Engineering transmembrane signal transduction in synthetic membranes using two-component systems.

Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step toward this goal, we investigated how bacterial two-component systems (TCSs) can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the TCS can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing.

A microfluidic platform for the synthesis of polymer and polymer-protein-based protocells.

In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells.

Tuning Targeted Liposome Avidity to Cells via Lipid Phase Separation.

The addition of both cell-targeting moieties and polyethylene glycol (PEG) to nanoparticle (NP) drug delivery systems is a standard approach to improve the biodistribution, specificity, and uptake of therapeutic cargo. The spatial presentation of these molecules affects avidity of the NP to target cells in part through an interplay between the local ligand concentration and the steric hindrance imposed by PEG molecules. Here, we show that lipid phase separation in nanoparticles can modulate liposome avidity by changing the proximity of PEG and targeting protein molecules on a nanoparticle surface. Using lipid-anchored nickel-nitrilotriacetic acid (Ni-NTA) as a model ligand, we demonstrate that the attachment of lipid anchored Ni-NTA and PEG molecules to distinct lipid domains in nanoparticles can enhance liposome binding to cancer cells by increasing ligand clustering and reducing steric hindrance. We then use this technique to enhance the binding of RGD-modified liposomes, which can bind to integrins overexpressed on many cancer cells. These results demonstrate the potential of lipid phase separation to modulate the spatial presentation of targeting and shielding molecules on lipid nanocarriers, offering a powerful tool to enhance the efficacy of NP drug delivery systems.

Lipid Phase Separation in Vesicles Enhances TRAIL-Mediated Cytotoxicity.

Ligand spatial presentation and density play important roles in signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Here, we harness lipid phase separation to spatially control the protein presentation on lipid vesicles. We use this system to improve the cytotoxicity of TNF-related apoptosis inducing ligand (TRAIL), a therapeutic anticancer protein. Vesicles with phase-separated TRAIL presentation induce more cell death in Jurkat cancer cells than vesicles with uniformly presented TRAIL, and cytotoxicity is dependent on TRAIL density. We assess this relationship in other cancer cell lines and demonstrate that phase-separated vesicles with TRAIL only enhance cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, is less sensitive to TRAIL density. This work demonstrates a rapid and accessible method to control protein conjugation and density on vesicles that can be adopted to other nanoparticle systems to improve receptor signaling by nanoparticles.

Elucidating Design Principles for Engineering Cell-Derived Vesicles to Inhibit SARS-CoV-2 Infection.

The ability of pathogens to develop drug resistance is a global health challenge. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody (mAb) therapies and vaccine-induced sera. Decoy nanoparticles-cell-mimicking particles that bind and inhibit virions-are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, quantitative understanding as to how design features impact performance of these therapeutics is lacking. To address this gap, this study presents a systematic, comparative evaluation of various biologically derived nanoscale vesicles, which may be particularly well suited to sustained or repeated administration in the clinic due to low toxicity, and investigates their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade mAbs and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.

PEO-b-PBD Diblock Copolymers Induce Packing Defects in Lipid/Hybrid Membranes and Improve Insertion Rates of Natively Folded Peptides.

Hybrid membranes assembled from biological lipids and synthetic polymers are a promising scaffold for the reconstitution and utilization of membrane proteins. Recent observations indicate that inclusion of small fractions of polymer in lipid membranes can improve protein folding and function, but the exact structural and physical changes a given polymer sequence imparts on a membrane often remain unclear. Here, we use all-atom molecular dynamics simulations to study the structure of hybrid membranes assembled from DOPC phospholipids and PEO-b-PBD diblock copolymers. We verified our computational model using new and existing experimental data and obtained a detailed picture of the polymer conformations in the lipid membrane that we can relate to changes in membrane elastic properties. We find that inclusion of low polymer fractions induces transient packing defects into the membrane. These packing defects act as insertion sites for two model peptides, and in this way, small amounts of polymer content in lipid membranes can lead to large increases in peptide insertion rates. Additionally, we report the peptide conformational space in both pure lipid and hybrid membranes. Both membranes support similar alpha helical peptide structures, exemplifying the biocompatibility of hybrid membranes.

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression.

