Accelerating SNARE‐Mediated Membrane Fusion by DNA–Lipid Tethers

SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid‐conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA–lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base‐pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE‐mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA–lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.     

Lateral mobility of tethered vesicle-DNA assemblies

Supported lipid membranes are particularly attractive for use in biochemical assays because of their resistance to nonspecific adsorption and their unique ability to host transmembrane proteins. Although ideal for use in many surface-based detection techniques, supported bilayers can make the incorporation of proteins problematic due to the steric constraints of the underlying substrate. A recently developed strategy overcomes this obstacle by tethering liposomes to supported lipid bilayers via cholesterol-tagged DNA. Due to the fluidity of the bilayer, the vesicle assemblies exhibited significant lateral mobility. The corresponding diffusion coefficients were then investigated using fluorescence recovery after photobleaching (FRAP). The diffusivity was neither sensitive to the size of the vesicles nor to the length of the DNA tether. However, changing from single cholesterol tethers to double cholesterol tethers caused a decrease in the diffusivity of the assemblies by a factor of 3. Perhaps even more notable was the fact that single cholesterol-DNA without vesicles diffused 6 times faster than the corresponding assemblies. Double cholesterol-DNA diffused 11 times faster. This discrepancy is believed to arise from the fact that each vesicle is tethered to the bilayer by multiple DNA pairs.     

A DNA‐Programmed Liposome Fusion Cascade

Chemically engineered and functionalized nanoscale compartments are used in bottom‐up synthetic biology to construct compartmentalized chemical processes. Progressively more complex designs demand spatial and temporal control over entrapped species. Here, we address this demand with a DNA‐encoded design for the successive fusion of multiple liposome populations. Three individual stages of fusion are induced by orthogonally hybridizing sets of membrane‐anchored oligonucleotides. Each fusion event leads to efficient content mixing and transfer of the recognition unit for the subsequent stage. In contrast to fusion‐protein‐dependent eukaryotic vesicle processing, this artificial fusion cascade exploits the versatile encoding potential of DNA hybridization and is generally applicable to small and giant unilamellar vesicles. This platform could thus enable numerous applications in artificial cellular systems and liposome‐based synthetic pathways.     

Supported Membranes with Well‐Defined Polymer Tethers—Incorporation of Cell Receptors

We report the design of supported lipid membranes attached to the surface by tailored lipopolymer tethers. A series of well‐defined lipopolymers were synthesized by means of living cationic polymerization of 2‐methyl‐2‐oxazolines. The polymers were equipped with a silane coupling group on the proximal, and lipid anchors on the distal chain ends. The length of the intermediate hydrophilic polymer tether was varied (n=14, 18, 33) to change the distance between the membrane and the substrate. Supported membranes have been prepared in two‐steps. First, a suitable lipopolymer/lipid mixture was deposited by Langmuir–Blodgett transfer, and annealed to establish the covalent coupling to the surface. On the dry lipopolymer/lipid monolayer, the upper leaflet was deposited by vesicle fusion. Optimization of both preparation steps resulted in the formation of stable and defect‐free membranes. Impacts of the spacer length and the lipopolymer fraction upon the lateral diffusivity of the lipids were systematically compared by fluorescence recovery after photobleaching (FRAP). First experiments on the incorporation of a large transmembrane cell receptor (integrin α_IIbβ₃) into the polymer‐tethered membrane suggested that the length of the polymer tether plays a crucial role in distribution of the proteins on the surface.     

Directed aggregation and fusion of lipid vesicles induced by DNA-surfactants

We investigated DNA-directed aggregation of vesicles using DNA-surfactants. Following tethering of single-stranded DNA oligonucleotides to vesicles using DNA-surfactant, the tethered vesicles were assembled with other vesicles bearing complementary strands. The vesicle aggregation was strongly affected by the salt concentration and by temperature according to the characteristics of DNA hybridization. Restriction enzyme, which can hydrolyze the double-stranded DNA used in the present study, dissociated the vesicle aggregates. Exploration using fluorescently labeled vesicles suggested that the DNA-directed vesicle aggregation took place in a sequence-specific manner through DNA-duplex formation. Interestingly, the DNA-directed aggregation using short DNA-surfactant induced the fusion of vesicles to produce giant vesicles, resulting in an enzymatic reaction in the giant vesicle.     

