论文标题
单个真核细胞中的分子拥挤：使用细胞环境生物传感和单分子光学显微镜来探测对细胞外离子强度，局部葡萄糖条件和传感器拷贝数的依赖性
Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number
论文作者
论文摘要
细胞内部的物理和化学环境对所有生命至关重要，但传统上很难在亚细胞上确定。在这里，我们将尖端的基因组整合的FRET生物传感与单个活酵母细胞中的局部分子拥挤结合在一起。共聚焦显微镜使我们能够使用比率测量品格建立亚细胞拥挤的热图，而全细胞分析表明，当葡萄糖浓度升高时，酵母菌种植时减少了拥挤。模拟表明，这些传感器在很大程度上无法访问细胞膜，并且在秒的时间范围内，每个细胞的胞质拥挤在每个细胞上都均匀。毫秒单分子光学显微镜用于跟踪分子并获得亮度估计值，以计算拥挤传感器拷贝数。扩散分子轨迹的定量为将亚细胞过程和细胞在压力下的物理化学环境相关联铺平了道路。
The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.
