Figure 3.

Widespread occurrence and anchorage of ZC3HC1 at the NEs of proliferating and non-dividing, terminally differentiated cells of different morphogenetic origin. (A) IFM of cryostat sections of the liver, the kidney’s renal pyramids and columns, and the forebrain’s prefrontal cortex from Macaca mulatta, representing tissues originating from the three germ layers. Overviews (left side) only show labelling for hsZC3HC1, while the micrographs at higher magnification also present the identical specimens’ triple-labelling with further antibodies, including such for TPR. For further comparison, liver and kidney sections were labelled with mAb414, while the cerebrum section was labelled for the neuronal cell marker RFOX3/NeuN to distinguish neuronal cells from glial cells. Note that ZC3HC1 and TPR were found colocalising at the NEs of cells present in these tissues. Bars, 100 µm (overview) and 10 µm, respectively. (B) IFM of non-dividing, mature X. laevis erythrocytes. The nuclei are shown in two focal planes, with the one on the nuclei’s equator and the other near the nuclei’s surface. Note the 4× enlarged line profile sections, showing ZC3HC1 at this level of resolution colocalising with TPR’s C-terminal domain, the latter known to be positioned in the TR region too. Bar, 10 µm. (C) IB of the LNN-enriched fraction of XL-177 cells and Xenopus erythrocytes obtained after extraction with TX-100, and of manually isolated and cleansed Xenopus oocyte NEs, obtained from a weight class 1 frog’s stage V oocytes and possessing intact NBs and NPCs, but hardly any of the different types of fibrillar appendices found appended in varying amounts to an oocyte NE from a weight class 3 frog. Loading amounts had been adjusted in such a manner that similar IB signal intensities were obtained for ZC3HC1. Labelling with indicated antibodies was performed on the upper and lower parts of the membrane stained with MemCode but here shown colour-converted from blue to red. Note that while the NE-associated amounts of TPR and ZC3HC1 relative to the reference NPC scaffold proteins NUP107 and NUP96 appeared higher in oocytes and XL-177 cells than in erythrocytes, the amount ratios between TPR and ZC3HC1 within the NB-enriched materials of these three different cell types appeared to be rather similar. (D) IB of cell extracts obtained from a confluent, not synchronised population of HeLa cells following fractionations of similar cell numbers performed in parallel, having applied either NB-s conditions, maintaining NB integrity, or NB-d conditions, causing partial or complete detachment of the one or other NB component from otherwise intact NPCs. Lanes were loaded with the non-fractionated cells’ total cell proteins (T), the soluble proteins released during extraction with TX-100 in NB-s or NB-d buffer (S), and the corresponding non-soluble pellet fractions (P). In addition, the same amounts of S and P materials from each fractionation were also loaded together in one lane to compare the resulting amount with that of the non-fractionated cells, demonstrating that hardly any amount of protein had been lost during the S and P fractions’ preparation. Labelling for hsZC3HC1, hsTPR, hsNUP153, and hsNUP107, and with mAb X223 cross-reactive with human LMNB2, the latter as an additional reference, was performed on the upper and lower parts of the MemCode-stained membrane shown colour-converted to red, and on an identical duplicate. Note that NUP153, anchored to the NPC’s NR, had remained bound to the LNN-enriched pelletable material, irrespective of whether NB-d or NB-s conditions had been applied, as it also was the case for NUP107 and LMNB2 (green arrowheads). Similarly, when having applied NB-s conditions, essentially all ZC3HC1 and TPR had remained bound to the LNN-enriched material too (arrowheads), even after the prolonged incubation at RT and despite efficient cell extraction and solubilisation of most of the cells’ other proteins. By striking contrast, essentially all ZC3HC1 had turned soluble (magenta-coloured large arrow) already after a short incubation at low temperature and in the absence of adequate amounts of divalent cations (for further details, see Materials and Methods). Further note that this had been accompanied by the loss of about half the cell’s total amount of TPR (magenta-coloured small arrow). As an aside, NUP107 polypeptides, also known to be part of ER-embedded individual pore complexes and small AL sheets, both of which are present in HeLa cells too (e.g., [99]), had contributed to the minor amounts of NUP107 in the soluble fractions following TX-100-extraction.