Reassembly of the nuclear pore complex (NPC) at the end of mitosis is an important event for eukaryotic nuclear function. In this study, we examined the dynamic behaviors of the endoplasmic reticulum (ER) by “Live CLEM” imaging. In metaphase, numerous fenestrations on the ER membrane were observed around chromosomes. In telophase, these fenestrations became filled at the region attached to chromosomes, whereas they remained open at the region unattached to chromosomes, suggesting that NPC assembly takes place at fenestrations on the membrane. To determine the roles of nucleoporins in postmitotic NPC formation, we used artificial beads conjugated with anti‐GFP antibody, which captures GFP‐fused proteins on the beads when incorporated into cells. Live CLEM imaging of telophase cells containing Nup133‐coated beads or Nup153‐coated beads showed that Nup133 and Nup153, as the sole effector molecules, assembled the NPC‐like structure on the membrane fenestrations. Indirect immunofluorescence staining of the Nup133‐coated beads showed that Nup133 effectively assembled Nup107 and ELYS, whereas minimal assembly of Nup98 and Nup62 was observed; the Nup153‐coated bead effectively assembled Nup98, Nup62 and Pom121, but assembled neither Nup107 nor ELYS. Our results suggest that Nup133 and Nup153 play different roles in assembling the NPC on membrane fenestrations.

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

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Reformation of a functional nucleus at the end of mitosis is crucial for normal cellular activity. Reconstitution approaches using artificial beads in frog egg extracts have clarified the molecules required for nuclear formation in vitro . However, the spatiotemporal regulation of these components, which is required for the formation of a functional nucleus in living embryos, remains unknown. Here we demonstrate that exogenous DNA introduced in the form of DNA-conjugated beads induces the assembly of an artificial nucleus in living mouse cleavage-stage embryos. Live-cell imaging and immunofluorescence studies revealed that core histones and regulator of chromosome condensation 1 (RCC1) assembled on the DNA, suggesting that nucleosomes were formed. Electron microscopy showed that double-membrane structures, partly extended from annulate lamellae, formed around the beads. Nuclear pore complex-like structures indistinguishable from those of native nuclei were also formed, suggesting that this membranous structure resembled the normal nuclear envelope (NE). However, the reconstituted NE had no nuclear import activity, probably because of the absence of Ras-related nuclear protein (Ran). Thus, DNA is necessary for NE reassembly in mouse embryos but is insufficient to form a functional nucleus. This approach provides a new tool to examine factors of interest and their spatiotemporal regulation in nuclear formation.

DNA transfection is an important technology in the life sciences, wherein nuclear entry of DNA is necessary to achieve expression of the exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe tools for transfection. However, they have no effects for transfection of non-proliferating cells, the reason for which remains unknown. This study aimed to elucidate the mechanism by which transfected DNA enters the nucleus for gene expression. To monitor the intracellular behavior of transfected DNA, we introduced a plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observed plasmid behavior and RFP expression. RFP expression appeared only after mitosis. Electron microscopy showed that plasmids were wrapped with nuclear envelope (NE)-like membranes or associated with chromosomes at telophase. Depletion of BAF, which is involved in NE reformation, delayed plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus via NE reformation at telophase.

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study investigates the functionality of geNOps - an iron-containing metalloprotein and NO biosensor - in cultured HEK293T epithelial and EA.hy926 endothelial cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate that cells adapted to physiological normoxia require significantly lower iron to achieve geNOps signals comparable to those measured in cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.

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