Abstract
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.
Highlights
Miniaturized bioassays of cellular signaling are of fundamental importance to increase both throughput and number of parameters evaluated, and substantially decrease sample consumption
We demonstrate the ability of single cell-derived plasma membrane vesicles to convert an external stimulus to an internal response by monitoring the different steps of G-protein-coupled receptors (GPCRs) mediated signaling, from initial signal detection to subsequent transmission of the external signal across the vesicle’s plasma membrane leading to intravesicular signaling reactions and receptor deactivation
Using fluorescence correlation spectroscopy (FCS), we determined the concentration and mobility of a prototypical GPCR, the adenosine A2A receptor fused to YFP (A2AR-YFP), in the plasma membrane of both individual native vesicles and their mother cells
Summary
Miniaturized bioassays of cellular signaling are of fundamental importance to increase both throughput and number of parameters evaluated, and substantially decrease sample consumption. Micrometer-sized lipid vesicles or polymeric containers have been used to observe simple biochemical reactions [8,9,10,11,12], the reliable reconstitution of complex transmembrane cellular signaling cascades into such artificial containers has never been shown and seems to be difficult to realize for the near future. In this context, plasma membrane vesicles, derived from living mammalian cells by chemical treatment [3,13,14] or opto-mechanical manipulation [15] are of utmost interest. As a native vesicle receives from its mother cell a portion of naturally oriented plasma membrane and part of the cytoplasm, it should be regarded as a single-cell biopsy, able to act as a miniaturized, minimal autonomous entity detecting external signals at and transmitting them across the vesicle plasma membrane, and activating signaling reaction cascades inside the vesicle similar to its mother cell
Sign-in/Register to view highlights & summary 
Published Version (
Free)
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 call