Giant Vesicles Research Articles

Chemical Communication between Giant Vesicles and Gated Nanoparticles for Strip-Based Sensing.

Inspired by nature, the development of artificial micro/nanosystems capable of communicating has become an emergent topic in nanotechnology, synthetic biology, and related areas. However, the demonstration of actual applications still has to come. Here, we demonstrate how chemical communication between micro- and nanoparticles can be used for the design of sensing systems. To realize this, we synergistically combine two different types of particles: i.e., giant unilamellar vesicles (GUVs) as senders and gated mesoporous nanoparticles as receivers. The use of engineered GUVs allows the detection of analytes based on responsive membranes, while the use of gated nanoparticles allows a straightforward application on test strips with smartphone-based detection. In addition, we demonstrate that the combined communication system exhibits signal amplification and its application in real samples employing the bacterial toxin α-hemolysin as target analyte. Altogether, our report presents a new route for engineering sensing systems based on the combination of communicative micro/nanoparticles.

Shape Analysis of Biomimetic and Plasma Membrane Vesicles

Giant membrane vesicles (GUVs) and Giant plasma membrane vesicles (GPMVs) are used as models to study membrane properties. We conducted a comparative study to examine how reducing the volume of vesicles with different lipid compositions, solution symmetries, solution asymmetries, and membrane charges affects their morphology. We used three‐dimensional visualization techniques to study the shape of the vesicles. Although the vesicles may not be perfectly spherical, they exhibit some fluctuations in their shape. To understand these variations, we used confocal image stacks for visualization. Our experimental observations show that the membrane's charge influences the deflation of the GUVs in the presence of trans‐bilayer sugar asymmetries. The lipid bilayers of our GUVs have a uniform distribution of lipids in both leaflets, indicating no asymmetry in lipid composition. However, we induce trans‐bilayer asymmetries by exposing each leaflet to different solution compositions, leading to asymmetric adsorption or desorption layers. We also estimated and compared the deformation of GPMV shapes extracted from cells with trans‐bilayer buffer asymmetries and composition asymmetries with biomimetic membranes.

Curvature fluctuations of fluid vesicles reveal hydrodynamic dissipation within the bilayer

The biological function of membranes is closely related to their softness, which is often studied through the membranes' thermally driven fluctuations. Typically, the analysis assumes that the relaxation rate of a pure bending deformation is determined by the competition between membrane bending rigidity and viscous dissipation in the surrounding medium. Here, we reexamine this assumption and demonstrate that viscous flows within the membrane dominate the dynamics of bending fluctuations of nonplanar membranes with a radius of curvature smaller than the Saffman-Delbrück length. Using flickering spectroscopy of giant vesicles made of dipalmitoylphosphatidylcholine, DPPC:cholesterol mixtures and pure diblock-copolymer membranes, we experimentally detect the signature of membrane dissipation in curvature fluctuations. We show that membrane viscosity can be reliably obtained from the short time behavior of the shape time correlations. The results indicate that the DPPC:cholesterol membranes behave as a Newtonian fluid, while the polymer membranes exhibit more complex rheology. Our study provides physical insights into the time scales of curvature remodeling of biological and synthetic membranes.

Insights into a clock's fidelity through vesicular encapsulation.

The single-celled cyanobacterium, Synechococcus elongatus , generates circadian rhythms with exceptional fidelity and synchrony despite their femtoliter volumes. Here, we explore the mechanistic aspects of this fidelity, by reconstituting the KaiABC post-translational oscillator (PTO) in cell-mimetic giant vesicles (GUVs) under well-defined conditions in vitro . PTO proteins were encapsulated with a coefficient of variation that closely matched protein variations observed in live cells. Using fluorescently labeled KaiB and confocal microscopy, we were able to measure circadian rhythms generated by thousands of encapsulated PTOs at the single-vesicle level for several days as a function of protein concentration and GUV size. We find that PTO fidelity decreased with decreasing levels of encapsulated PTO proteins and in smaller GUVs. We also observed that in encapsulated PTOs, a significant fraction of KaiB localized to GUV membranes like it does in cyanobacteria. A mathematical model that uses empirical bulk concentration and stoichiometry limitations suggests that cyanobacteria overcome challenges to fidelity by expressing high levels of PTO proteins along with the CikA and SasA proteins, which buffer stochastic variations in the levels of KaiA and KaiB, respectively. Further, the model suggests that the transcription-translation feedback loop (TTFL) contributes at most a small percentage to the overall fidelity of the cyanobacterial circadian clock under constant conditions but is essential for maintaining phase synchrony. Our results are the first experimental demonstration of populations of synthetic cells that can autonomously keep circadian time. Additionally, the approach of using bulk relationships to understand complex phenomena in cell-like systems could be useful for understanding other collective behavior important in biology, such as liquid-liquid phase separation.

