Constructing Supported Cell Membranes with Controllable Orientation

Scientific Reports
Shao-Wei LyuLing Chao

Abstract

Membrane proteins play important roles in various cellular processes. Methods that can retain their structure and membrane topology information during their characterization are desirable for understanding their structure-function behavior. Here, we use giant plasma membrane vesicles (GPMVs) to form the supported cell membrane and develop a blotting method to control the orientation of the deposited cell membrane in order to study membrane proteins from either the extracellular or the cytoplasmic sides. We show that the membrane orientation can be retained in the directly-deposited membrane and the deposited membrane on mica can be blotted onto glass to reverse the membrane orientation. We used Aquaporin 3 (AQP3), an abundant native transmembrane protein in Hela cells, as a target to examine the cell membrane orientation in the directly-deposited and reversed membrane platforms. The immunostaining of antibodies targeting either the cyto-domain or ecto-domain of AQP3 shows that the intracellular side of the cell membrane faced the bulk aqueous environment when the GPMVs spontaneously ruptured on the support and that the membrane orientation was reversed after blotting. With this blotting method, we can thus control the orientati...Continue Reading

References

Mar 12, 2004·Journal of the American Chemical Society·Motomu TanakaStefan Kaufmann
Nov 3, 2004·Langmuir : the ACS Journal of Surfaces and Colloids·Martin BenesWim Th Hermens
Nov 3, 2004·Biochimica Et Biophysica Acta·Annela M SeddonPaula J Booth
May 20, 2005·Nature·Guillaume T CharrasT J Mitchison
Jul 6, 2005·Trends in Biochemical Sciences·Dov LichtenbergHeiko Heerklotz
Sep 30, 2005·Nature·Motomu Tanaka, Erich Sackmann
Dec 2, 2006·Nature Reviews. Molecular Cell Biology·Gunnar von Heijne
Apr 23, 2009·Proceedings of the National Academy of Sciences of the United States of America·Joseph D HoRobert M Stroud
Mar 2, 2010·Nature Reviews. Drug Discovery·UNKNOWN International Transporter ConsortiumLei Zhang
Mar 23, 2011·Journal of Controlled Release : Official Journal of the Controlled Release Society·Pille SäälikMargus Pooga
Jul 27, 2011·Journal of the American Chemical Society·Lisa SimonssonFredrik Höök
Nov 12, 2013·Current Topics in Membranes·Francis J Alenghat, David E Golan
Apr 25, 2014·ACS Applied Materials & Interfaces·Chung-Ta Han, Ling Chao
Jan 27, 2016·Langmuir : the ACS Journal of Surfaces and Colloids·Mark J RichardsSusan Daniel
Feb 7, 2016·Protein Expression and Purification·Lynette Sin Yee LiewCongBao Kang
Nov 15, 2006·Surface Science Reports·Edward T Castellana, Paul S Cremer

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Methods Mentioned

BETA
fluorescence recovery after photobleaching
fluorescence recovery after

Software Mentioned

ImageJ
Matlab

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