Investigating cellular signaling reactions in single attoliter vesicles.
Understanding cellular signaling mediated by cell surface receptors is key to modern biomedical research and drug development. The discovery of a growing number of potential molecular targets and therapeutic compounds requires downscaling and accelerated functional screening. Receptor-mediated cellular responses are typically investigated on single cells or cell populations. Here, we show how to monitor cellular signaling reactions at a yet unreached miniaturization level. On the basis of our observations, cytochalasin induces mammalian cells to extrude from their plasma membrane submicrometer-sized native vesicles. They comprise functional cell surface receptors correctly exposing their extracellular ligand binding sites on the outer vesicle surface and retaining cytosolic proteins in the vesicle interior. As a prototypical example, ligand binding to the ionotropic 5-HT(3) receptor and subsequent transmembrane Ca(2+) signaling were monitored in single attoliter vesicles. Thus, native vesicles are the smallest autonomous containers capable of performing cellular signaling reactions under physiological conditions. Because a single cell delivers about 50 native vesicles, which can be isolated and addressed as individuals, our concept allows multiple functional analyses of individual cells having a limited availability and opens new vistas for miniaturized bioanalytics.
Pick
Horst
H
Laboratory of Physical Chemistry of Polymers and Membranes, Swiss Federal Institute of Technology in Lausanne (EPFL), 1015 Ecublens, Switzerland.
Schmid
Evelyne L
EL
Tairi
Ana-Paula
AP
Ilegems
Erwin
E
Hovius
Ruud
R
Vogel
Horst
H
D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D003571:Cytochalasin B / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008024:Ligands; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology
D008024:Ligands; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D003571:Cytochalasin B; D066246:ErbB Receptors