Controlled and Tunable Loading and Release of Vesicles by Using Gigahertz Acoustics

Controllable exchange of molecules between the interior and the external environment of vesicles is critical in drug delivery and micro/nano‐reactors. While many approaches exist to trigger release from vesicles, controlled loading remains a challenge. Herein, we show that gigahertz acoustic streaming generated by a nanoelectromechanical resonator can control the loading and release of cargo into and from vesicles. Polymer‐shelled vesicles showed loading and release of molecules both in solution and on a solid substrate. We observed deformation of individual giant unilamellar vesicles and propose that the shear stress generated by gigahertz acoustic streaming induces the formation of transient nanopores, with diameters on the order of 100 nm, in the vesicle membranes. This provides a non‐invasive method to control material exchange across membranes of different types of vesicles, which could allow site‐specific release of therapeutics and controlled loading into cells, as well as tunable microreactors.     

Diffusive dynamics of vesicles tethered to a fluid supported bilayer by single-particle tracking

We recently introduced a method to tether intact phospholipid vesicles onto a fluid supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). Once tethered, the vesicles can diffuse in two dimensions parallel to the supported membrane surface. The average diffusion coefficient, D, is typically 0.2 microm(2)/s; this is 3-5 times smaller than for individual lipid or DNA-lipid conjugate diffusion in supported bilayers. In this article, we investigate the origin of this difference in the diffusive dynamics of tethered vesicles by single-particle tracking under collision-free conditions. D is insensitive to tethered vesicle size from 30 to 200 nm, as well as a 3-fold change in the viscosity of the bulk medium. The addition of macromolecules such as poly(ethylene glycol) reversibly stops the motion of tethered vesicles without causing the exchange of lipids between the tethered vesicle and supported bilayer. This is explained as a depletion effect at the interface between tethered vesicles and the supported bilayer. Ca ions lead to transient vesicle-vesicle interactions when tethered vesicles contain negatively charged lipids, and vesicle diffusion is greatly reduced upon Ca ion addition when negatively charged lipids are present both in the supported bilayer and tethered vesicles. Both effects are interesting in their own right, and they also suggest that tethered vesicle-supported bilayer interactions are possible; this may be the origin of the reduction in D for tethered vesicles. In addition, the effects of surface defects that reversibly trap diffusing vesicles are modeled by Monte Carlo simulations. This shows that a significant reduction in D can be observed while maintaining normal diffusion behavior on the time scale of our experiments.     

Construction of macroscopic cytomimetic vesicle aggregates based on click chemistry: controllable vesicle fusion and phase separation

Vesicle-vesicle aggregation to mimic cell-cell aggregation has attracted much attention. Here, hyperbranched polymer vesicles (branched-polymersomes, BPs) with a cell-like size were selected as model membranes, and the vesicle aggregation process, triggered by click chemistry of the copper-catalysed azide-alkyne cycloaddition reaction, was systematically studied. For this purpose, azide and alkynyl groups were loaded on the membranes of BPs through the co-assembly method to obtain N(3)-BPs and Alk-BPs, respectively. Subsequently, macroscopic vesicle aggregates were obtained when these two kinds of functional BPs were mixed together with the ratio of azide to alkynyl groups of about 1:1. Both the vesicle fusion events and lateral phase separation on the vesicle membrane occurred during such a vesicle aggregation process, and the fusion rate and phase-separation degree could be controlled by adjusting the clickable group content. The vesicle aggregation process with N(3) -micelles as desmosome mimics to connect with Alk-BPs through click-chemistry reaction was also studied, and large-scale vesicle aggregates without vesicle fusion were obtained in this process. The present work has extended the controllable cytomimetic vesicle aggregation process with the use of covalent bonds, instead of noncovalent bonds, as the driving force.     

Determinants for membrane fusion induced by cholesterol-modified DNA zippers

Intracellular membrane fusion is coordinated by membrane-anchored fusion proteins. The cytosolic domains of these proteins form a specific complex that pulls the membranes into close proximity. Although some results indicate that membrane merger can be accomplished solely on the basis of proximity, others emphasize the importance of bilayer stress exerted by transmembrane peptides. In a reductionist approach, we recently introduced a fusion machinery built from cholesterol-modified DNA zippers to mimic fusion protein function. Aiming to further optimize DNA-mediated fusion, we varied in this work length and number of DNA strands and used either one or two cholesterol groups for membrane anchoring of DNA. The results reveal that the use of two cholesterol anchors is essential to prevent cDNA strands from shuttling to the same membrane, which leads to vesicle release instead of membrane merger. A surface coverage of 6-13 DNA strands was a precondition for efficient fusion, whereas fusion was insensitive to DNA length within the tested range. Besides lipid mixing, we also demonstrate DNA-induced content mixing of large unilamellar vesicles composed of the most abundant cellular lipids phosphatidylcholine, phosphatidylethanolamine, cholesterol, and sphingomyelin. Taken together, DNA-mediated fusion emerges as a promising tool for the functionalization of artificial and biological membranes and may help to dissect the functional role of fusion proteins.     

