Mass determination of the nuclear pore complex and its sub-complexesl by high-resolution Scanning Transmission Electron Microscopy (Stem)

Abstract

The nuclear pore complex (NPC) is an elaborate membrane-bound molecular machine residing in the nuclear envelope of all eukaryotic cells and acting as a passageway for molecular transport between the nucleus and the cytoplasm. Complementary to transmission electron microscopic studies of negatively stained and frozen hydrated NPCs yielding detailed information about their overall size, shape and substructure, the high-resolution scanning transmission electron microscope (STEM) was used to measure the mass, to map the radial mass distribution, and to evaluate the rotational symmetry of unstained native NPCs and of several distinct sub-complexes. “Native” NPCs were prepared for STEM as follows: the nucleus of Xenopus laevis oocytes was placed on a 4 nm carbon film supported by a holey film mounted on a copper mesh grid, and its nuclear envelope ruptured and spread out using fine glass needles. The grid was washed with low salt buffer to remove macroscopic contaminants, and excess liquid was withdrawn with filter paper. Next, tobacco mosaic virus (TMV; serves as internal mass standard) was co-adsorbed, before the grid was washed for 10 sec each on 3 drops of bi-destilled H2O. To disintegrate the NPC into distinct sub-complexes, nuclei were placed on a 4-nm thick carbon film supported by a thick holey carbon foil on a copper grid, ruptured and spread as above, and then detergent extracted. After washing, the samples were frozen in liquid N2, transferred to a VG HB-5 STEM and freeze-dried at 153 K. Low-dose annular dark field (AD) images (50 to 300 e/nm2) were acquired digitally, corrected for nonlinearities in the AD signal due to high mass thickness, and stored on tape for further processing.Native NPCs, with and without plug, are displayed in Fig. 1. As shown in Fig. 2, two types of rings can be distinguished after detergent extraction: bright rings (“outer” rings; thick arrow) and dimmer rings (“inner” rings; thin arrow). In the same preparations particle “pairs” consisting of a ring (Fig. 3, thin arrow) and a plug-spoke complex (Fig. 3, thick arrow) can often be depicted. From these data averaged and 8-fold symmetrized mass maps of the native NPC with plug (Fig. 4; ˜50 NPCs) and without plug (Fig. 5; ˜50 NPCs), the outer ring (Fig. 6; ˜50 rings) and the inner ring (Fig. 7; ˜15 rings) were computed. Corresponding radial mass distributions are displayed at the same scale for comparison (Figs. 8 to 11). The average masses of the different structures are summarized in Table 1 together with a tentative model. These measurements bring speculations about the mass of the NPC to an end which have stated values ranging from 20 MDa to over 100 MDa, and they suggest that the NPC could easily be made of the order of 100 different polypeptides.