Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes.

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.

Rapid Generation of Therapeutic Nanoparticles Using Cell-Free Expression Systems.

The surface modification of membrane-based nanoparticles, such as liposomes, polymersomes, and lipid nanoparticles, with targeting molecules, such as binding proteins, is an important step in the design of therapeutic materials. However, this modification can be costly and time-consuming, requiring cellular hosts for protein expression and lengthy purification and conjugation steps to attach proteins to the surface of nanocarriers, which ultimately limits the development of effective protein-conjugated nanocarriers. Here, the use of cell-free protein synthesis systems to rapidly create protein-conjugated membrane-based nanocarriers is demonstrated. Using this approach, multiple types of functional binding proteins, including affibodies, computationally designed proteins, and scFvs, can be cell-free expressed and conjugated to liposomes in one-pot. The technique can be expanded further to other nanoparticles, including polymersomes and lipid nanoparticles, and is amenable to multiple conjugation strategies, including surface attachment to and integration into nanoparticle membranes. Leveraging these methods, rapid design of bispecific artificial antigen presenting cells and enhanced delivery of lipid nanoparticle cargo in vitro is demonstrated. It is envisioned that this workflow will enable the rapid generation of membrane-based delivery systems and bolster our ability to create cell-mimetic therapeutics.

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.

Engineered membrane receptors with customizable input and output functions.

Morsut et al. reported a synthetic receptor system, based on the natural Notch receptor, with customizable input and output functions. Their work on advanced receptor design expands the reach of synthetic receptor systems. Incorporating new protein design tools with better-understood membrane biophysics will create the next generation of engineered receptors.

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.

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.

EMG characteristics of the external anal sphincter guarding reflex and effects of a unilateral ventral root avulsion injury in rhesus macaques ( Macaca mulatta).

The external anal sphincter (EAS) is important for the maintenance of bowel continence and may be compromised by a variety of neuropathic conditions. However, large animal models for the study of EAS functions have been sparse. The EAS guarding reflex was examined by electromyography (EMG) in neurologically intact rhesus macaques ( n = 6) and at 4-6 wk after a unilateral EAS denervation from an L6-S3 ventral root avulsion (VRA) injury ( n = 6). Baseline EAS EMG recordings were quiescent in all subjects, and evoked responses showed an initial large-amplitude EMG activity, which gradually returned to baseline within 1-2 min. At 4-6 wk postoperatively, the EAS guarding reflex showed a significantly reduced EMG response duration of 47 ± 15 s and area under the curve (AUC) of 0.198 ± 0.097 mV·s compared with the corresponding evoked EAS EMG duration of 102 ± 19 s and AUC of 0.803 ± 0.225 mV·s ( P < 0.05) in the control group. Detailed time- and frequency-domain analysis of the evoked EAS EMG responses for the first 40 s showed no difference between groups for the maximum amplitude but a significant decrease for the mean amplitude across the study period and an early AUC reduction for the first 10 s in the VRA injury group. Time-frequency analysis and power spectrum plots indicated decreased intensity and a narrower midrange of frequencies in the VRA injury group. We conclude that the EAS guarding reflex in rhesus macaques shows characteristic EMG features in control subjects and signs of partial target denervation after a unilateral L6-S3 VRA injury. NEW & NOTEWORTHY The external anal sphincter guarding reflex showed initial large-amplitude peaks and a gradual return to a quiescent baseline after a rectal probe stimulus in rhesus macaques. At 4-6 wk after a unilateral ventral root avulsion (VRA) injury, the electromyography duration, mean amplitude, and area under the curve measurements were decreased. Time-frequency analysis and power spectrum plots indicated decreased intensity and a narrowed midrange of frequencies in the VRA injury cohort.

Bridging the gap: microfluidic devices for short and long distance cell-cell communication.

Cell-cell communication is a crucial component of many biological functions. For example, understanding how immune cells and cancer cells interact, both at the immunological synapse and through cytokine secretion, can help us understand and improve cancer immunotherapy. The study of how cells communicate and form synaptic connections is important in neuroscience, ophthalmology, and cancer research. But in order to increase our understanding of these cellular phenomena, better tools need to be developed that allow us to study cell-cell communication in a highly controlled manner. Some technical requirements for better communication studies include manipulating cells spatiotemporally, high resolution imaging, and integrating sensors. Microfluidics is a powerful platform that has the ability to address these requirements and other current limitations. In this review, we describe some new advances in microfluidic technologies that have provided researchers with novel methods to study intercellular communication. The advantages of microfluidics have allowed for new capabilities in both single cell-cell communication and population-based communication. This review highlights microfluidic communication devices categorized as "short distance", or primarily at the single cell level, and "long distance", which mostly encompasses population level studies. Future directions and translation/commercialization will also be discussed.

Microfluidics Cell Loading-Dock System: Ordered Cellular Array for Dynamic Lymphocyte-Communication Study.

It remains a great challenge to establish a high-throughput platform that can explore the interactions among multiple lymphocytes (>2 cells) and retrieve the interested cells for downstream analysis. This study demonstrates a microfluidics cell loading-dock system (Cell-Dock) to enclose multiple cells in 1D, 2D, and 3D chambers with high throughput and efficiency and single-cell accuracy. The loading efficiencies of 95%, 85%, and 74% for one-, three-, and five-cell systems are achieved, respectively. The Cell-Dock system provides precise and dynamic cell packing models to facilitate lymphocyte-interaction studies. The results demonstrate that individual natural killer (NK) cells may function independently rather than cooperate to lyse target cells in the defined microenvironment. Furthermore, the strong/weak NK cells are retrieved based on their on-chip cytotoxicity and mRNA sequencing is conducted to find the possible mechanisms for "serial killing," an important but unsolved issue. This study finds that the stronger NK cells overexpress multiple genes involved in cytotoxicity and adhesion molecules (including the well-known ICAM1 and seldom reported B4GALT1) might play important roles in the regulation of NK cytolysis.

A microfabricated, optically accessible device to study the effects of mechanical cues on collagen fiber organization.

As the primary structural protein of our bodies, fibrillar collagen and its organizational patterns determine the biomechanics and shape of tissues. While the molecular assembly of individual fibrils is well understood, the mechanisms determining the arrangement of fibers and thus the shape and form of tissues remain largely unknown. We have developed a cell culture model that successfully recapitulates early tissue development and the de novo deposition of collagen fibers to investigate the role of mechanical cues on collagen fiber alignment. The devices used a thin, collagen-coated deformable PDMS membrane inside a tissue culture well built on microscope-grade coverslips. Deformations and strains in the PDMS membrane were quantified by tracking fluorescent bead displacement and through the use of a COMSOL model. Cyclical strains were applied to serum-cultured rabbit corneal cells at 0.5 Hz for 24-48 h and showed a preferred alignment after 36 h of cyclical loading. Cells cultured with ascorbic acid under methylcellulose serum-free conditions deposited a collagenous matrix that was visible under live second harmonic generation microscopy at week 4. Our microfabricated tissue culture system allows for the controllable application of a wide range of stress profiles to cells, and for the observation and quantification of cells and de novo collagen formation in vitro. Future studies will involve the fabrication of models to study the formation and organization of collagen in ocular diseases.