The expression and integration of membrane proteins into vesicle membranes is a critical step in the design of cell-mimetic biosensors, bioreactors, and artificial cells. While membrane proteins have been integrated into a variety of nonnatural membranes, the effects of the chemical and physical properties of these vesicle membranes on protein behavior remain largely unknown. Nonnatural amphiphiles, such as diblock copolymers, provide an interface that can be synthetically controlled to better investigate this relationship. Here, we focus on the initial step in a membrane protein's life cycle: expression and folding. We observe improvements in both the folding and overall production of a model mechanosensitive channel protein, the mechanosensitive channel of large conductance, during cell-free reactions when vesicles containing diblock copolymers are present. By systematically tuning the membrane composition of vesicles through incorporation of a poly(ethylene oxide)-b-poly(butadiene) diblock copolymer, we show that membrane protein folding and production can be improved over that observed in traditional lipid vesicles. We then reproduce this effect with an alternate membrane-elasticizing molecule, C12E8 Our results suggest that global membrane physical properties, specifically available membrane surface area and the membrane area expansion modulus, significantly influence the folding and yield of a membrane protein. Furthermore, our results set the stage for explorations into how nonnatural membrane amphiphiles can be used to both study and enhance the production of biological membrane proteins.

EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol.

Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (Ka), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet, the impact of PUFAs on membrane Ka has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent Ka (Kapp) of phospholipid model membranes containing nonesterified fatty acids. First, we measured membrane Kapp as a function of the location of the unsaturated bonds and degree of unsaturation in the incorporated fatty acids and found that Kapp generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the Kapp of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the Kapp in comparison to EPA. We also measured how these ω-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane Kapp changes in relation to these other physical properties. Our study shows that PUFAs generally decrease the Kapp of membranes and that EPA and DHA have differential effects on Kapp when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior and the distribution of membrane-elasticizing amphiphiles impact the ability of a membrane to stretch.

Barcoding Biological Reactions with DNA-Functionalized Vesicles.

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase-segregated membranes, promote fusion between specific vesicle populations. Membrane phase-segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA-mediated fusion events. The orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. Vesicle fusion between DNA-tethered vesicles can be used to initiate in vitro protein expression to produce model soluble and membrane proteins. Engineering orthogonal fusion events between DNA-tethered vesicles provides a new strategy to control the spatiotemporal dynamics of cell-free reactions, expanding opportunities to engineer artificial cellular systems.

Visualizing Tension and Growth in Model Membranes Using Optical Dyes.

Cells dynamically regulate their membrane surface area during a variety of processes critical to their survival. Recent studies with model membranes have pointed to a general mechanism for surface area regulation under tension in which cell membranes unfold or take up lipid to accommodate membrane strain. Yet we lack robust methods to simultaneously measure membrane tension and surface area changes in real time. Using lipid vesicles that contain two dyes isolated to spatially distinct parts of the membrane, we introduce, to our knowledge, a new method to monitor the processes of membrane stretching and lipid uptake in model membranes. Laurdan, located within the bilayer membrane, and Förster resonance energy transfer dyes, localized to the membrane exterior, act in concert to report changes in membrane tension and lipid uptake during osmotic stress. We use these dyes to show that membranes under tension take up lipid more quickly and in greater amounts compared to their nontensed counterparts. Finally, we show that this technique is compatible with microscopy, enabling real-time analysis of membrane dynamics on a single vesicle level. Ultimately, the combinatorial use of these probes offers a more complete picture of changing membrane morphology. Our optical method allows us to remotely track changes in membrane tension and surface area with model membranes, offering new opportunities to track morphological changes in artificial and biological membranes and providing new opportunities in fields ranging from mechanobiology to drug delivery.

Copying of Mixed-Sequence RNA Templates inside Model Protocells.

The chemical replication of RNA inside fatty acid vesicles is a plausible step in the emergence of cellular life. On the primitive Earth, simple protocells with the ability to import nucleotides and short oligomers from their environment could potentially have replicated and retained larger genomic RNA oligonucleotides within a spatially defined compartment. We have previously shown that short 5'-phosphoroimidazolide-activated "helper" RNA oligomers enable the nonenzymatic copying of mixed-sequence templates in solution, using 5'-phosphoroimidazolide-activated mononucleotides. Here, we report that citrate-chelated Mg2+, a catalyst of nonenzymatic primer extension, enhances fatty acid membrane permeability to such short RNA oligomers up to the size of tetramers, without disrupting vesicle membranes. In addition, selective permeability of short, but not long, oligomers can be further enhanced by elevating the temperature. The ability to increase the permeability of fatty acid membranes to short oligonucleotides allows for the nonenzymatic copying of RNA templates containing all four nucleotides inside vesicles, bringing us one step closer to the goal of building a protocell capable of Darwinian evolution.

