Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

Journal name:
Nature Chemistry
Volume:
8,
Pages:
881–889
Year published:
DOI:
doi:10.1038/nchem.2537
Received
Accepted
Published online
Corrected online

Abstract

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid–lipid and lipid–membrane protein interactions involved in the regulation of cellular functions.

At a glance

Figures

  1. Schematic representation of asymmetric GV formation from a planar lipid bilayer using pulsed microfluidic jet flow.
    Figure 1: Schematic representation of asymmetric GV formation from a planar lipid bilayer using pulsed microfluidic jet flow.

    (1) Formation of a lipid microtube from the planar lipid bilayer by the jet-flow method, (2) deformation of a sinusoidal undulation on the lipid microtube and (3) generation of vesicles from the unstable break-up of the deformed lipid microtube. The generated vesicles were categorized mainly into two sizes, with diameters of approximately 100–200 µm and 3–20 µm. The two differently sized vesicles were presumably generated based on a Rayleigh--Plateau instability of the tubular jet. We observed lipid–lipid interaction (lipid flip-flop) and lipid–membrane protein interaction (reconstitution of membrane proteins) on the asymmetric GVs.

  2. Formation of GVs via inhomogeneous break-up of a lipid microtube by a jet flow.
    Figure 2: Formation of GVs via inhomogeneous break-up of a lipid microtube by a jet flow.

    a, Structures of DOPC (1) and DPPC (2). b, A photo of vesicles formed by our method (hole size, 200 µm in diameter; pressure (P), 300 kPa; application time (t), 2 ms). Scale bar, 10 µm. c, Typical CLSM fluorescence images of vesicles that contain calcein formed from a planar lipid bilayer using pulsed jet flow after ten minutes and after 24 h (we observed a new GV at each time point). Green fluorescence indicates calcein encapsulated in the GV and red fluorescence indicates rhodamine labelled with a lipid molecule. The size distribution of the vesicles was determined after 10 min (n = 80) and after 24 h (n = 87) (hole size, 200 µm in diameter; P, 300 kPa; t, 4 ms). Scale bar, 5 μm. d, Size-controlled formation of the vesicles using a separator hole with different sizes (100, 200 and 500 µm) and a different application time (2, 4 and 6 ms). Average values were obtained by measuring over 70 single vesicles for each hole size and application time (t, 2, 4 and 6 ms). Error bars, s.d. Ave, average.

  3. Characterization of GV membranes.
    Figure 3: Characterization of GV membranes.

    a, We measured Raman intensity ratios of four different GV samples (mean ± s.d.): (1) GVs formed by our method (hole size, 200 µm in diameter; P, 300 kPa; t, 2 ms), (2) GVs formed by gentle hydration, (3) GVs formed by jet flow (previous method)8 and (4) GVs formed by the droplet phase-transfer method6. b, The distribution of the Raman ratios obtained by evaluating ∼51 single vesicles formed by the various formation methods. **P < 0.01 compared with our method. c, Vesicles were reconstituted with αHL to investigate their unilamellarity. Typical CLSM fluorescence images at different times for vesicles that contain calcein and reconstituted with αHL after 0, 7 and 14 min (hole size, 200 µm in diameter; P, 300 kPa; t, 2 ms). Scale bar, 10 µm. The graph shows the relative fluorescence intensity inside the GVs with αHL (diamonds) or without αHL (squares) (n = 12). Error bars, s.e.m.

  4. Formation and flip-flop measurement of DOPS asymmetric GVs.
    Figure 4: Formation and flip-flop measurement of DOPS asymmetric GVs.

    Asymmetric vesicles were prepared with DOPC (1) and DOPS (3) (hole size, 200 µm in diameter; P, 300 kPa; t, 2 ms). a,b, GVs with DOPS in the outer (a) or inner (b) leaflet. PS was detected by annexin V labelled with AlexaFluor 488. CLSM images of GVs with DOPS—the green fluorescence represents annexin V (AlexaFluor 488) and the red fluorescence represents the lipid membrane (rhodamine). Scale bar, 10 µm. c,d, Fluorescence CLSM images of asymmetric GVs with DOPS in the outer (c) or inner (d) leaflet obtained after incubation at 37 °C for 0 and 12 h (we observed a new GV at each time point). PS was detected by annexin V labelled with AlexaFluor 488. Scale bar, 10 μm. e,f, DOPS flip-flop behaviour of GVs that contain DOPS in the outer (e) or inner (f) leaflet after incubation at 37 °C for 1–24 h. Average values were obtained by measuring 12–17 single GVs at each parameter. Error bars, s.e.m.

