Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M University Health Science Center, College Station, United States.
Department of Physics, University of Toronto, Toronto, Canada.
Elife. 2017 Sep 26;6:e28716. doi: 10.7554/eLife.28716.
The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC's internal organization consists of multiple dynamic environments with different local properties.
核孔复合体（NPC）的关键组成部分控制着核质转运的通透性、选择性和速度，它是一组天然无规多肽的组装体，其中包含与核转运受体结合的苯丙氨酸-甘氨酸（FG）结合位点。由于缺乏能够探测完整 NPC 内 FG-多肽和嵌入大分子的流动性和密度的实验方法，FG 网络的结构和动力学一直难以描述。我们结合荧光偏振、超分辨率显微镜和数学分析，在不同条件下检查了 FG 网络中不同位置处荧光探针的旋转流动性。我们证明，偏振 PALM（p-PALM）提供了大量关于低旋转流动性的信息，而这些信息是使用体荧光各向异性方法无法获得的，并且预计 p-PALM 非常适合探索众多拥挤的细胞环境。总的来说，我们的研究结果表明，NPC 的内部组织由多个具有不同局部性质的动态环境组成。
