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Layer-by-layer cell membrane assembly - PubMed

Layer-by-layer cell membrane assembly

Sandro Matosevic et al. Nat Chem. 2013 Nov.

Abstract

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

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Figures

Figure 1
Figure 1. Layer-by-layer assembly schematic

a, Capture cups each retain a single droplet that contains aqueous cytoplasmic material (AQcy, blue) in a mixture of oil-lipid 1 (yellow). A monolayer of lipid 1 stabilizes the droplets. b, Oil-lipid 2 (green) mixture replaces oil-lipid 1. c, Extracellular aqueous phase (AQex, blue) replaces oil-lipid 2. As the AQex/oil interface traverses the array, it envelopes each trapped droplet and lipids in the interfacial monolayer composed of oil-lipid 2 deposit on the trapped droplets. d, Complete exterior phase exchange transforms the droplets into unilamellar vesicles. e, Exchanging AQex with oil-lipid 3 deposits a third lipid leaflet onto the unilamellar vesicle to produce a triple monolayer droplet. f–g, Introducing oil-lipid 4 followed by AQex deposits a final fourth leaflet of lipids. h, The final product after three phase exchanges is a double bilayer vesicle.

Figure 2
Figure 2. Vesicle assembly circuit schematic and imaging

Three fluidic inputs deliver extracellular aqueous medium (AQex,TAE buffer), oil-lipid mixture (DOPC in squalene, CMC = 2 mM), and cytoplasmic aqueous medium (AQcy,TAE buffer) to the circuit and one output removes fluids to waste. The capture array chamber is 1.2 mm wide. Each 120-μm-diameter cup contains a 30-μm-wide drain. All other channels are 120 μm wide. Channel depth is 50 μm. DOPC-stabilized water-in-oil droplets emerge from the flow-focusing junction (green, scale = 100 μm), travel through a delay line (violet, scale = 100 μm) and arrive at the droplet capture chamber (red, scale = 100 μm) where an array of cups captures single droplets. Driving a phase boundary over the trapped droplets deposits an external monolayer of lipids, transforming the water-in-oil droplets into vesicles (dashed red, scale = 100 μm). High-resolution DIC imaging of a trapped droplet displays strong DIC between the internal aqueous and external oil phase (black left, scale = 20 μm), but after a single monolayer deposition, the refractive indices of the external and internal aqueous phase contrast weakly (black center, scale = 20 μm). High-resolution confocal fluorescence imaging of the same vesicle labeled with fluorescent lipid 2 yielded clean annular sections (black right, scale = 20 μm).

Figure 3
Figure 3. Membrane protein synthesis and function in LbL vesicles

a, Symmetric vesicles were assembled with DOPC (1), loaded with fluorescein/10-kDa dextran size exclusion markers, perfused with purified hemolysin and intravesicular fluorescence monitored for multiple vesicles (n=7). Time-dependent loss of fluorescein fluorescence (green) and invariance in dextran fluorescence (blue) signified selective membrane permeabilization toward fluorescein, confirming that the product membrane is unilamellar and that the membrane protein is functionally reconstituted. b, Vesicles loaded with DNA encoding the hemolysin gene, RNA transcription components, protein translation components, and fluorescein/10-kDa dextran size exclusion markers were incubated to allow in vitro transcription/translation (IVTT) of hemolysin monomers, membrane incorporation of monomers, pore complex assembly and function. Pore function was followed as in a.

Figure 4
Figure 4. Double bilayer assembly intermediate and final product chemical probing

Fluorescent reporter lipids used for chemical probing included N-NBD-labeled dioleolylphosphatidylethanolamine (2) and 12-NBD-dodecyl-labeled oleoylphosphatidylcholine (3). Stars (✹) indicate the position of the NBD fluorophore within the reporter lipid structures. a, Treating unilamellar vesicles assembled with 2 in either the cytoplasmic L1 leaflet (black) or the extracellular L2 leaflet (red) with membrane-impermeable reductive quenching agent S2O42- (▼) selectively quenches the membrane fluorescence of L2-labeled vesicles (n = 3). b, Treating triple monolayer water-in-oil droplets assembled with 3 in either the internal leaflet L2 (black) or the oil-exposed leaflet L3 (red) with the oil-soluble collisional quenching agent Co(II) (▼) selectively quenches the membrane fluorescence of L3-labeled vesicles (n = 3). c, Treating double bilayer vesicles assembled with 2 in either the penultimate leaflet L3 (black) or the extracellular leaflet L4 (red) with S2O42- (▼) selectively quenches the membrane fluorescence of L4-labeled vesicles (n = 3). Insets schematize the proposed assembly intermediates and products, leaflet localization of reporter lipids, and location of NBD fluorophore.

Figure 5
Figure 5. Quantitative layer-by-layer deposition

Layer-by-layer deposition of lipid monolayers containing 1 mol% 2 yielded vesicles with membrane fluorescence that varied in agreement with predicted models of deposition-dependent fluorescence. Membrane fluorescence normalized to the intensity of structures generated after 3 monolayer depositions (4 putative monolayers) and plotted as a function of the number of depositions increases linearly (left axis, red circles and trend line; slope = 0.247 ± .007, R2 = 0.998, 5 experiments). Membrane fluorescence after quenching the external leaflet and normalizing to the intensity of the unquenched construct increases asymptotically, proportional to the inverse of the number of depositions (right axis, open gray circles and trend line, X intercept = 0.97 ± .05, R2 = 0.995, 6 experiments). X axis insets schematize the LbL assembly intermediates en route to a double bilayer membrane with equimolar 2 in each monolayer.

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