  5. Membrane dynamics of asymmetric GVs with the outer leaflet composed of pure DOPC and the inner leaflet composed of DOPS/DOPE/DOPC in the presence of cinnamycin.
    Figure 5: Membrane dynamics of asymmetric GVs with the outer leaflet composed of pure DOPC and the inner leaflet composed of DOPS/DOPE/DOPC in the presence of cinnamycin.

    a, Schematic of cinnamycin (Ro 09-0198)31 (4) (top) and structure of DOPE (5). b, Schematic structure of the stimulation of flop in DOPS/DOPE/DOPC asymmetric GVs by cinnamycin. c, CLSM images at different time points for asymmetric GVs composed of DOPS/DOPE/DOPC in the inner leaflet in the presence of cinnamycin after incubation at 37 °C for 10, 30 and 60 min (hole size, 200 µm in diameter; P, 300 kPa; t, 2 ms). The CLSM image on the right represents asymmetric GVs composed of DOPS/DOPC in the inner leaflet (without DOPE) in the presence of cinnamycin after incubation at 37 °C for 60 min. Scale bar, 10 µm. d, Time-lapse imaging of annexin V fluorescence intensity in a DOPS/DOPE/COPC asymmetric GV membrane in the presence of cinnamycin. Fluorescence intensity values (arbitrary units (a.u.)) were obtained for three areas (A, B and C) of a GV membrane. e, Flop ratio of asymmetric vesicles composed of DOPS/DOPE/DOPC or DOPS/DOPC in the presence of cinnamycin after 60 min of incubation (n = 7). Error bars, s.e.m.

  6. Reconstituted amount and function of membrane proteins into asymmetric GVs.
    Figure 6: Reconstituted amount and function of membrane proteins into asymmetric GVs.

    a, CLSM images of asymGVps-pc/pc and symGVps-pc (hole size, 200 µm in diameter; P, 300 kPa; t, 2 ms). Scale bar, 10 µm. b, Fluorescence intensity of CX43-eGFP on the GVs (n = 19–23 at each parameter). Error bars, s.e.m. **P < 0.01 compared with asymGVps-pc/pc. c, Fluorescence intensity of Cx43-eGFP on the GVs with four concentrations of DOPS in the outer leaflet (DOPC 100%, DOPS/DOPC (1:3, 1:1 and 3:1 molar ratios); n = 20–23 at each parameter). **P < 0.01 compared with asymGVps-pc/pc. d, Normalized fluorescence intensity of the leakage of rhodamine 101 from GVs (n = 11–12 at each parameter). Control represents symGVpc in the absence of DNA coding for Cx43-eGFP. Dextran represents asymGVps-pc/pc that contains rhodamine-labelled dextran (molecular weight, 4,400 g mol–1). Error bars, s.e.m. e, Example of the single-channel current recordings of Cx43-eGFP proteins (+50 mV and −25 mV). f, IV relationship of the single Cx43-eGFP pore. Error bars, s.d. g, Histogram of the amplitude of Cx43-eGFP conductance from 609 open events.

Change history

Corrected online 22 June 2016
In the version of this Article originally published, in Fig. 1 some of the lipid heads in the bottom right panel were mistakenly coloured grey and in Fig. 5b the red arrow labelling ‘flop’ in the right panel was out of place. These errors have been corrected in all versions of the Article.

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Author information

  1. Present address: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan

    • Ryuji Kawano

Affiliations

  1. Artificial Cell Membrane Systems Group, Kanagawa Academy of Science and Technology, KSP East 303, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa 213-0012, Japan

    • Koki Kamiya,
    • Ryuji Kawano,
    • Toshihisa Osaki &
    • Shoji Takeuchi
  2. Japan Science of Technology Agency, PRESTO, KSP East 303, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa 213-0012, Japan

    • Koki Kamiya
  3. Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan

    • Toshihisa Osaki &
    • Shoji Takeuchi
  4. Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University JST ERATO, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

    • Kazunari Akiyoshi

Contributions

K.K. and S.T. designed the research; K.K. performed the research and analysed data; K.K., K.A. and S.T. designed the electrophysiological measurement of a single Cx43-eGFP channel current; R.K. and T.O. contributed to the device concept and fabrication and K.K. and S.T. co-wrote the paper. All the authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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