Goulian M, Simon S M
Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York 10021, USA.
Biophys J. 2000 Oct;79(4):2188-98. doi: 10.1016/S0006-3495(00)76467-8.
We present experiments in which single proteins were imaged and tracked within mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged by epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/s. We acquired two-dimensional trajectories of proteins (corresponding to the projection of three-dimensional trajectories onto the plane of focus) for an average of 17 frames in the cytoplasm and 16 frames in the nucleus. Diffusion constants were determined from linear fits to the mean square displacement and from the mean displacement squared per frame. We find that the distribution of diffusion constants for RPE within cells is broader than the distributions obtained from RPE in a glycerol solution, from a Monte Carlo simulation, and from the theoretical distribution for simple diffusion. This suggests that on the time scales of our measurements, the motion of single RPE proteins in the cytoplasm and nucleoplasm cannot be modeled by simple diffusion with a unique diffusion constant. Our results demonstrate that it is possible to follow the motion of single proteins within cells and that the technique of single molecule tracking can be used to probe the dynamics of intracellular macromolecules.
我们展示了在哺乳动物细胞内对单个蛋白质进行成像和追踪的实验。通过落射荧光显微镜对核质和细胞质中的单个藻红蛋白（RPE）进行成像，成像速度为每秒71帧。我们获取了蛋白质的二维轨迹（对应于三维轨迹在焦平面上的投影），在细胞质中平均获取了17帧，在细胞核中平均获取了16帧。扩散常数通过对均方位移的线性拟合以及每帧位移平方的平均值来确定。我们发现，细胞内RPE的扩散常数分布比在甘油溶液中的RPE、蒙特卡罗模拟以及简单扩散的理论分布所得到的分布更宽。这表明在我们测量的时间尺度上，细胞质和核质中单个RPE蛋白的运动不能用具有唯一扩散常数的简单扩散来建模。我们的结果表明，追踪细胞内单个蛋白质的运动是可能的，并且单分子追踪技术可用于探测细胞内大分子的动力学。
