UNIFORM-SIZED PROTEOLIPOSOME FORMATION BY USING ELECTROSPRAY FOR MICROSCOPIC MEMBRANE PROTEIN ASSAYS

T. Osaki,1,2 K. Kamiya,1 R. Kawano,1 R. Iino,3,4 H. Noji,3,4 and S. Takeuchi1,2 1 Kanagawa Academy of Science and Technology, JAPAN

2 Institute of Industrial Science, The University of Tokyo, JAPAN 3 Department of Applied Chemistry, The University of Tokyo, JAPAN

4 CREST, Japan Science and Technology Agency, JAPAN ABSTRACT

This paper describes an instant formation method of uniform-sized proteoliposomes for membrane protein assays using fluorescence microscopy. The applied electrospray technique was the same as our previous works [1], although here we directly sprayed proteovesicles (<1 μm size) dispersed in “an aqueous buffer” and micropatterned them on a substrate; technical problems/conditions of the electrospray using aqueous buffers were accommodated (Fig. 2d). Hydration of the protein/lipid micropattern instantly produced a uniform-sized proteoliposome array. Moreover, the assay results indicated that the target protein FoF1 was integrated on the liposomal membrane and its transport function may be preserved after the rehydration. KEYWORDS: Liposome, Membrane Protein, FoF1 ATP-synthase, Electrospray

INTRODUCTION Giant Liposomes

Giant liposomes or giant lipid vesicles are one of the self-assembled structures with amphiphilic lipid molecules in aqueous media, encapsulating an aqueous solution within a vesicular-shape of bilayer membranes, whose diameter distributes between a few micrometers and a few hundred micrometers [2]. Since resembling cells, giant liposomes have been studied as a cell membrane model in various fields such as biomimetic chemistry, biomembrane physics, and synthetic biology [2,3].

Selective Patterning and Size Uniformity of Formed Giant Liposomes

Gentle hydration and electroformation are the most common methods for the giant liposome preparation. The gentle hydration simply takes advantage of a hydration process of a dried lipid film under an aqueous solution while the electroformation applies a low-frequency AC voltage during the hydration [2]. Although these methods have been widely used, they both have difficulty in controlling the shape and/or the size distribution of the formed liposomes [2,4,5]. Patterning of dried lipids prior to the hydration process will be one of the solutions of those problems. There are several techniques reported to obtain such lipid patterns, for instance, chemical hydrophilic/hydrophobic patterning of a substrates’ surface [5]; a lift-off process of a patterned polymer film after spin-coating of a lipid solution [6]; an application of a microcontact printing [7]. The results in these works indicated that the selective patterning would improve the uniformity of the formed liposomes, yet neither method has successfully regulated the lipid patterning/deposition process in an appropriate manner.

In the previous works, we developed an alternative method that enables the precise control of the patterning process by the integration of an electrospray deposition (ESD) technique and a microfabrication process [1]; by the ESD, the spray of lipids was electrically led only to the bottom of microwells which was a conductive ITO-glass slide (Fig. 2a). With a simple hydration process of these dried patterns, we succeeded in the formation of giant liposomes on top of the

Figure 1: Conceptual diagrams of FoF1-reconstituted giant liposome array on a composite substrate, aiming for obser-vation of proton transport kinetics of the FoF1-ATP synthase. (a) Uniform-sized liposomes on a substrate become femto-liter chambers that would enable quantitative evaluation of transported protons by using fluorescent microscopy.(b) Schematics of proton pumping into a liposome. Driven by ATP, FoF1 transports protons into the liposome and in-duces decrease of pH over time. The pH change can be detected by using a fluorescent pH indicator.

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1698 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

microwells with a narrow range of the size distribution (CV of less than 10%) and easily obtained the desired sizes of liposomes by changing the microwell diameter, between 5 and 30 μm in diameter, close to common cellular sizes. The method achieved more than 1,000 cell-sized liposomes arrayed on the substrate.

