms of the membrane sculpting ESCRT pathway Growing functions of the ESCRT machinery in cell biology and viral replication The ESCRT-machinery: closing holes and expanding roles and dynamics of ESCRT-III/Vps4 membrane remodeling and fission complexes How to get out: ssRNA enveloped viruses and membrane fission Reverse-topology membrane scission by the ESCRT proteins ESCRT-III is required for scissioning new peroxisomes from the endoplasmic reticulum Ordered assembly of the ESCRT-III complex on endosomes is required to sequester cargo during MVB formation Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding Structural basis for budding by the ESCRT-III factor CHMP3 Structural basis for ESCRT-III protein autoinhibition Structural basis of Ist1 function and Ist1-Did2 interaction in the multivesicular body pathway and cytokinesis Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-Terminal domain Structural basis for autoinhibition of ESCRT-III CHMP3 A crescent-shaped ALIX dimer targets ESCRT-III CHMP4 filaments Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly Relaxation of loaded ESCRT-III spiral springs drives membrane deformation Helical structures of ESCRT-III are disassembled by VPS4 The Endosomal Sorting Complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding Structure and disassembly of filaments formed by the ESCRT-III subunit Vps24 Plasma membrane deformation by circular arrays of ESCRT-III protein filaments Structure of cellular ESCRT-III spirals and their relationship to HIV budding Charged multivesicular body protein 2B (CHMP2B) of the endosomal sorting complex required for transport-III (ESCRT-III) polymerizes into helical structures deforming the plasma membrane Structure and membrane remodeling activity of ESCRT-III helical polymers assembly and membrane binding of ESCRT-III Snf7 filaments The ESCRT protein CHMP2B acts as a diffusion barrier on reconstituted membrane necks ATP-dependent force generation and membrane scission by ESCRT-III and Vps4 Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly Association of ESCRT-II with VPS20 generates a curvature sensitive protein complex capable of nucleating filaments of ESCRT-III The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis VPS4 triggers constriction and cleavage of ESCRT-III helical filaments ESCRT-III Protein Requirements for HIV-1 Budding Pfitzner, A.-K., Mercier, V. & Roux, A. Vps4 triggers sequential subunit exchange in ESCRT-III polymers that drives membrane constriction and fission. Preprint at https://www.biorxiv.org/content/10.1101/718080v1 (2019) Divergent pathways lead to ESCRT-III catalyzed membrane fission Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission Resolving ESCRT-III spirals at the intercellular bridge of dividing cells using 3D STORM Membrane buckling Induced by curved filaments Vesicle fusion triggered by optically heated gold nanoparticles Nature of curvature-coupling of amphiphysin with membranes depends on its bound density Septin-based readout of PI(4,5)P2 incorporation into membranes of giant unilamellar vesicles Helical crystallization on nickel–lipid nanotubes: Perfringolysin O as a model protein Molecular mechanisms of membrane deformation by I-BAR domain proteins Ezrin enrichment on curved cell membranes requires phosphorylation or interaction with a curvature-sensitive partner Alqabandi, M. et al. The ESCRT-III isoforms CHMP2A And CHMP2B display different effects on membranes upon polymerization. Preprint at https://www.biorxiv.org/content/10.1101/756403v1 (2019) von Filseck, J. M. et al. Anisotropic ESCRT-III architecture governs helical membrane tube formation. Nat. Commun. 11, https://doi.org/10.1038/s41467-020-15327-4 (2020) An ESCRT–spastin interaction promotes fission of recycling tubules from the endosome Membrane constriction and thinning by sequential ESCRT-III polymerization ESCRT-II coordinates the assembly of ESCRT-III filaments for cargo sorting and multivesicular body vesicle formation ALIX-CHMP4 interactions in the human ESCRT pathway Electrostatic lateral interactions drive ESCRT-III heteropolymer assembly Changes in ESCRT-III filament geometry drive membrane remodelling and fission in silico Computer visualization of three-dimensional image data using IMOD Automated electron microscope tomography using robust prediction of specimen movements Automated tilt series alignment and tomographic reconstruction in IMOD user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments Dynamo catalogue: geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms Gel-assisted formation of Giant Unilamellar Vesicles Dynamic and sequential protein reconstitution on negatively curved membranes by giant vesicles fusion Download references The authors thank Daniel Levy for support and insightful discussions Michael Henderson for carefully reading the manuscript We thank Eric Nicolau for his drawing skills This work was initiated with a grant from FINOVI (W B.) and was supported by the ANR (ANR-14-CE09-0003-01) (W.W. by