PH-induced morphological reversible transition from microparticles to vesicles for effective bacteria entrapment

Polymer particles with stimuli-responsive properties offer promising applications in healthcare, chemical reactors, development of artificial cells and organelles, as well as in the entrapment of bacteria. In this study, a novel biocompatible, biodegradable, and pH-responsive diblock copolymer based on polylactide (PLA) and poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) was synthesized via a metal-free one-pot/simultaneous ring-opening polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT) approach (ROP/RAFT). This copolymer was then employed to produce highly monodisperse microparticles using microfluidics droplet generation technique. Utilizing confocal microscopy imaging, a reversible pH-induced morphological transition from microparticles to vesicle-like structures was observed at pH 5.1. The reversible shift in morphology from microparticles to giant vesicles (Vs) in acidic environments is triggered by the protonation of amino groups of the PDPA block, rendering vesicle surfaces positively charged − an advantageous feature for attracting and engulfing negatively charged bacteria. Initial validation involved electrostatic interactions with negatively charged latex resin beads followed by assessing interaction capabilities with gram-negative bacteria, Escherichia coli (E. coli). Additionally, the reversible morphological transition of microparticles-to-vesicles was employed to study drug release at different pHs. This approach proven to be a promising strategy for targeted drug delivery and bacteria entrapment using smart pH-responsive microparticles.

Topological and Morphological Membrane Dynamics in Giant Lipid Vesicles Driven by Monoolein Cubosomes

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically‐active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group Im3m incorporate into 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time‐resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life‐like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Topological and Morphological Membrane Dynamics in Giant Lipid Vesicles Driven by Monoolein Cubosomes.

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically-active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group Im3m incorporate into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time-resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life-like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Mixtures of Intrinsically Disordered Neuronal Protein Tau and Anionic Liposomes Reveal Distinct Anionic Liposome-Tau Complexes Coexisting with Tau Liquid-Liquid Phase-Separated Coacervates.

Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein that binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full-length human protein tau and other negatively charged binding substrates, as revealed by differential interference contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase-separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes (i.e. tau-lipoplexes) exhibited distinct types of morphologies. This included large ∼20-30 μm tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles and tau-mediated finite-size assemblies of small liposomes. As the salt concentration was increased to near and above 150 mM for 1:1 electrolytes, AL-tau complexes remained stable, while tau self-coacervate droplets were found to dissolve, indicative of the breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen-facing monolayer of the axon's plasma membrane, suggesting the possibility of transient yet robust interactions near relevant ionic strengths found in neurons.

A molecular mechanism for how pressure induces interdigitation of phospholipid bilayer membranes

The phase transition from the ripple gel phase to the interdigitated gel phase of bilayers of phosphatidylcholines (PCs) with two saturated long-chain fatty acids under high pressure was investigated by pressure-scanning microscopy, fluorometry, and dynamic light scattering (DLS) measurements. Microscopic observation for giant vesicles (GVs) of distearoyl-PC (DSPC) under high pressure showed that spherical GVs transforms significantly into warped and distorted spherical ones instantaneously at the pressure-induced interdigitation. The fluorescence intensities of amphiphilic probe Prodan and hydrophobic probe Laurdan in the dipalmitoyl-PC (DPPC) bilayer steeply decreased and increased, respectively, at the interdigitation, suggesting that the conformational change of the polar head group of DPPC molecule in the bilayer transiently occurred at the interdigitation. Further, it was found from the high-pressure DLS measurements that the size of the vesicle particles of the DPPC and DSPC transiently increases near the interdigitation pressure, whereas the chemically induced interdigitation by adding ethanol to the DSPC bilayer membrane under atmospheric pressure produce no such change in the particle size. Taking account of the critical packing parameter of the PC molecule, the above experimental results would lead us to the conclusion that the pressure-induced interdigitation is attributable to the increase in repulsive interaction between the polar head groups of the PC molecules resulting from the orientational change of the head group from a parallel alignment to a perpendicular one with respect to the bilayer surface by applying pressure, namely the transient state: it occurs when the repulsive interaction exceeds a threshold value for the balance between the repulsive interaction and the attractive interaction among the hydrophobic acyl chains.