Experimental evidence to support a theory of lipid membrane fusion

Membrane fusion between two lipid membranes with different curvatures was measured by using a fluorescence fusion assay for lipid vesicle systems and was also obtained by measuring lipid monolayer surface tension upon the fusion of vesicles to monolayer membranes. For such membrane systems, it was found that when lysolipid was incorporated only in the membrane with a greater curvature, membrane fusion was more suppressed than those for the case where the same amount (molar ratio of lysolipid to non-lysolipids) of lysolipid was incorporated only in the membrane with a lower curvature. When lysolipid was incorporated only in a flat membrane (e.g., monolayer) and the fusion of small vesicles (SUV) to the monolayer was measured, suppression of membrane fusion by lysolipid was minimal. It is known that lysolipid lowers the surface energy of curved membranes, which stabilizes energetically such membrane surfaces, and thus suppresses membrane fusion. Our results support our theory of lipid membrane fusion where the membrane fusion occurs through the most curved membrane region at the contact area of two interacting membranes.     

Vesicle Tubulation with Self‐Assembling DNA Nanosprings

A major goal of nanotechnology and bioengineering is to build artificial nanomachines capable of generating specific membrane curvatures on demand. Inspired by natural membrane‐deforming proteins, we designed DNA‐origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA‐coated membrane tubules emerge from spherical vesicles when DNA‐origami polymerization or high membrane‐surface coverage occurs. Unlike many previous methods, the DNA self‐assembly‐mediated membrane tubulation eliminates the need for detergents or top‐down manipulation. The DNA‐origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphology, underscoring the intricate interplay between lipid bilayers and vesicle‐deforming DNA structures.     

Controlling two-dimensional tethered vesicle motion using an electric field: interplay of electrophoresis and electro-osmosis

We recently introduced methods to tether phospholipid vesicles or proteoliposomes onto a fluid-supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L.; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). These intact tethered vesicles diffuse in two dimensions parallel to the supporting membrane surface. In this article, we report the dynamic response of individual tethered vesicles to an electric field applied parallel to the bilayer surface. Vesicles respond to the field by moving in the direction of electro-osmotic flow, and this can be used to reversibly concentrate tethered vesicles against a barrier. By adding increasing amounts of negatively charged phosphatidylserine to the supporting bilayer to increase electro-osmosis, the electrophoretic mobility of the tethered vesicles can be increased. The electro-osmotic contribution can be modeled well by a sphere connected to a cylindrical anchor in a viscous membrane with charged headgroups. The electrophoretic force on the negatively charged tethered vesicles opposes the electro-osmotic force. By increasing the amount of negative charge on the tethered vesicle, drift in the direction of electro-osmotic flow can be slowed; at high negative charge on the tethered vesicle, motion can be forced in the direction of electrophoresis. The balance between these forces can be visualized on a patterned supporting bilayer containing negatively charged lipids that reorganize in an externally applied electric field to create a gradient of charge within a corralled region. The charge gradient at the surface creates a gradient of electro-osmotic flow, and vesicles carrying similar amounts of negative charge can be focused to a region perpendicular to the applied field where electrophoresis is balanced by electro-osmosis, away from the corral boundary. Electric fields are effective tools to direct tethered vesicles and concentrate them and to measure the tethered vesicle's electrostatic properties.     

Real-time membrane fusion of giant polymer vesicles

Membrane fusion is very important for the formation of many complex organs in metazoans throughout evolution, such as muscles, bones, and placentae. Lipid vesicles (liposomes) are frequently used as model membranes to study the fusion process. This work demonstrates for the first time the real-time membrane fusion of giant polymer vesicles by directly displaying a series of high-resolution and real-time transformation images of individual vesicles. The fusion process includes the sequential steps of membrane contact, forming the center wall, symmetric expansion of fusion pore and complete fusion, undergoing the intermediates of "8" shape with a protruding rim at the contact site, peanut (pear) shape, and oblate sphere. The vesicle swells during fusion, and the fusing vesicle only deforms in the neck domain around the fusion pore in the lateral direction, which verifies the importance of the lateral tension on the fusion pore at the vesicle deformation level. The successful fusion of the synthetic and protein-free polymer vesicles reported here also supports that vesicle proximity combined with membrane perturbation suffices to induce membrane fusion, and that the protein is not necessary for the fusion process.     

