Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
PLoS One. 2013 Aug 5;8(8):e70929. doi: 10.1371/journal.pone.0070929. Print 2013.
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.
细胞信号转导通常通过测量单个或群体活细胞的光学或电学性质来进行研究。在这里，我们展示了可以使用单分子灵敏度在单个（亚）微米大小的天然囊泡中监测配体与细胞表面受体的结合以及随后的信号级联的激活。这些囊泡使用化学物质或光学镊子从活的哺乳动物细胞中提取。它们包含细胞膜和细胞质的部分，代表执行细胞信号反应的最小自主容器，因此功能类似于最小化的细胞。使用荧光显微镜，我们在单个囊泡中测量了 G 蛋白偶联受体介导的信号转导的不同步骤，如配体与受体的结合、随后的 G 蛋白激活以及最后指示受体失活的 arrestin 易位。在单个囊泡中观察细胞信号转导反应为将细胞功能的生物分析缩小到阿特升范围、对单细胞分析进行多路复用以及以多阵列格式研究受体介导的信号转导开辟了大门。
