Biomimetic behaviors in hydrogel artificial cells through embedded organelles.

Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom-up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.

Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation.

Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.

Magnetic Modulation of Biochemical Synthesis in Synthetic Cells.

Synthetic cells can be constructed from diverse molecular components, without the design constraints associated with modifying 'living' biological systems. This can be exploited to generate cells with abiotic components, creating functionalities absent in biology. One example is magnetic responsiveness, the activation and modulation of encapsulated biochemical processes using a magnetic field, which is absent from existing synthetic cell designs. This is a critical oversight, as magnetic fields are uniquely bio-orthogonal, noninvasive, and highly penetrative. Here, we address this by producing artificial magneto-responsive organelles by coupling thermoresponsive membranes with hyperthermic Fe3O4 nanoparticles and embedding them in synthetic cells. Combining these systems enables synthetic cell microreactors to be built using a nested vesicle architecture, which can respond to alternating magnetic fields through in situ enzymatic catalysis. We also demonstrate the modulation of biochemical reactions by using different magnetic field strengths and the potential to tune the system using different lipid compositions. This platform could unlock a wide range of applications for synthetic cells as programmable micromachines in biomedicine and biotechnology.

Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells.

To date, reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnology. Here, we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle-cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic molecules difficult to introduce to biological cells. This work represents a foundation for the engineering of multicompartment-spanning designer pathways that can be utilized to control downstream events inside an AC, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.

The membrane transporter lactose permease increases lipid bilayer bending rigidity.

Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.

New Directions for Artificial Cells Using Prototyped Biosystems.

Microfluidics has has enabled the generation of  a range of single compartment and multicompartment vesicles and bilayer-delineated droplets that can be assembled in 2D and 3D. These model systems are becoming increasingly used as artificial cell chassis and as biomimetic constructs for assembling tissue models, engineering therapeutic delivery systems, and screening drugs. One bottleneck in developing this technology is the time, expertise, and equipment required for device fabrication. This has led to interest across the microfluidics community in using rapid prototyping to engineer microfluidic devices from computer-aided-design (CAD) drawings. We highlight how this rapid-prototyping revolution is transforming the fabrication of microfluidic devices for artificial cell construction in bottom-up synthetic biology. We provide an outline of the current landscape and present how advances in the field may give rise to the next generation of multifunctional biodevices, particularly with Industry 4.0 on the horizon. Successfully developing this technology and making it open-source could pave the way for a new generation of citizen-led science, fueling the possibility that the next multibillion-dollar start-up could emerge from an attic or a basement.

Engineering Swollen Cubosomes Using Cholesterol and Anionic Lipids.

Dispersions of nonlamellar lipid membrane assemblies are gaining increasing interest for drug delivery and protein therapeutic application. A key bottleneck has been the lack of rational design rules for these systems linking different lipid species and conditions to defined lattice parameters and structures. We have developed robust methods to form cubosomes (nanoparticles with porous internal structures) with water channel diameters of up to 171 Å, which are over 4 times larger than archetypal cubosome structures. The water channel diameter can be tuned via the incorporation of cholesterol and the charged lipid DOPA, DOPG, or DOPS. We have found that large molecules can be incorporated into the porous cubosome structure and that these molecules can interact with the internal cubosome membrane. This offers huge potential for accessible encapsulation and protection of biomolecules and development of confined interfacial reaction environments.

Mask-Free Laser Lithography for Rapid and Low-Cost Microfluidic Device Fabrication.

Microfluidics has become recognized as a powerful platform technology associated with a constantly increasing array of applications across the life sciences. This surge of interest over recent years has led to an increased demand for microfluidic chips, resulting in more time being spent in the cleanroom fabricating devices using soft lithography-a slow and expensive process that requires extensive materials, training and significant engineering resources. This bottleneck limits platform complexity as a byproduct of lengthy delays between device iterations and affects the time spent developing the final application. To address this problem, we report a new, rapid, and economical approach to microfluidic device fabrication using dry resist films to laminate laser cut sheets of acrylic. We term our method laser lithography and show that our technique can be used to engineer 200 µm width channels for assembling droplet generators capable of generating monodisperse water droplets in oil and micromixers designed to sustain chemical reactions. Our devices offer high transparency, negligible device to device variation, and low X-ray background scattering, demonstrating their suitability for real-time X-ray-based characterization applications. Our approach also requires minimal materials and apparatus, is cleanroom free, and at a cost of around $1.00 per chip could significantly democratize device fabrication, thereby increasing the interdisciplinary accessibility of microfluidics.

Structure and function of PspA and Vipp1 N-terminal peptides: Insights into the membrane stress sensing and mitigation.