Differences in permeability of dioleoylphosphatidylcholine model membranes containing saturated or unsaturated phosphatidic acids

At low millimolar Ca²⁺ concentrations, large unilamellar dioleoylphosphatidylcholine vesicles containing dimyristoylphosphatidate (20 mol%) release enclosed solutes like sulphate, but influx of Ca²⁺ is not demonstrable. Coincident with the permeability change, vesicle aggregation and membrane fusion are observed. These results contrast with those for dioleoylphosphatidate-containing vesicles under the same conditions, which show Ca²⁺ influx and Ca²⁺ chelator efflux, but no sulphate efflux, vesicle aggregation or membrane fusion. The observed differences in permeability behaviour of membranes containing these two phosphatidate molecular species are discussed with respect to the differences in their phase behaviour.     

Solid-supported biomimetic membranes with tailored lipopolymer tethers

Stable lipid membranes with controlled substrate-membrane spacing can be prepared using well-defined lipopolymers as a tether. Based on the living cationic ring-opening polymerization of 2-methyl- or 2-ethyl-2-oxazoline, lipopolymers can be synthesized bearing a lipid head group as well as a silanol reactive coupling end group. Using a “grafting onto” procedure these polymers can form dense, brush like monolayers, whose layered structures can be obtained by x-ray reflectivity measurements. By transfer of a pre-organized monolayer that is followed by vesicle fusion, stable polymer supported lipid membranes can be prepared. The substrate-membrane spacing can be controlled via the degree of polymerization, while the lateral diffusion of lipids within the membrane depends on the density of polymer tethers. Preliminary experiments implied that the membrane with long (N = 40) polymer tethers could reside trans-membrane receptors homogeneously, suggesting a large potential of this strategy.     

Optical measurement of the deformation of giant lipid vesicles driven by a micropipet electrode

We investigate the deformation of giant lipid vesicles driven by a micropipet electrode by use of differential confocal microscopy. This optical technique provides nanometer depth resolution without mechanical contact and hence prevents large tension or perforation of the soft membrane. For dipalmitoyl phosphatidylcholine (DPPC) membranes in the gel phase, we observed deformations of several hundreds of nanometers when the driving voltage was about 0.1 V. The voltage and frequency responses of the vesicle deformation can be explained by the balance between the electroosmotic force inside the micropipet and the membrane tension. We also used DPPC:cholesterol vesicles to check the validity of this model. In the fluid phase, however, the deformation is independent of the modulation signal because micrometer-scale thermal fluctuations dominate the membrane motion.     

Surface response methodology for the study of supported membrane formation

We report on the investigations of the formation of the tethered lipid bilayer by vesicle deposition on amine-functionalized surfaces. The tethered bilayer was created by the deposition of egg-PC vesicles containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-(ethyleneglycol)-N-hydroxysuccinimide as anchoring molecules on an amine-coated surface. This approach is an easy route for the formation of a biomimetic-supported membrane. A Doelhert experimental design was applied to determine the conditions leading to the formation of a continuous and defect-free tethered bilayer on different surfaces (gold and glass). Doehlert designs allow modeling of the experimental responses by second-order polynomial equations as a function of experimental factors. Four factors expected to influence bilayer formation were studied: the lipid concentration in the vesicle suspension, the mass percentage of anchoring molecules in the vesicles, the contact time between the vesicles and the surface, and the resting time of the membrane after buffer rinse. The optimization of the membrane preparation parameters was achieved by monitoring lipid assembly formation using surface plasmon resonance spectroscopy on gold and by fluorescence recovery after photobleaching on glass. Three characteristic responses were systematically measured: the bilayer thickness, the lipid diffusion coefficient, and the lipid mobile fraction. The simultaneous inspection of the three characteristics revealed that a restricted experimental domain leads to properties that are in accordance with a bilayer presence. The factors of this domain are a lipid concentration from 0.1 to 1 mg/mL, 4-8% of anchoring molecules in the vesicles, 1-4 h of contact time between vesicles and surface, and 21-24 h of resting time after buffer rinse. Under these conditions, a membrane having a lipid mass per surface between 545 +/- 5 and 590 +/- 10 ng/cm2, a diffusion coefficient of between 2.5 +/- 0.3 x 10(-8) and 3.60 +/- 0.5 x 10(-8) cm2/s, and a mobile fraction between 94 +/- 2 and 99 +/- 1% was formed. These findings were confirmed by atomic force microscopy observations, which showed the presence of a continuous and homogeneous bilayer in the determined experimental domain. This formation procedure presents many advantages; it provides an easily obtainable biomimetic membrane model for proteins studies and offers a versatile tethered bilayer because it can be adapted easily to various types of supports.     