In this work, we further aim to integrate membrane proteins, the major players for cell membrane functionalities, into the liposome systems. We propose a method that instantly produces uniform-sized, giant proteoliposomes (protein-incorporated liposomes, Fig. 1). The method is based on the previous platform for formation of a uniform-sized liposome array [1]. The key is the electrospray conditions where volatile solvent for lipids has to be replaced to an aqueous buffer to preserve membrane protein functions; electrolytes in aqueous solutions inhibit convergence of the electric field at the tip of the capillary and disturb the spray (Fig. 2b).

EXPERIMENTAL Protein/Lipid Micropatterning

The schematic diagrams of the proteoliposome micropatterning is shown in Fig. 2. The method consists of two steps: 1) Fabrication process of the patterned substrate by using a common photolithography, and 2) Protein/lipid deposition process with the ESD technique. First, a poly(chloro-p-xylylene) (parylene C) thin film was coated on an ITO-glass slide by a chemical vapor deposition method (PDS-2010, Specialty Coating Systems, IN, USA). Then, thin layers of aluminum and positive photoresist were respectively deposited and coated on the parylene C coated ITO-glass slides. By a standard UV-lithography process, we obtained an array of microwells (10 μm in diameter). The ESD technique applies a relatively high DC voltage between a proteovesicle solution filled in a thin capillary and the ITO-glass slide (i.e. target substrate) to perform a selective deposition of the vesicles at a conductive surface.

Here, we chose ATP-synthase FoF1 as a model membrane protein (Fig. 1b, c). FoF1 was expressed in E. coli and reconstituted into small lipid vesicles [8]. The FoF1 vesicles were then dispersed in the aqueous buffer, consisting of 5 mM NaCl and 1% glycerol. As a control condition, we used DOPE-DOPG lipid (1:1-mixture, 0.5 mg/mL) in a solvent consisting of chloroform. A rhodamine-labeled lipid was also mixed for fluorescent observations (Rhod-DOPE, 1 wt%). After the deposition, the samples were kept in vacuum before use. The ESD conditions were adjusted to obtain a stable/fine mist of spray for the aqueous buffer. A higher voltage seemed crucial to form sufficiently fine spray (Fig. 2c, d).

Fluorescence Imaging after Proteoliposome Formation

Hydration of the protein/lipid micropattern was performed by infusion of a sucrose solution. Same as previous works, liposome formation completed within a several minutes [1]. Fluorescence images were taken through the ITO-glass slide with a confocal inverted microscope (SP5, Leica Microsystems, Germany).

Figure 2: (a) Fabrication flow of the conductive/non-conductive substrate. (b) Schematic of protein/lipid pat-terning on the substrate by using electrospray deposition. (c, d) Conditions for the optimal electrospray of an aque-ous buffer containing proteoliposomes. Contrast within the circle was adjusted.

Figure 3: (a) FoF1-reconstituted proteoliposomes were instantly formed by hydration of a protein/lipid pattern sprayed with an aqueous buffer that is required to pre-serve membrane protein activity. (b) A liposome for-mation result previously reported [1], in which the sprayed lipids were dispersed in chloroform. BF or con-focal FL images were shown.

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RESULTS AND DISCUSSION As shown in Fig. 3, proteoliposome formation instantly occurred

by hydration of the protein/lipid pattern although the size uniformity and reproducibility were not yet sufficient compared with the organic solvent condition.

We further confirmed the existence of FoF1 at the liposomal membrane surface by using the specific binding between fluorescent-labeled protein (streptavidin) and biotinylated β-subunits of F1 (Fig. 4).

Finally, proton transport by FoF1 was attempted on the formed proteoliposomes. The result indicated that proton transport could occur only when Mg-ATP was added outside the liposomes as a driving force for proton pumping. More detailed results and discussion will be presented on the poster presentation.

