TY - JOUR TI - Investigating molecular crowding within nuclear pores using polarization-PALM AU - Fu, Guo AU - Tu, Li-Chun AU - Zilman, Anton AU - Musser, Siegfried M A2 - Weis, Karsten VL - 6 PY - 2017 DA - 2017/09/26 SP - e28716 C1 - eLife 2017;6:e28716 DO - 10.7554/eLife.28716 UR - https://doi.org/10.7554/eLife.28716 AB - 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. KW - nuclear pores KW - super-resolution microscopy KW - PALM KW - polarization PALM KW - rotational diffusion JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -