Cell-derived plasma membrane vesicles as minimal cells for analyzing transmembrane signaling

Cellular signalling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show how cell-derived vesicles can be used for anlaysing transmembrane signalling. The vesicles are derived from live mammalian cells by using either chemicals, or by optical tweezers and they comprise parts of the plasma membrane and cytosol of the mother cell. We measured in vesicles derived from individual cells with single molecule sensitivity the different steps of G protein-coupled receptor mediated signalling like ligand binding to receptors, subsequent G protein activation and finally receptor deactivation by interaction with arrestin. Cell-derived plasma membrane vesicles represent the smallest autonomous containers capable of performing cellular signaling reactions thus functioning like minimal cells. Observing cellular signalling 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 signalling in multiarray format

GPCR desensitization in cell-derived plasma membrane vesicles.

<p>(A) Scheme: Binding of agonists to GPCRs initiates receptor phosphorylation, which in turn leads to binding of arrestin to the GPCRs preventing further activation of G proteins. Here, we monitor by confocal microscopy in an individual native vesicle the translocation of fluorescent arrestin (arrestin-GFP) from the lumen to the membrane containing NK1R. (B) Confocal micrograph of a particular single plasma membrane vesicle showing the fluorescence of arrestin-GFP. The agonist substance P added to the bulk binds to the NK1R and induces rapid recruitment of arrestin-GFP at the vesicle membrane (scale bar: 2 µm) as shown in as a time course of the luminal fluorescence of arrestin-GFP. (C) Confocal micrographs of a plasma membrane vesicle and its mother cell expressing NK1R and arrestin-GFP, showing the fluorescence signal of arrestin-GFP before (left) and after (right) substance P perfusion (scale bar: 5 µm) and the time course of both the cytosolic fluorescence of arrestin-GFP in the cell and in the vesicle.</p

Transmembrane signaling in cell-derived plasma membrane vesicles.

<p>(A) Scheme of the activation of G-proteins inside a single native vesicle. Upon binding to an agonist A2AR-YFP receptor forms a complex with the heterotrimeric Gα_sβ₁γ₂-CFP measured by FRET between CFP of Gγ₂ and YFP of A2AR. (B) Confocal micrograph of a typical plasma membrane vesicle derived from a HEK cell expressing heterologously Gγ₂-CFP (blue, left) and A2AR-YFP (yellow, right); scale bar: 1 µm. (C) FRET is detected as fluorescence intensity ratio <i>F_YFP/F_CFP</i> within a single native vesicle; addition of 1 µM agonist APEC significantly increases FRET and subsequent addition of excess of antagonist XAC (10 µM) results in a decrease of the FRET signal. (D) The FRET signal increases with the concentration of agonist APEC added to the bulk solution (concentrations indicated above the bars in nM). (E) A dose-dependent increase of the FRET yields <i>EC₅₀</i> = 100±11 nM; shown are averages and standard deviations of the mean of nine titration experiments each performed on different, individual vesicles.</p

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