Fatty Acid/Phospholipid Blended Membranes: A Potential Intermediate State in Protocellular Evolution.

Prior to the evolution of membrane proteins, intrinsic membrane stability and permeability to polar solutes are essential features of a primitive cell membrane. These features are difficult to achieve simultaneously in model protocells made of either pure fatty acid or phospholipid membranes, raising the intriguing question of how the transition from fatty acid to phospholipid membranes might have occurred while continuously supporting encapsulated reactions required for genomic replication. Here, the properties of a blended membrane system composed of both oleic acid (OA), a monoacyl fatty acid, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a diacyl phospholipid are described. This hybrid vesicle system exhibits high stability to divalent cations (Mg2+ ), while simultaneously maintaining its permeability to small charged molecules such as nucleotides and divalent ions such as Mg2+ . This combination of features facilitates key reactions expected to occur during a transition from primitive to modern cells, including nonenzymatic RNA replication, and is also compatible with highly evolved functions such as the ribosomal translation of a protein. The observations support the hypothesis that the early transition from fatty acid to phospholipid membranes could be accomplished through intermediate states in which membranes are composed of amphiphile mixtures, and do not require protein transporters.

N-Carboxyanhydride-Mediated Fatty Acylation of Amino Acids and Peptides for Functionalization of Protocell Membranes.

Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid N-carboxyanhydrides (NCAs). Here, we show that the polymerization of valine-NCA in the presence of fatty acids yields acylated amino acids and peptides via a mixed anhydride intermediate. Notably, Nα-oleoylarginine, a product of the reaction between arginine and oleic acid in the presence of valine-NCA, partitions spontaneously into vesicle membranes and mediates the association of RNA with the vesicles. Our results suggest a potential mechanism by which activated amino acids could diversify the chemical functionality of fatty acid membranes and colocalize RNA with vesicles during the formation of early protocells.

Electrostatic Localization of RNA to Protocell Membranes by Cationic Hydrophobic Peptides.

Cooperative interactions between RNA and vesicle membranes on the prebiotic earth may have led to the emergence of primitive cells. The membrane surface offers a potential platform for the catalysis of reactions involving RNA, but this scenario relies upon the existence of a simple mechanism by which RNA could become associated with protocell membranes. Here, we show that electrostatic interactions provided by short, basic, amphipathic peptides can be harnessed to drive RNA binding to both zwitterionic phospholipid and anionic fatty acid membranes. We show that the association of cationic molecules with phospholipid vesicles can enhance the local positive charge on a membrane and attract RNA polynucleotides. This phenomenon can be reproduced with amphipathic peptides as short as three amino acids. Finally, we show that peptides can cross bilayer membranes to localize encapsulated RNA. This mechanism of polynucleotide confinement could have been important for primitive cellular evolution.

Sensing membrane stress with near IR-emissive porphyrins.

Probes embedded within a structure can enable prediction of material behavior or failure. Carefully assembled composites that respond intelligently to physical changes within a material could be useful as intrinsic sensors. Molecular rotors are one such tool that can respond optically to physical environmental changes. Here, we propose to use molecular rotors within a polymersome membrane to report membrane stress. Using supermolecular porphyrin-based fluorophores as rotors, we characterize changes in the optical emission of these near-infrared (NIR) emissive probes embedded within the hydrophobic core of the polymersome membrane. The configuration of entrapped fluorophore depends on the available space within the membrane; in response to increased volume, emission is blue shifted. We used this feature to study how shifts in fluorescence correlate to membrane integrity, imparted by membrane stress. We monitored changes in emission of these porphyrin-based fluorophores resulting from membrane stress produced through a range of physical and chemical perturbations, including surfactant-induced lysis, hydrolytic lysis, thermal degradation, and applied stress by micropipette aspiration. This paper comprehensively illustrates the potential for supermolecular porphyrin-based fluorophores to detect intrinsic physical changes in a wide variety of environments, and suggests how molecular rotors may be used in soft materials science and biology as sensors.

A synthetic method to assay polycystin channel biophysics.

Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive states are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here we describe a fully synthetic method to assay the functional properties of the polycystin subfamily of transient receptor potential (TRP) channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their membrane integration, orientation and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate other channels resistant to biophysical analysis and pharmacological characterization.

Improving Cell-Free Expression of Model Membrane Proteins by Tuning Ribosome Cotranslational Membrane Association and Nascent Chain Aggregation.

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE.

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions.

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.