CONCLUSION

We presented an instant giant-proteoliposome formation method. A micrometer-sized proteoliposome becomes a femtoliter chamber that is suitable for microscopic quantification of a molecular influx carried by membrane transport proteins (transporters). We now set a goal to advance to practical protein assays using the platform through further optimization. ACKNOWLEDGEMENTS

The authors acknowledge the technical support provided by Ms. Utae Nose (KAST) and Ms. Maiko Onuki (IIS, UT). This work was partly supported by JSPS (Grant-in-Aid for Young Scientists A; 25706015, and B; 23710154), Japan. REFERENCES [1] T. Osaki, K. S. Kuribayashi, R. Kawano, H. Sasaki, S. Takeuchi, “Uniformly-Sized Giant Liposome Formation

with Gentle Hydration”, Proc. IEEE MEMS 2011, Cancun, 103-106; T. Osaki, K. Kamiya, R. Kawano, H. Sasaki, S. Takeuchi, “Towards Artificial Cell Array System: Encapsulation and Hydration Technologies Integrated in Lip-osome Array”, Proc. IEEE MEMS 2012, Paris, 333-3366; T. Osaki, K. Kamiya, R. Kawano, S. Takeuchi, “Uniform Size Liposomes on a Chip: Observation of Transport Kinetics through Nanopore Membrane Protein”, Proc. Mi-croTAS 2012, Okinawa, 94-6.

[2] P. L. Luisi, P. Walde, Giant Vesicles, John Willy and Sons Inc., New York, 2000; N. Duzgunes, Methods in Enzy-mology Volume 367 Liposomes Part A, Academic Press, California, 2003.

[3] A. Karlsson, R. Karlsson, M. Karlsson, A-S. Cans, A. Strömberg, F. Ryttsén, O. Orwar, “Networks of Nanotubes and Containers”, Nature 2001, 409, 150- 152; I. A. Chen, K. Salehi-Ashtiani, J. W. Szostak, “RNA Catalysis in Model Protocell Vesicles”, J. Am. Chem. Soc. 2005, 127, 13213-13219; G. Tresset, S. Takeuchi, “Utilization of Cell-sized Lipid Containers for Nanostructure and Macromolecule Handling in Microfabricated Devices”, Anal. Chem. 2005, 77, 2795- 2801.

[4] K. Kuribayashi, G. Tresset, H. Fujita, S. Takeuchi, “Electroformation of Giant Liposomes in Microfluidic Chan-nels”, Meas. Sci. Technol. 2006, 17, 3121-3126.

[5] M. Le Berre, A. Yamada, L. Rech, Y. Chen, D. Baigl, “Electroformation of Giant Phospholipid Vesicles on a Sili-con Substrate: Advantages of Controllable Surface Properties”, Langmuir 2008, 24, 2643-2649.

[6] K. Kuribayashi, S. Takeuchi, “Electroformation of Solvent-Free Lipid Membranes over Microaperture Array”, Proc. IEEE MEMS 2008, Tucson, 296-299.

[7] P. Taylor, C. Xu, P. D. I. Fletcher, V. Paunov, “Fabrication of 2D Arrays of Giant Liposomes on Solid Substrates by Microcontact Printing”, Phys. Chem. Chem. Phys. 2003, 5, 4918-1922.

[8] R. Watanabe, K. V. Tabata, R. Iino, H. Ueno, M. Iwamoto, S. Oiki, H. Noji, “Biased Brownian Stepping rotation of FoF1-ATP synthase driven by proton motive force”, Nat. Commun. 2013, 4, 1631.

CONTACT *T. Osaki, Phone: +81-3-5452-6650; tosaki@iis.u-tokyo.ac.jp

Figure 4: (a, b) Streptavidin labeled with AlexaFluor488 (SA) showed specific binding only toFoF1-liposomes. For visualization, a labeled lipid was mixed with PE/PG liposomes (b, right). (c) β-subunits of F1 were biotinylated as SA-binding sites. (d) Comparison of the fluorescence intensi-ty from SA at the membrane surfaces.

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