Coarse-grained transmembrane proteins: hydrophobic matching, aggregation, and their effect on fusion

Molecular transport between organelles is predominantly governed by vesicle fission and fusion. Unlike experimental vesicles, the fused vesicles in molecular dynamics simulations do not become spherical readily, because the lipid and water distribution is inappropriate for the fused state and spontaneous amendment is slow. Here, we study the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles. This is done by adding several kinds of model proteins to fusing vesicles. The results show that equilibration of both water and lipid content is a requirement for spherical vesicles. In addition, the effect of these transmembrane proteins is studied in bilayers and vesicles, including investigations into hydrophobic matching and aggregation. Our simulations show that the level of aggregation does not only depend on hydrophobic mismatch, but also on protein shape. Additionally, one of the proteins promotes fusion by inducing pore formation. Incorporation of these proteins allows even flat membranes to fuse spontaneously. Moreover, we encountered a novel spontaneous vesicle enlargement mechanism we call the engulfing lobe, which may explain how lipids added to a vesicle solution are quickly incorporated into the inner monolayer.     

Incorporation of Na(+),K(+)-ATP-ase into the thiolipid biomimetic assemblies via the fusion of proteoliposomes

The black lipid membranes (BLMs) are artificial membrane systems that have been widely used in the study of different biological processes. In this paper the planar bilayer lipid membranes have been used to study the behavior of thiolipid molecules-dipalmitoyl-phosphatidyl-ethanolamine-mercaptopropionamide (DPPE-MPA) and cholesteryl 3-mercaptopropionate (Chs-MPA)-as compared to classical BLM made of natural lipids. We present our experiments on black thiolipid bilayer (BTM) formation from a thiolipid solution and basic results of pump currents generated by sodium-potassium pump-Na(+),K(+)-ATP-ase-introduced to such bilayer systems via proteoliposome adsorption with subsequent fusion. Our results imply that no substantial difference exists between BLMs formed from classical lipids and those made from thiolipids used in this study. The same thiolipid molecules were subsequently used for the formation of covalently bound, tethered bilayer lipid membranes (t-BLMs) on polycrystalline gold electrodes. Similarly, as in the case of BLMs, we took advantage of proteoliposome adsorption/fusion to obtain a t-BLM system with reconstituted enzyme. The vesicle fusion on hydrophobic or hydrophilic substrates is one of the main ways to obtain a bilayer system with incorporated biological species. In this paper we present also our preliminary results of electrochemical experiments using rapid solution exchange technique on such t-BLMs systems and their comparison with painted solid supported membranes (SSMs) and BLMs. We have also followed the process of vesicles fusion onto thiolipid monolayer by means of in situ atomic force microscopy in tapping mode (TM-AFM). On the basis of these experiments, we conclude that DPPE-MPA and Chs-MPA molecules used in our experiments preserve lipid properties, allowing for at least partial reconstitution of Na(+),K(+)-ATP-ase into such t-BLMs. On the other hand, the relatively compact organization on polycrystalline gold and the hydrophobic nature of the first monolayer of tethered thiolipids slows down the proteoliposome fusion onto such monolayers and consequently hinders the protein insertion. However, this effect can be overcome by mechanical stimulus that facilitates proteoliposome delamination onto the self-assembled monolayer.     

Programmed vesicle fusion triggers gene expression

The membrane properties of phospholipid vesicles can be manipulated to both regulate and initiate encapsulated biochemical reactions and networks. We present evidence for the inhibition and activation of reactions encapsulated in vesicles by the exogenous addition of charged amphiphiles. While the incorporation of cationic amphiphile exerts an inhibitory effect, complementation of additional anionic amphiphiles revitalize the reaction. We demonstrated both the simple hydrolysis reaction of β-glucuronidase and the in vitro gene expression of this enzyme from a DNA template. Furthermore, we show that two vesicle populations decorated separately with positive and negative amphiphiles can fuse selectively to supply feeding components to initiate encapsulated reactions. This mechanism could be one of the rudimentary but effective means to regulate and maintain metabolism in dynamic artificial cell models.