Abstract
Molecular networks on the cytoplasmic faces of cellular plasma membranes are critical research topics in biological sciences and medicinal chemistry. However, the selective permeability of the cell membrane restricts the researchers from accessing to the intact intracellular factors on the membrane from the outside. Here, a microfluidic method to prepare cell membrane sheets was developed as a promising tool for direct examination of the cytoplasmic faces of cell membranes. Mammalian cells immobilized on a poly(ethylene glycol)-lipid coated substrate were rapidly and efficiently fractured, with the sheer stress of laminar flow in microchannels, resulting in isolation of the bottom cell membrane sheets with exposed intact cytoplasmic faces. On these faces of the cell membrane sheets, both ligand-induced phosphorylation of receptor tyrosine kinases and selective enzymatic modification of a G-protein coupling receptor were directly observed. Thus, the present cell membrane sheet should serve as a unique platform for studies providing new insights into juxta-membrane molecular networks and drug discovery.
Highlights
Molecular networks around the cell membrane are critical interfaces between the extracellular environments and the intracellular living systems and have been actively studied in a variety of research fields, from fundamental molecular biology to drug discovery[1,2]
Our strategy for rapidly preparing intact cell membrane sheets is as follows (Fig. 1b): (1) the bottom glass surfaces of microchannels were coated by lipids with a poly(ethylene glycol) (PEG) linker; (2) cells were immobilized on these surfaces through interactions between the lipid moieties and cell membranes[15,16]; (3) the immobilized cells were fractured using laminar microchannel flow, resulting in preparation of intact cell membrane sheets
Double stained Ba/F3 cells, with cytoplasm and plasma membrane fluorescently stained with CalceinAM and Alexa Fluor 647 (AF647)labeled PEG–lipid (Supplementary Fig. S1), respectively, were immobilized on the lipid modified surface
Summary
Preparation and validation of cell membrane sheets. Double stained Ba/F3 cells (a murine pro-B cell line), with cytoplasm and plasma membrane fluorescently stained with CalceinAM and Alexa Fluor 647 (AF647)labeled PEG–lipid (Supplementary Fig. S1), respectively, were immobilized on the lipid modified surface. Ligand-induced activation was detected on almost half of the cells (Fig. 6a and b) Such information about receptor state, at a single cell level, is important for gaining new insights into diversity among individual cells, not possible with conventional methods of bulk analysis. On the photo-responsive surface, prepared cell membrane sheets are potentially detached by light-induced release of the anchoring lipid moiety from the substrate for further analysis and use of the membrane molecules. These cell membrane sheets should serve as a unique platform for studies providing new insights into molecular networks on the cytoplasmic face of the cell membrane
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
