Visualization of maltose uptake in living yeast cells by fluorescent nanosensors
Visualization of maltose uptake in living yeast cells by fluorescent nanosensors
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DOI：
10.1073/pnas.142089199
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发表时间：
2002-07-23
影响因子：
11.1
通讯作者：
Lalonde, S
中科院分区：
文献类型：
--
作者：
Fehr, M;Frommer, WB;Lalonde, S
只有了解基于无损动态监测的亚细胞分布，才能理解代谢反应的区隔以及细胞内和细胞间的转运。目前，还没有在细胞或亚细胞水平上进行体内代谢物成像的方法。有限的信息来自需要固定或分离组织的方法（1,2）。因此，我们开发了一种灵活的策略来设计基于蛋白质的纳米传感器，用于广泛的溶质光谱，允许分析活细胞中溶质浓度的变化。我们利用细菌周质结合蛋白（PBPs），在与底物结合时，PBPs将其铰链弯曲运动转化为两个耦合绿色荧光蛋白之间增加的荧光共振能量转移（FRET）。以麦芽糖结合蛋白为原型，构建了纳米传感器，可以在体外以浓度依赖的方式测定FRET的变化。在生理应用方面，产生了具有不同结合亲和力的突变体，使单个酵母细胞中细胞质麦芽糖浓度增加的动态体内成像成为可能。控制传感器允许排除其他细胞或环境参数对比率成像的影响。因此，无数的PBPs识别广泛的不同底物，适用于基于fret的体内检测，提供了许多科学，医学和环境应用。
Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing protein-based nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells. We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. By using the maltose-binding protein as a prototype, nanosensors were constructed allowing in vitro determination of FRET changes in a concentration-dependent fashion. For physiological applications, mutants with different binding affinities were generated, allowing dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells. Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging. Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based in vivo detection, providing numerous scientific, medical, and environmental applications.