The phage shock protein (Psp) response maintains integrity of the inner membrane (IM) in response to extracytoplasmic stress conditions and is widely distributed amongst enterobacteria. Its central component PspA, a member of the IM30 peripheral membrane protein family, acts as a major effector of the system through its direct association with the IM. Under non-stress conditions PspA also negatively regulates its own expression via direct interaction with the AAA+ ATPase PspF. PspA has a counterpart in cyanobacteria called Vipp1, which is implicated in protection of the thylakoid membranes. PspA's and Vipp1's conserved N-terminal regions contain a putative amphipathic helix a (AHa) required for membrane binding. An adjacent amphipathic helix b (AHb) in PspA is required for imposing negative control upon PspF. Here, purified peptides derived from the putative AH regions of PspA and Vipp1 were used to directly probe their effector and regulatory functions. We observed direct membrane-binding of AHa derived peptides and an accompanying change in secondary structure from unstructured to alpha-helical establishing them as bona fide membrane-sensing AH's. The peptide-binding specificities and their effects on membrane stability depend on membrane anionic lipid content and stored curvature elastic stress, in agreement with full length PspA and Vipp1 protein functionalities. AHb of PspA inhibited the ATPase activity of PspF demonstrating its direct regulatory role. These findings provide new insight into the membrane binding and function of PspA and Vipp1 and establish that synthetic peptides can be used to probe the structure-function of the IM30 protein family.

Mechanical Characterization of Ultralow Interfacial Tension Oil-in-Water Droplets by Thermal Capillary Wave Analysis in a Microfluidic Device.

Measurements of the ultralow interfacial tension and surfactant film bending rigidity for micron-sized heptane droplets in bis(2-ethylhexyl) sodium sulfosuccinate-NaCl aqueous solutions were performed in a microfluidic device through the analysis of thermally driven droplet interface fluctuations. The Fourier spectrum of the stochastic droplet interface displacement was measured through bright-field video microscopy and a contour analysis technique. The droplet interfacial tension, together with the surfactant film bending rigidity, was obtained by fitting the experimental results to the prediction of a capillary wave model. Compared to existing methods for ultralow interfacial tension measurements, this contactless, nondestructive, all-optical approach has several advantages, such as fast measurement, easy implementation, cost-effectiveness, reduced amount of liquids, and integration into lab-on-a-chip devices.

Microfluidic SAXS Study of Lamellar and Multilamellar Vesicle Phases of Linear Sodium Alkylbenzenesulfonate Surfactant with Intrinsic Isomeric Distribution.

The structure and flow behavior of a concentrated aqueous solution (45 wt %) of the ubiquitous linear sodium alkylbenzenesulfonate (NaLAS) surfactant is investigated by microfluidic small-angle X-ray scattering (SAXS) at 70 °C. NaLAS is an intrinsically complex mixture of over 20 surfactant molecules, presenting coexisting micellar (L1) and lamellar (Lα) phases. Novel microfluidic devices were fabricated to ensure pressure and thermal resistance, ability to handle viscous fluids, and low SAXS background. Polarized light optical microscopy showed that the NaLAS solution exhibits wall slip in microchannels, with velocity profiles approaching plug flow. Microfluidic SAXS demonstrated the structural spatial heterogeneity of the system with a characteristic length scale of 50 nL. Using a statistical flow-SAXS analysis, we identified the micellar phase and multiple coexisting lamellar phases with a continuous distribution of d spacings between 37.5 and 39.5 Å. Additionally, we showed that the orientation of NaLAS lamellar phases is strongly affected by a single microfluidic constriction. The bilayers align parallel to the velocity field upon entering a constriction and perpendicular to it upon exiting. On the other hand, multilamellar vesicle phases are not affected under the same flow conditions. Our results demonstrate that despite the compositional complexity inherent to NaLAS, microfluidic SAXS can rigorously elucidate its structure and flow response.

Optically assembled droplet interface bilayer (OptiDIB) networks from cell-sized microdroplets.

We report a new platform technology to systematically assemble droplet interface bilayer (DIB) networks in user-defined 3D architectures from cell-sized droplets using optical tweezers. Our OptiDIB platform is the first demonstration of optical trapping to precisely construct 3D DIB networks, paving the way for the development of a new generation of modular bio-systems.

Light-activated control of protein channel assembly mediated by membrane mechanics.

Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

Transcription Regulation and Membrane Stress Management in Enterobacterial Pathogens.

Transcription regulation in a temporal and conditional manner underpins the lifecycle of enterobacterial pathogens. Upon exposure to a wide array of environmental cues, these pathogens modulate their gene expression via the RNA polymerase and associated sigma factors. Different sigma factors, either involved in general 'house-keeping' or specific responses, guide the RNA polymerase to their cognate promoter DNAs. The major alternative sigma54 factor when activated helps pathogens manage stresses and proliferate in their ecological niches. In this chapter, we review the function and regulation of the sigma54-dependent Phage shock protein (Psp) system-a major stress response when Gram-negative pathogens encounter damages to their inner membranes. We discuss the recent development on mechanisms of gene regulation, signal transduction and stress mitigation in light of different biophysical and biochemical approaches.

Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays.

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 µm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Buffer-induced swelling and vesicle budding in binary lipid mixtures of dioleoylphosphatidylcholine:dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine:lysophosphatidylcholine using small-angle X-ray scattering and ³¹P static NMR.

A large variety of data exists on lipid phase behavior; however, it is mostly in nonbuffered systems over nonbiological temperature ranges. We present biophysical data on lipid mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and lysophosphatidylcholine (LysoPC) examining their behaviors in excess water and buffer systems over the temperature range 4-34 °C. These mixtures are commonly used to investigate the effects of spontaneous curvature on integral membrane proteins. Using small-angle X-ray scattering (SAXS) and (31)P NMR, we observed lamellar and vesicle phases, with the buffer causing an increase in the layer spacing. Increasing amounts of DOPE in a DOPC bilayer decreased the layer spacing of the mesophase, while the opposite trend was observed for increasing amounts of LysoPC. (31)P static NMR was used to analyze the DOPC:LysoPC samples to investigate the vesicle sizes present, with evidence of vesicle budding observed at LysoPC concentrations above 30 mol %. NMR line shapes were fitted using an adapted program accounting for the distortion of the lipids within the magnetic field. The distortion of the vesicle, because of magnetic susceptibility, varied with LysoPC content, and a discontinuity was found in both the water and buffer samples. Generally, the distortion increased with LysoPC content; however, at a ratio of DOPC:LysoPC 60:40, the sample showed a level of distortion of the vesicle similar to that of pure DOPC. This implies an increased flexibility in the membrane at this point. Commonly, the assumption is that for increasing LysoPC concentration there is a reduction in membrane tension, implying that estimations of membrane tension based on spontaneous curvature assumptions may not be accurate.

Pressure-temperature phase behavior of mixtures of natural sphingomyelin and ceramide extracts.

Ceramides are a group of sphingolipids that act as highly important signaling molecules in a variety of cellular processes including differentiation and apoptosis. The predominant in vivo synthetic pathway for ceramide formation is via sphingomyelinase catalyzed hydrolysis of sphingomyelin. The biochemistry of this essential pathway has been studied in detail; however, there is currently a lack of information on the structural behavior of sphingomyelin- and ceramide-rich model membrane systems, which is essential for developing a bottom-up understanding of ceramide signaling and platform formation. We have studied the lyotropic phase behavior of sphingomyelin-ceramide mixtures in excess water as a function of temperature (30-70 °C) and pressure (1-200 MPa) by small- and wide-angle X-ray scattering. At low ceramide concentrations the mixtures form the ripple gel phase (P(ß)') below the gel transition temperature for sphingomyelin, and this observation has been confirmed by atomic force microscopy. Formation of the ripple gel phase can also be induced at higher temperatures via the application of hydrostatic pressure. At high ceramide concentration an inverse hexagonal phase (HII) is formed coexisting with a cubic phase.

Structural studies of the lamellar to bicontinuous gyroid cubic (Q(G)(II)) phase transitions under limited hydration conditions.

Non-equilibrium pathways of lyotropic phase transitions such as the lamellar to inverse bicontinuous cubic phase transition are important dynamical processes resembling cellular fusion and fission processes which can be exploited in biotechnological processes such as drug delivery. However, utilising and optimising these structural transformations for applications require a detailed understanding of the energetic pathways which drive the phase transition. We have used the high pressure X-ray diffraction technique to probe the lamellar to Q(G)(II) phase transition in limited hydration monolinolein on the millisecond time scale. Our results show that the phase transition goes via a structural intermediate and once the Q(G)(II) phase initially forms the elastic energy in the bilayer drives this structure to its equilibrium lattice parameter.

Electrostatic swelling of bicontinuous cubic lipid phases.

Lipid bicontinuous cubic phases have attracted enormous interest as bio-compatible scaffolds for use in a wide range of applications including membrane protein crystallisation, drug delivery and biosensing. One of the major bottlenecks that has hindered exploitation of these structures is an inability to create targeted highly swollen bicontinuous cubic structures with large and tunable pore sizes. In contrast, cubic structures found in vivo have periodicities approaching the micron scale. We have been able to engineer and control highly swollen bicontinuous cubic phases of spacegroup Im3m containing only lipids by (a) increasing the bilayer stiffness by adding cholesterol and (b) inducing electrostatic repulsion across the water channels by addition of anionic lipids to monoolein. By controlling the composition of the ternary mixtures we have been able to achieve lattice parameters up to 470 Å, which is 5 times that observed in pure monoolein and nearly twice the size of any lipidic cubic phase reported previously. These lattice parameters significantly exceed the predicted maximum swelling for bicontinuous cubic lipid structures, which suggest that thermal fluctuations should destroy such phases for lattice parameters larger than 300 Å.

Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity.

In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.