the Institut Curie and the Centre National de la Recherche Scientifique (CNRS) acknowledges the Institute Universitaire de France (IUF) and the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB) Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) For cryo-electron microscopy we acknowledge the support of G Schoehn at the Grenoble FRISBI/Instruct-ERIC electron microscopy platform Hagen of the cryo-electron microscopy platform of the European Molecular Biology Laboratory (EMBL Nilges from the UBI facility (Institut Pasteur The Falcon II detector at the UBI facility was financed by the “Equipement d’excellence CACSICE” and the Grenoble Instruct-ERIC EM platform acknowledges support from the FRM and GIS IBiSA Access to the EMBL cryo-electron microscopy facility was supported by iNEXT (project number 653706) funded by the Horizon 2020 program of the European Union We further acknowledge the Cell and Tissue Imaging (PICT IBiSA Institut Curie) platform supported by France-BioImaging (ANR10-INBS-04) N.D.F was funded by post-doctoral fellowships from the Institut Curie the Fondation pour la Recherche Médicale and Marie Curie actions (MSCA-IF-2016 #751715 (ESCRT model)) E.M.L was supported by a post-doctoral fellowship from ANR (ANR-15-CE11-0027-02) was funded by the Université Pierre et Marie Curie/Sorbonne Université Doctoral school “Physique en Ile de France” (ED-564) and the Fondation pour la Recherche Médicale is a member of the CNRS consortium CellTiss are members of the Labex CelTisPhyBio (ANR-11-LABX0038) and Paris Sciences et Lettres (ANR-10-IDEX-0001-02) These authors contributed equally: Aurélie Bertin Stéphanie Mangenot & Patricia Bassereau performed tube pulling experiments and analyzed data supervised HS-AFM work and S.Mai performed HS-AFM experiments and analyzed data optimized the characteristics of the membrane samples and S.Man equally contributed to this work The authors declare no competing interests Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-020-16368-5 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article npj Biological Physics and Mechanics (2024) Nature Structural & Molecular Biology (2023) Sign up for the Nature Briefing newsletter — what matters in science Verena passed away peacefully with her family at her side on the 10th of January 2020 at the age of 88 She is survived by her loving husband of 67 years Kurt and her three children Ingrid (Tyler) predeceased by her daughter in law Robyn and son in law Gord A special thank you to the staff at Sun Pointe Village for the loving care that was shown to our mom over the past few years There will be a private family service held at a later date donations may be made to the charity of one’s choice please scroll down the page to the area called “Condolences.” » Condolences sent through this page can be seen by the public. 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Metrics details Matrix proteins from enveloped viruses play an important role in budding and stabilizing virus particles In order to assess the role of the matrix protein M1 from influenza C virus (M1-C) in plasma membrane deformation we have combined structural and in vitro reconstitution experiments with model membranes We present the crystal structure of the N-terminal domain of M1-C and show by Small Angle X-Ray Scattering analysis that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers Using negatively charged giant unilamellar vesicles (GUVs) we demonstrate that M1-C full-length binds to and induces inward budding of membrane tubules with diameters that resemble the diameter of viruses Membrane tubule formation requires the C-terminal domain of M1-C corroborating its essential role for M1-C polymerization Our results indicate that M1-C assembly on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress we combine structural studies and GUV-based experiments to understand the M1-C induced membrane deformation We show that the N-terminal domain of M1-C adopts a similar fold as M1-A despite the low sequence homology of both proteins but coordinates two Mg2+ ions which increases the positive surface charge and may thus facilitate electrostatic interactions with negatively charged membranes Full length M1-C adopts an elongated structure that tends to polymerize into ring-like or filamentous structures via lateral interactions of M1-C protomers M1-C interaction with GUVs containing negatively charged lipids leads to inward membrane tubulation Although the N-terminal domain on its own interacts with GUVs Our study thus confirms that M1-C assembly on membranes constitutes the driving force for influenza C virus bud formation (a) M1-C forms polymers in vitro at low pH conditions as shown by negative staining electron microscopy The width of the circular or spiral filaments is approximately 10 nm The inset shows a close-up of some rod-like structures that associate laterally to form the filament (b) M1-C also forms monomers at low pH conditions that produced the experimental SAXS