Forming giant vesicles with controlled membrane composition, asymmetry, and contents

Abstract

Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.

Fig. 1.

GUVs with oil-insoluble lipids were formed by SUV incorporation into planar bilayers followed by microfluidic jetting. (A) A custom acrylic chamber was used to form giant vesicles by microfluidic jetting with a piezoelectric inkjet. The chamber was mounted on a microscope stage, and the inkjet device was inserted from a port in the side of the chamber. For image clarity, this chamber does not contain oil or aqueous droplets. Scale bar, 4 mm. (B) Aqueous droplets containing SUVs with oil-insoluble lipids (red) were incubated in the acrylic chamber containing oil. A thin acrylic divider separates the two aqueous droplets. (C) SUVs diffuse within the water droplet until they contact and fuse to the oil/water interface, forming a continuous lipid monolayer around each droplet. Removal of the thin acrylic divider allows the two droplets to move together and exclude oil between them. When the two lipid monolayers come into contact, they form a planar lipid bilayer. GUVs were formed by microfluidic jetting with the inkjet device that deforms the planar bilayer into a vesicle. Repeated pulsing of the inkjet results in the formation of multiple monodisperse vesicles. (D) TMR-PIP₂ was incorporated into a GUV by this method and imaged by confocal microscopy. Scale bar, 50 μm.

Fig. 2.

GUVs with asymmetric lipid composition can be formed by controlling the SUV content of each reservoir. (A) SUVs containing Ni-chelating lipids were incubated in the droplet nearest the inkjet (inner droplet). His-GFP (green star) was either encapsulated in a GUV by microfluidic jetting (Left), or added to the outer droplet (Right), and the distribution of His-GFP was observed by confocal microscopy. (B) SUVs containing Ni-chelating lipids were incubated in the droplet furthest from the inkjet (outer droplet). His-GFP was either encapsulated in a GUV by microfluidic jetting (Left), or added to the outer droplet after vesicle formation (Right). All scale bars, 50 μm.

Fig. 3.

Membrane proteins can be incorporated into GUVs with controlled orientation. (A) GFP-Syb was incorporated into GUVs and imaged by confocal microscopy. (B) SybSN-GFP (lacking the transmembrane domain) was encapsulated into (Upper) GUVs lacking tSNARE, and (Lower) GUVs containing tSNAREs. (C) (Top) Cartoons of the experimental setup. (Middle) GUVs made by microfluidic jetting of planar bilayers generated by GFP-Syb SUVs incubated in (i) the inner droplet, (ii) both droplets, or (iii) the outer droplet. (Bottom) The same GUVs were imaged again after the addition of Protease K, which degrades exposed protein, to the external medium. (D) GUVs made by incubating GFP-Syb SUVs in (i) the inner droplet had 91 ± 5% (SEM, n = 4 bilayers) GFP-Syb molecules oriented inward, (ii) both droplets had 49 ± 3% (SEM, n = 4 bilayers) GFP-Syb molecules oriented inward, and (iii) the outer droplet had 10 ± 2% (SEM, n = 6 bilayers) GFP-Syb molecules oriented inward. All scale bars, 50 μm.

Fig. 4.

Membrane mixing leads to transfer of SNARE proteins from SUVs to GUV membranes. (A) GUVs were formed with incorporated tSNAREs and encapsulated Doc2 and vSNARE-SUVs. Ca²⁺ was entrained from the aqueous droplets during the formation process. (B) Confocal image of vSNARE-SUVs encapsulated in a tSNARE-GUV (C) Docking of an SUV cluster to a GUV. Diffusion of an encapsulated SUV cluster was tracked for 10 min. The location of the SUV cluster in the first frame is denoted by “x.” The track is separated into before (red) and after (green) docking to the GUV membrane. Fluorescence intensity of the GUV membrane at the docking location (yellow box) is shown for 2 min around the docking event. (D) Membrane transfer from an SUV cluster to a GUV membrane. The path of an SUV cluster was tracked (red) for 5 min, until it contacted the GUV membrane. The location of the SUV cluster in the first frame is denoted by “x.” Fluorescence intensity of the GUV membrane at the contact location (yellow box) is shown for 2 min around the time of contact. Fluorescence decay is fit by a 2D diffusion model (R² = 0.81). (E) Transfer of GFP-Syb to the external leaflet of the GUV membrane was confirmed by addition of a fluorescently labeled antibody against GFP to the external solution. Fluorescence intensity of GUV membranes containing syntaxin + SNAP25 (tSNAREs) increased by 5922 ± 2108 a.u. (SEM, n = 6 bilayers), whereas GUV membranes containing only syntaxin, or no SNARE proteins, increased by 816 ± 193 a.u. (SEM, n = 7 bilayers) and 506 ± 419 a.u. (SEM, n = 5 bilayers), respectively. ^∗p < 0.05 (Student’s t test). All scale bars, 25 μm.