New Technologies for detecting extracellular fluxes

摘要： 这项重点技术和发展赠款的目标是重新利用细胞内 荧光离子和代谢物传感器来检测细胞外通量。三个正交 这种方法直接针对细胞的糖萼：纳米厚的糖涂层， 和代谢物浓度变化很大， 活动目的1描述了一种将任何荧光蛋白传感器附着到细胞上的方法 用非天然叠氮糖标记的代谢物。目标2是一种非遗传方法， 用小分子和蛋白质标记所有哺乳动物细胞，包括人类细胞 荧光传感器AIM3是一种实现细胞类型特异性标记的化学遗传学方法 小分子和蛋白质荧光传感器。目标的实现将使这些 使用三种主要类型的荧光传感器（小分子， 蛋白质，和FRET）的技术，使可视化的离子和代谢物在 原代细胞、组织的表面，并可能存在于活体动物中。

Abstract: The goal of this Focused Technology and Development grant is to repurpose intracellular fluorescent ion and metabolite sensors to detect extracellular fluxes. The three orthogonal approaches directly target the cell’s glycocalyx: the nanometer thick, sugar‐coating where ion and metabolite concentrations vary greatly during ion channel and membrane transporter activity. Aim 1 describes an approach to attach any fluorescent protein sensor to cells metabolically-labeled with an unnatural azidosugar. Aim 2 is a non-genetic approach that labels all mammalian cells—including human cells—with both small molecule and protein fluorescent sensors. Aim 3 is a chemical genetic approach to achieve cell-type-specific labeling with small molecule and protein fluorescent sensors. Completion of the aims will convert these working prototypes that utilize three major classes of fluorescent sensors (small molecule, protein, and FRET) into technologies that enable the visualization of ions and metabolites at the surface of a primary cells, tissues, and potentially in living animals.