data (red); the scattering pattern computed from the Dammin model shown in d is drawn in green (c) Kratky plots for M1-C (red) and M1-A (blue) (d) Structural model of M1-C produced at low pH and reconstructed ab initio (a) Ribbon diagram of M1-C composed of two four-helical bundles connected by helix 5. Alpha helices are labeled. (b) Close up of the Mg2+ binding sites. M1-C binds two Mg2+ ions coordinated by residues from helices 5 and 8 and six water molecules. Alpha helices are labeled. (c) Superposing of the Cα atoms of M1-A and M1-C reveals an overall similar fold and the displacement of helix 6. Electrostatic potential map of M1-C (a) compared to the M1-A map (b) The electrostatic potential was calculated from −3.000 KbT/ec (red) to +3.000 KbT/ec (blue) The interaction between M1-C and lipid membranes was investigated using GUVs of two different lipid compositions The first composition contained 33 mol% of the negatively charged lipid DOPS in addition to DOPE (33 mol%) and DOPC (33 mol%) (herein referred to as DOPS-GUV) The second composition consisted of lipids extracted from a natural tissue (porcine brain extract) which contains approximately 10 wt% PS lipids to which 5 mol% PI(4,5)P2 was added (herein referred to as TBE-GUVs) we worked with two extreme cases: a very simple composition a “minimal model of the cell membrane” consisting of a protein-binding lipid and background non-interacting lipids and a more complex possibly reflecting the properties of native membranes more closely (a) Representative confocal images at the vesicles equator of (i) TBE-GUVs (ii) DOPS-GUVs and (ii) DOPC-GUVs after incubation with M1 The red signal corresponds to bodipy-ceramide lipids incorporated into the GUV membrane and the green signal to Alexa-488 M1-C Protein-induced membrane tubules are visible for the negatively charged vesicles of both compositions No binding is observed in absence of negatively charged lipids The protein concentration was 1.9 μM for the TBE-GUV experiment and 2.8 μM for the DOPS-GUVs and the DOPC-GUVs The fraction of vesicles with tubulation in independent experiments for (b) TBE-GUVs and (c) DOPS-GUVs as a function of protein bulk concentration (d) Tubule density at the equator as a function of the average intensity of the vesicle rim for TBE-GUVs (crosses) and DOPS-GUVs (circles) together with their respective linear fits (dotted line: TBE-GUVs; full line: DOPS-GUVs) Each data point corresponds to one analyzed vesicle and only vesicles with at least one tubule at the equator were taken into account (a) Projections of TBE-GUVs with a negatively charged fluorescently-labeled lipid (Bodipy TMR PI(4,5)P2) The red signal corresponds to Bodipy TMR PI(4,5)P2 incorporated to the GUV membrane and the green signal to Alexa-488 M1-C The negatively charged fluorescent lipids co-localize with the protein network (b) (i) Projections of TBE-GUVs containing small amounts of bodipy-ceramide The red signal corresponds to bodipy-ceramide lipids incorporated to the GUV membrane and the green signal to Alexa-488 M1-C (ii) Control experiment using vesicles without fluorescent lipids at maximum laser power: only signal noise is detected showing the absence of bleed-through between the fluorescence channels from local membrane deformations at the vesicle surface the fluorescence increase reflects the surface projection of the curved membrane Further analysis of the ratio of the lipid signal under the M1-C protein clusters and around them reveals that this effect dominates over local recruitment of PIP(4,5)P2 since the ratios are similar in both experiments with fluorescent labeled lipids (ratios: 1.75 ± 0.42 and 1,79 ± 0,32 for fluorescent PIP(4,5)P2 and for the fluorescent ceramide These ratios further indicate that the deformation corresponds to less than half-cylinder with a diameter below optical resolution since in this case the fluorescence would be locally increased by a factor π Confocal images at the equator of TBE-GUVs after incubation with the M1C-NTD although supported bilayers that prevent membrane deformation may not be ideal to study M1 polymerization thus for systems where protein-protein interactions were absent no simulation or experimental data on network assembly has been reported for polymerizing proteins such as M1-C it remains to be elucidated whether the network-like morphology represents a precursor state in the tubulation process or whether an alternative dead-end polymerization state although it is likely that protein clustering and tubulation are related processes as further suggested by the results with M1-NTD where no clustering or tubulation is observed it is important to note that there is no direct correlation between size and amount of clusters and the amount of tubules on the GUVs Although DOPS-GUVs contain 33% DOPE while the TBE-GUVs contain only ~17% of PE-lipids it is important to mention that about 60% of the TBE lipid composition is not known thus it is possible that other lipids than PE with a negative