Architecting Multicompartmentalized, Giant Vesicles with Recombinant Fusion Proteins.

We present a straightforward strategy for constructing giant, multicompartmentalized vesicles using recombinant fusion proteins. Our method leverages the self-assembly of globule-zipper-elastin-like polypeptide fusion protein complexes in aqueous conditions, eliminating the need for organic solvents and chemical conjugation. By employing the thin-film rehydration method, we have successfully encapsulated a diverse range of bioactive macromolecules and engineered organelle-like compartments─ranging from soluble proteins and coacervate droplets to vesicles─within these protein-assembled giant vesicles. This approach also facilitates the integration of water-soluble block copolymers, enhancing the structural stability and functional versatility of the vesicles. Our results suggest that these multicompartment giant protein vesicles not only mimic the complex architecture of living cells but also support biochemically distinct reactions regulated by functionally folded proteins, providing a robust model for studying cellular processes and designing microreactor systems. This work highlights the transformative potential of self-assembling recombinant fusion proteins in artificial cell design.

Droplet Microfluidics for High-Throughput Screening and Directed Evolution of Biomolecules.

Directed evolution is a powerful technique for creating biomolecules such as proteins and nucleic acids with tailor-made properties for therapeutic and industrial applications by mimicking the natural evolution processes in the laboratory. Droplet microfluidics improved classical directed evolution by enabling time-consuming and laborious steps in this iterative process to be performed within monodispersed droplets in a highly controlled and automated manner. Droplet microfluidic chips can generate, manipulate, and sort individual droplets at kilohertz rates in a user-defined microchannel geometry, allowing new strategies for high-throughput screening and evolution of biomolecules. In this review, we discuss directed evolution studies in which droplet-based microfluidic systems were used to screen and improve the functional properties of biomolecules. We provide a systematic overview of basic on-chip fluidic operations, including reagent mixing by merging continuous fluid streams and droplet pairs, reagent addition by picoinjection, droplet generation, droplet incubation in delay lines, chambers and hydrodynamic traps, and droplet sorting techniques. Various microfluidic strategies for directed evolution using single and multiple emulsions and biomimetic materials (giant lipid vesicles, microgels, and microcapsules) are highlighted. Completely cell-free microfluidic-assisted in vitro compartmentalization methods that eliminate the need to clone DNA into cells after each round of mutagenesis are also presented.

Assessing the mechanism of facilitated proton transport across GUVs trapped in a microfluidic device

Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H+ permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin.

Continuous Transformation from Membrane‐Less Coacervates to Membranized Coacervates and Giant Vesicles: Toward Multicompartmental Protocells with Complex (Membrane) Architectures

AbstractThe membranization of membrane‐less coacervates paves the way for the exploitation of complex protocells with regard to structural and cell‐like functional behaviors. However, the controlled transformation from membranized coacervates to vesicles remains a challenge. This can provide stable (multi)phase and (multi)compartmental architectures through the reconfiguration of coacervate droplets in the presence of (bioactive) polymers, bio(macro)molecules and/or nanoobjects. Herein, we present a continuous protocell transformation from membrane‐less coacervates to membranized coacervates and, ultimately, to giant hybrid vesicles. This transformation process is orchestrated by altering the balance of non‐covalent interactions through varying concentrations of an anionic terpolymer, leading to dynamic processes such as spontaneous membranization of terpolymer nanoparticles at the coacervate surface, disassembly of the coacervate phase mediated by the excess anionic charge, and the redistribution of coacervate components in membrane. The diverse protocells during the transformation course provide distinct structural features and molecular permeability. Notably, the introduction of multiphase coacervates in this continuous transformation process signifies advancements toward the creation of synthetic cells with different diffusible compartments. Our findings emphasize the highly controlled continuous structural reorganization of coacervate protocells and represents a novel step toward the development of advanced and sophisticated synthetic protocells with more precise compositions and complex (membrane) structures.

