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. 2021 Jul 30;10(8):1937. doi: 10.3390/cells10081937

Figure 1.

Figure 1

ZC3HC1 is a NE-associated protein in Xenopus laevis oocytes and cultured cells. (A) SEM micrographs of manually isolated and then differently treated X. laevis NEs from a weight class 1 frog’s late-stage V oocytes (for weight class definition, see Material and Methods). (A1) Nuclear and cytoplasmic face of NEs that had been isolated under NB-stabilising conditions (NB-s) preserving NPC and NB integrity while largely removing other NE-associated materials. (A2) Nuclear face of NEs treated with TX-100 and RNases, after having been isolated like the NEs presented in (A1), maintaining NB integrity. (A3) Nuclear and cytoplasmic face of the same NE, which had been treated with TX-100 but not with nucleases, following an NB-removal procedure that nonetheless allowed for maintaining the integrity of the lamina, the actual NPCs and the appendices at the NPCs’ cytoplasmic side. One of those sporadically observed NBs that had been disassembled only partially (arrowhead) is shown as a point of reference for the NE’s nuclear side. Bars, 100 nm; same magnification for all SE micrographs. (B) IB of subcellular fractions of X. laevis oocytes. (B1) IB of NEs that had been manually isolated from late-stage V oocytes, then incubated as two separate batches in parallel, either under NB integrity (+NB) or NB removal (-NB) conditions, subsequently extracted with TX-100, and finally sedimented by centrifugation. Labelling with indicated antibodies (target regions in parentheses) was on the upper and lower parts of the same membrane and on an identical duplicate, respectively. Since the total amount of loaded NE proteins did not exceed 500 ng per lane, resulting in polypeptide patterns not visible after staining by dyes like Ponceau S (PS), the stained membranes are not shown here. Note that ZC3HC1 was no longer present among the NE proteins after having applied conditions that removed most of the NB scaffold protein TPR and other NB proteins, like MAD1 and MAD2. By contrast, components of the NPC, like NUP62, had remained largely unaffected, and the NE-associated amount of NUP153 had only been slightly reduced in the absence of the NBs. (B2) IB of the soluble cytosolic proteins and particulate cytoplasmic materials (C) that after low-speed centrifugation had remained in the supernatant, the AL-containing membrane fraction, which still contained some contaminating yolk protein (asterisk marks major representative), the second wash of this latter fraction (W), and the total of proteins from the intact nuclei (N). All fractions stemmed from a batch of a weight class 3 frog’s stage VI oocytes that had been manually enucleated first. The oocytes’ other fractions, including other organelles and membranes, pigment granules, and the bulk of yolk, all of which were devoid of ZC3HC1 and TPR, are not shown here. Labelling for xlZC3HC1 and reference proteins xlTPR and xlNUP153, the latter recurrently detected also within the Xenopus oocyte’s AL fraction in trace amounts (blue arrowhead), and xlNUP62, as a regular part of the ALPCs, was on the upper and lower parts of the PS-stained membrane shown here, and on an identical duplicate. Each lane was loaded with the respective fraction or washing solution volume corresponding to only one oocyte, explaining why materials in lane N were hardly visible after PS-staining. Note that ZC3HC1, in stark contrast to NUP62, was not detectable in the AL fraction and instead turned out to be a primarily nuclear protein, just like TPR. (C) Triple-labelling IFM of cryostat sections of Xenopus oocytes with guinea pig, rabbit, and mouse antibodies for xlZC3HC1, xlTPR, and xlNUP62, respectively. Note that while NUP62 was seen both at the NE (arrowhead) and the cytoplasmic AL (some marked by arrows), ZC3HC1 and TPR were only detectable at the NE. The nucleus (N) and cytoplasmic compartment (C) of the stage VI oocyte are marked, as is the nucleus (asterisk) of an early-stage oocyte in which colocalisation at the NE occurred too. Oocytes were from a weight class 2 frog, with commonly less AL material than in class 3 frog oocytes. White dashed lines demark the cell boundaries of the early- and late-stage oocyte. Bar, 50 µm. (D) Double-labelling IFM of XL-177 cells for xlZC3HC1 and xlTPR, with the focal plane at the polar region of a representative nucleus. Note that both proteins at this resolution appeared to largely colocalise in dots, which for TPR were already known to represent NPC-associated NBs. Bar, 5 µm. (E) IFM of XL-177 cells for xlZC3HC1 and xlTPR and with a monoclonal antibody (mAb), mAb414, which binds to several NPC-located FG-repeat nucleoporins (NUPs). The focal plane was at the equator of a representative nucleus. Rectangles in the overlay micrographs were analysed by the ImageJ software, with line profiles plotted. Note the 4× enlarged line profile sections, showing almost complete overlap of IF-labelling for NE-associated ZC3HC1 and TPR, and an offset location of ZC3HC1 towards the nuclear interior relative to the FG-repeat NUPs, with approximate distances of around 150 nm between the line profile peaks for the labellings with mAb414 and for ZC3HC1. Note also the inverse order of labelling for ZC3HC1 and the FG-repeat NUPs at an NE invagination (arrowhead), compared to the NE’s not folded parts. Bar, 5 µm.