curvature are present in this lipid mixture and contribute to facilitate tubulation Another hypothesis is that PI(4,5)P2 lipids amplify membrane tubulation Although they do not increase M1 binding to the membrane they may induce an arrangement of the proteins more favorable for M1-C polymerization and thus membrane tubulation We therefore propose that the presence of PI(4,5)P2 in the bilayer may increase M1-C filament assembly required for budding A detailed investigation on the influence of different membrane components on the tubulation process will be the subject of future investigations 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate) (PI(4,5)P2) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) were purchased from avanti lipids Fluorescent BODIPY-Texas Red Ceramide (bodipy-ceramide) was from molecular probes and the BODIPY-TMR-PI(4,5)P2 from Tebu-bio All other chemicals were purchased from Sigma-Aldrich The M1 protein of Influenza (strain C/Ann Arbor/1/1950) was cloned into a pET21d vector using the NdeI and XhoI restriction sites Expression was performed in BL21 RIL (DE3) cells for 18 h at 20 °C Cells were lysed by sonication in lysis buffer containing 150 mM NaCl The cell lysate was centrifuged at 20 000 g and 277 K The pellet fraction containing the protein in inclusion bodies (IBs) was washed five times in lysis buffer without lysozyme but with 2.0% (v/v) TritonX-100 and then twice in lysis buffer without detergent The purified IBs were solubilized in 6 M guanidinium hydrochloride (GdnHCl) and then diluted with water to a final concentration of 4 M GdnHCl Refolding of M1-C was performed by the rapid dilution method with 100 mM NaCl 100 mM Tris pH 8.0 and 0.8 M l-arginine as the refolding buffer The refolded protein was further dialyzed in 20 mM MES pH 5.7 10 mM NaCl and loaded onto a heparin column The protein was eluted by applying a salt gradient between 10 mM and 1 M NaCl Fractions containing M1-C were concentrated and further purified on a S75 (100/300) size exclusion chromatography column equilibrated in 10 mM MES pH 5.7 M1-C was adsorbed to the clean side of a carbon film on mica stained with 1% sodium silicotungstate pH 7.0 attached to a 400-mesh copper grid and transferred into a JEOL1200 EX II operating at 100 kV The images were taken on a 2.7 k by 2.7 k Gatan ORIUS CCD camera at a nominal magnification of x20000 Coordinates and structure factors have been deposited in the Protein Data Bank with accession ID 5M1M M1-C containing an extra C-terminal cystein was labeled with Alexa Fluor 488 C5 maleimide (Invitrogen) pH 7,5 at 0.5 mg/ml was incubated for 15 min in fresh TCEP (tris(2-carboxyethyl)phosphine) (40 ug/ml) followed by addition of the dye with a 1:1 protein:label ratio free label was removed by size exclusion chromatography Two different lipid compositions were used to form the GUVs One natural composition (referred herein as TBE-GUVs) consisting of (in molar %): 94% BTE 5% PI(4,5)P2 and 1% BODIPY®Texas Red ceramide The other composition consisted of (in molar %): 33.2% DOPC 33.2% DOPS and 0.5% BODIPY®Texas Red Ceramide (DOPS-GUVs) vesicles consisting of 99.5% DOPC and 0.5% BODIPY®Texas Red Ceramide (DOPC-GUV) (in molar %) was used For the lipid colocalization experiments the concentration of BODIPY-Texas Red Ceramide was lowered to 0.15% (in molar %) or exchanged for the negatively charged lipid BODIPY®TMR PI(4,5)P2 at 0.1% The TBE-GUVs were obtained by electroformation on platinum wires were mounted in an in-house made teflon chamber The lipids were dissolved in chloroform to a concentration of 3 mg/ml premixed and deposited on the wires in small droplets to a total volume of 3 μl The chamber and wires were then dried in vacuum for 30 min before being rehydrated in buffer containing 10 mM TRIS The chamber was sealed with sigillum wax (Vitrex USA) and two glass cover slips (Menzel-Gläser A function generator was then connected to the wires with a sinusoidal wave of 500 Hz and 280 mV RMS-voltage The GUVS were left to grow overnight (12–15 hours) at 4 °C and extracted through gentle pipetting directly above the wires 1 mm apart and a sucrose solution (100 mM) was added to a chamber made with sigillum wax (Vitrex A sinus wave with RMS-voltage of 1 V and frequency of 10 Hz was then applied over the ITO -plates for 30–45 min (DOPS-GUV) or 60–120 min (DOPC-GUV) The vesicles were then extracted by pipetting directly from the chamber The tubulation or protein binding behavior was found to be the same regardless of the electroformation method chosen Protein binding experiments were performed using observation chambers produced in house from coverslips and coated with β-casein by incubation for 15–30 min (5 mg/ml 100 mM NaCl at pH 7.5) followed by rinsing with experiment buffer Protein binding experiments were carried out in TRIS buffer (10 mM Tris 50 mM NaCl at pH 7.5 and adjusted to the same osmolarity as the vesicle growth medium by adding glucose) The vesicles were injected into the observation chamber filled with