Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes.
Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.
Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Luigino
L
Grasso
Romain
R
Wyss
Joachim
J
Piguet
Michael
M
Werner
Ghérici
G
Hassaïne
Ruud
R
Hovius
Horst
H
Vogel
D019393:Arrestin / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D052898:Optical Tweezers; D021381:Protein Transport; D043705:Receptor, Adenosine A2A / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*
D019393:Arrestin; D043705:Receptor, Adenosine A2A; D018040:Receptors, Neurokinin-1