Dataset Information

Constructing Supported Cell Membranes with Controllable Orientation.

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 orientation of the supported cell membrane to study membrane protein functions and structures from either side of the cell plasma membrane.

SUBMITTER: Lyu SW

PROVIDER: S-EPMC6391389 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Constructing Supported Cell Membranes with Controllable Orientation.

Lyu Shao-Wei SW Wang Jou-Fang JF Chao Ling L

Scientific reports 20190226 1

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 cytoplasmi ...[more]

PMID: 30808885

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Project description:Protein-reinforced supported bilayer membranes (rSBMs) composed of phosphatidylcholine (PC), biotin-PE and Neutravidin were used to coat hybrid microchips composed of polydimethylsiloxane (PDMS) and glass. Since the coatings required a freshly oxidized, hydrophilic substrate, a novel method to rapidly connect reservoirs using plasma oxidation was first developed and found to support up to 5.2 N cm(-2) (1.5 N) pull-off force. rSBMs were then assembled in the oxidized hydrophilic channels. The electroosmotic mobility (mu(eo)) of rSBM-coated channels was measured over a 3 h time to evaluate the stability of the coatings for microchip electrophoresis. rSBM-coated microchips with a simple cross-design had excellent properties for microchip separations, yielding efficiencies of up to 700,000 plates m(-1) for fluorescent dyes and peptides. The separation performance of rSBM and PC-coated channels was evaluated after repeatedly drying and rehydrating the channels. The separation efficiency of fluorescein on PC-coated devices decreased by 40% after one dehydration cycle and nearly 75% after 3 cycles. In contrast for rSBM-coated devices there was no significant change in the fluorescein efficiency until the third cycle (10% decreased efficiency). rSBM-coated channels were also markedly more stable when placed in a dehydrated state during long-term storage compared to PC-coated channels, and showed reduced chip failure and no reduction in performance for up to one month of dehydrated storage. Finally, rSBM-coated devices were used to perform single-cell cytometry. Microchips that had been dehydrated, stored two weeks, and rehydrated prior to use demonstrated similar performance to newly coated devices for the separation of fluorescein and carboxyfluorescein from single cells. Thus rSBM-coated devices were rugged withstanding electric fields, prolonged storage under dehydrated conditions, and biofouling by cellular constituents while maintaining excellent separation performance.

Dataset Information

Constructing Supported Cell Membranes with Controllable Orientation.

Publications

Constructing Supported Cell Membranes with Controllable Orientation.

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