protein solution at required concentration leading in a 4:1 protein:GUV volume ratio and incubated for at least 30 min before observation The vesicles were imaged using a spinning disk system set up on inverted Nikon eclipse Ti-E at The BioImaging Cell and Tissue Core Facility of the Institut Curie (PICT-IBiSA) member of the France-BioImaging national research infrastructure The images were recorded on a CoolSNAP HQ2 camera Some images were also recorded with an EMCCD iXon 897 Andor camera All data for quantification based on fluorescence intensity were taken on the same microscope with the same camera (CoolSNAP HQ2 camera) The only parameter that was changed between samples was the exposure time This was necessary to allow imaging of both low intensity samples and high intensity samples with the same laser power without saturating the detector the standard deviation z-projection from the top or bottom plane of the vesicle to the vesicle equator plane was used the number of vesicles that displayed tubulation and the number of tubules at the equatorial plane of the vesicle were counted manually The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Membrane curvature and mechanisms of dynamic cell membrane remodelling Clinical features of influenza C virus infection in children Age distribution of the antibody to type C influenza virus doi: 10.1016/j.virusres.2004.08.012 (2004) Spherical influenza viruses have a fitness advantage in embryonated eggs while filament-producing strains are selected in vivo Cryotomography of budding influenza A virus reveals filaments with diverse morphologies that mostly do not bear a genome at their distal end Influenza virus M2 protein mediates ESCRT-independent membrane scission Mechanisms for enveloped virus budding: can some viruses do without an ESCRT Conformational plasticity of the Ebola virus matrix protein Architecture of respiratory syncytial virus revealed by electron cryotomography More than one door - Budding of enveloped viruses through cellular membranes Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein Influenza virus assembly: effect of 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VP40 Selectively Induces Vesiculation from Phosphatidylserine-enriched Membranes Linear aggregation of proteins on the membrane as a prelude to membrane remodeling Membrane tension controls the assembly of curvature-generating proteins Membrane tubule formation by banana-shaped proteins with or without transient network structure How curvature-generating proteins build scaffolds on membrane nanotubes Membrane tension and peripheral protein density mediate membrane shape transitions doi: 10.1016/j.virusres.2009.05.010 (2009) Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension Role of curvature and phase transition in lipid sorting and fission of membrane tubules In Vitro Reconstitution of Membrane Budding by Influenza A Virus Matrix Protein 1 PHENIX: a comprehensive Python-based system for macromolecular structure solution Refinement of Macromolecular Structures by the Maximum-Likelihood Method Overview of the CCP4 suite and current developments a Program Package for Small-Angle Scattering Data Analysis X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing PRIMUS: a Windows PC-based system for small-angle scattering data analysis Dark pixel intensity determination and its applications in normalizing different exposure time and autofluorescence removal doi: 10.1111/j.1365-2818.2011.03581.x (2012) Download references This work was supported by the Marie Curie actions (FP7-PEOPLE-2012-IEF the Swedish Research Council (621-2012-5024) (MB) the Labex GRAL (ANR-10-LABX-49-01) and the Institut Universitaire de France (WW) We acknowledge the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) supported by the French Infrastructure for Integrated Structural Biology Initiative FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB) We also thank the ESRF-EMBL Joint Structural Biology Group for access and support at the ESRF beam lines and J Marquez (EMBL) from the crystallization platform We further thank François Waharte at the microscopy center PICT-IBiSA (Institut Curie member of the France-BioImaging national research infrastructure (ANR-10-INSB-04) for assistance with fluorescence microscopy group belongs to the CNRS consortium CellTiss to the Labex CelTisPhyBio (ANR-11-LABX0038) and to Paris Sciences et Lettres (ANR-10-IDEX-0001-02) Mijo Simunovic and Feng Tsing Tsai (Institut Curie) are acknowledged for general discussions and help with setting up the experiments Nicola De Franceschi is acknowledged for help with data evaluation David Saletti and Jens Radzimanowski: These authors contributed equally to this work Gregory Effantin & Winfried Weissenhorn Protein purification and structural characterization was performed by J.R Colocalization analysis was performed by D.M. The manuscript was read and approved by all co-authors The authors declare no competing financial interests Download citation