Continuous Transformation from Membrane-Less Coacervates to Membranized Coacervates and Giant Vesicles: Toward Multicompartmental Protocells with Complex (Membrane) Architectures.

The membranization of membrane-less coacervates paves the way for the exploitation of complex protocells with regard to structural and cell-like functional behaviors. However, the controlled transformation from membranized coacervates to vesicles remains a challenge. This can provide stable (multi)phase and (multi)compartmental architectures through the reconfiguration of coacervate droplets in the presence of (bioactive) polymers, bio(macro)molecules and/or nanoobjects. Herein, we present a continuous protocell transformation from membrane-less coacervates to membranized coacervates and, ultimately, to giant hybrid vesicles. This transformation process is orchestrated by altering the balance of non-covalent interactions through varying concentrations of an anionic terpolymer, leading to dynamic processes such as spontaneous membranization of terpolymer nanoparticles at the coacervate surface, disassembly of the coacervate phase mediated by the excess anionic charge, and the redistribution of coacervate components in membrane. The diverse protocells during the transformation course provide distinct structural features and molecular permeability. Notably, the introduction of multiphase coacervates in this continuous transformation process signifies advancements toward the creation of synthetic cells with different diffusible compartments. Our findings emphasize the highly controlled continuous structural reorganization of coacervate protocells and represents a novel step toward the development of advanced and sophisticated synthetic protocells with more precise compositions and complex (membrane) structures.

Mixtures of Intrinsically Disordered Neuronal Protein Tau and Anionic Liposomes Reveal Distinct Anionic Liposome-Tau Complexes Coexisting with Tau Liquid-Liquid Phase Separated Coacervates.

Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein, which binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full length human protein tau and other negatively charged binding substrates, as revealed by differential-interference-contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes exhibited distinct types of morphologies. This included, large ≈20-30 micron tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles, and tau-mediated finite-size assemblies of small liposomes. As the ionic strength of the solution was increased to near and above physiological salt concentrations for 1:1 electrolytes (≈150 mM), AL-tau complexes remained stable while tau self-coacervate droplets were found to dissolve indicative of breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen facing monolayer of the axon plasma membrane suggesting the possibility of transient yet robust interactions at physiologically relevant ionic strengths.

Ready, Set, Grow: From Micelles to Giant Vesicles via Biocatalytic Activation.

Controlling physicochemical processes that drive changes in supramolecular aggregates is an important objective toward creating artificial soft micro- and nanomachines. Previous research explored the morphology control of membrane-based materials subjected to externally imposed chemical stimuli. Here, we modulate the microscale morphology of pH-responsive assemblies by using biocatalysis to internally generate changes in global pH. Catalytic reactions offer flexibility in the mechanism and rate at which stimuli are introduced to responsive assemblies, ultimately enabling precision and control over size and morphology. We observed, by dynamic light scattering and fluorescence microscopy, substantial microscale differences between assemblies subjected to manually titrated pH changes compared to biocatalytically activated pH changes, including the growth of giant vesicles from micelles. Coarse-grained molecular dynamics simulations of these metastable self-assembled structures provided insight into the thermodynamics and kinetics of the preferred structures. These results demonstrate the feasibility of using biocatalytic reactions to modulate the size and morphology of supramolecular assemblies, from micelles to giant vesicles.

Membrane nanotubes transform into double-membrane sheets at condensate droplets

Cellular membranes exhibit a multitude of highly curved morphologies such as buds, nanotubes, cisterna-like sheets defining the outlines of organelles. Here, we mimic cell compartmentation using an aqueous two-phase system of dextran and poly(ethylene glycol) encapsulated in giant vesicles. Upon osmotic deflation, the vesicle membrane forms nanotubes, which undergo surprising morphological transformations at the liquid-liquid interfaces inside the vesicles. At these interfaces, the nanotubes transform into cisterna-like double-membrane sheets (DMS) connected to the mother vesicle via short membrane necks. Using super-resolution (stimulated emission depletion) microscopy and theoretical considerations, we construct a morphology diagram predicting the tube-to-sheet transformation, which is driven by a decrease in the free energy. Nanotube knots can prohibit the tube-to-sheet transformation by blocking water influx into the tubes. Because both nanotubes and DMSs are frequently formed by cellular membranes, understanding the formation and transformation between these membrane morphologies provides insight into the origin and evolution of cellular organelles.

