Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

Abstract

The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes.

Extending resolution of structured illumination microscopy with sparse deconvolution

“…2e, 2f). These estimations nicely agreed with previous results obtained with different SR methods in fixed cells [32][33][34] .…”

Applications of Super Resolution Expansion Microscopy in Yeast

“…The error close to the size of the GFP protein and the antibodies is ∼15 nm. However, we located the position of the center of the pore with intensity information over five points from the same nuclear pore, which reduces the error to below 7 nm [37]. For further improvements in resolution, small tags such as nanobody or snap [38] can be used in the future to obtain more accurate data in combination with our protocol.…”

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

“…Accumulation of several thousand images of individual nuclear pores has enabled a structural analysis of the pore with an accuracy of more than 1 nm [ 136 ]. The localization and distribution of individual nuclear pore subunits over the membrane have been determined [ 137 - 141 ]. Using SRM, the contacts between nucleoporins and transport receptors have been studied [ 142 ], and contact between the nuclear pore and the active locus has been demonstrated [ 143 ].…”