Absolute Stoichiometry of the Nuclear Pore Complex

Abstract

The nuclear pore complex (NPC) is one of the largest macromolecular assemblies in eukaryotic cells and tightly regulates the transport between the cytoplasm and the nucleus. In order to unravel the structural details of the NPC, an exact stoichiometry of its constituent proteins (NUPs) is needed. Mass spectrometry can be exploited to establish stoichiometric ratios of the NUPs, but in order to trace absolute copy numbers it is necessary to quantify the number of instances of at least one NUP. To achieve this goal, a gene replacement strategy was combined with a quantitative PALM approach: one of the core proteins of the NPC, namely NUP107, was genetically tagged with the photo-activatable protein mEos2 and expressed in HEK293 cells where native NUP107 was knocked down using micro-RNAs. For optimal signal to noise, NUP107-mEos2 containing NPCs from functional purified nuclei were placed in the evanescent field of a highly sensitive microscope. We then performed PALM imaging of mEos2-NUP107 with an optimized photo-conversion scheme for iterative activation and bleaching cycles assuring complete photo-conversion of the sample. In order to retrieve the average number of fluorophores in each pore, a computational workflow was designed to select isolated pores from the resulting super-resolution images. The counted number of apparent photo-conversion events in each pore was further corrected for fluorophore blinking effect by statistical methods. Taking protein and mEos2 maturation profiles into account, this allowed us to calculate the lower limit for the exact copy number of NUP107 in each pore establishing the baseline for the stoichiometry of the entire NPC.