Poly(Sitosterol)-Based Hydrophobic Blocks in Amphiphilic Block Copolymers for the Assembly of Hybrid Vesicles.

Amphiphilic block copolymer and lipids can be assembled into hybrid vesicles (HVs), which are an alternative to liposomes and polymersomes. Block copolymers that have either poly(sitostryl methacrylate) or statistical copolymers of sitosteryl methacrylate and butyl methacrylate as the hydrophobic part and a poly(carboxyethyl acrylate) hydrophilic segment are synthesized and characterized. These block copolymers assemble into small HVs with soybean L-α-phosphatidylcholine (soyPC), confirmed by electron microscopy and small-angle X-ray scattering. The membrane's hybrid nature is illustrated by fluorescence resonance energy transfer between labeled building blocks. The membrane packing, derived from spectra when using Laurdan as an environmentally sensitive fluorescent probe, is comparable between small HVs and the corresponding liposomes with molecular sitosterol, although the former show indications of transmembrane asymmetry. Giant HVs with homogenous distribution of the block copolymers and soyPC in their membranes are assembled using the electroformation method. The lateral diffusion of both building blocks is slowed down in giant HVs with higher block copolymer content, but their permeability toward (6)-carboxy-X-rhodamine is higher compared to giant vesicles made of soyPC and molecular sitosterol. This fundamental effort contributes to the rapidly expanding understanding of the integration of natural membrane constituents with designed synthetic compounds to form hybrid membranes.

Alkyl anchor–modified artificial viral capsid budding outside-to-inside and inside-to-outside giant vesicles

ABSTRACT The budding of human immunodeficiency virus from an infected host cell is induced by the modification of structural proteins bearing long-chain fatty acids, followed by their anchoring to the cell membrane. Although many model budding systems using giant unilamellar vesicles (GUVs) induced by various stimuli have been developed, constructing an artificial viral budding system of GUVs using only synthesized molecules remains challenging. Herein, we report the construction of an artificial viral capsid budding system from a lipid bilayer of GUV. The C-terminus of the β-annulus peptide was modified using an octyl chain as an alkyl anchor via a disulfide bond. The self-assembly of the β-annulus peptide with an octyl chain formed an artificial viral capsid aggregate. The fluorescence imaging and transmission electron microscopy observations revealed that the addition of the tetramethylrhodamine (TMR)-labeled octyl chain–bearing β-annulus peptide to the outer aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle outside-to-inside the mother GUV. Conversely, the encapsulation of the TMR-labeled octyl chain–bearing β-annulus peptide in the inner aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle inside-to-outside the mother GUV. Contrarily, the addition of the TMR-labeled β-annulus peptide to GUV barely induced budding. It was demonstrated that the higher the membrane fluidity of GUV, the more likely budding would be induced by the addition of the alkyl anchor–modified artificial viral capsid. The simple virus-mimicking material developed in this study, which buds off through membrane anchoring, can provide physicochemical insights into the mechanisms of natural viral budding from cells.

CaV1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

A key player of excitable cells in the heart and brain is the L-type calcium channel CaV1.3. In the heart, it is required for voltage-dependent Ca2+-signaling, i.e., for controlling and modulating atrial cardiomyocyte excitation-contraction coupling. The clustering of CaV1.3 in functionally relevant channel multimers has not been addressed due to a lack of stoichiometric labeling combined with high-resolution imaging. Here, we developed a HaloTag-labeling strategy to visualize and quantify CaV1.3 clusters using STED nanoscopy to address the questions of cluster size and intra-cluster channel density. Channel clusters were identified in the plasma membrane of transfected live HEK293 cells as well as in giant plasma membrane vesicles derived from these cells that were spread on modified glass support to obtain supported plasma membrane bilayers (SPMBs). A small fraction of the channel clusters was colocalized with early and recycling endosomes at the membranes. STED nanoscopy in conjunction with live-cell and SPMB imaging enabled us to quantify CaV1.3 cluster sizes and their molecular density revealing significantly lower channel densities than expected for dense channel packing. CaV1.3 channel cluster size and molecular density were increased in SPMBs after treatment of the cells with the sympathomimetic compound isoprenaline, suggesting a regulated channel cluster condensation mechanism.