Nuclear formation induced by DNA-conjugated beads in living fertilised mouse egg

Abstract

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.

Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes

“…This hypothesis is also supported by the result that the degree of nuclear formation was relatively high in injection at metaphase II and anaphase II, slightly lower at telophase II, and none in interphase (Figure 2). This result is consistent with previous findings that that λDNA injected into metaphase Xenopus oocytes formed functional nuclei with nuclear transport activity (Forbes et al, 1983), whereas DNA-conjugated beads injected into interphase mouse fertilized-egg did not (Suzuki et al, 2019). However, abnormal polar body extrusion was observed in almost all oocytes injected with DNA at metaphase II.…”

“…This notion is supported by our results that T4 DNA artificial nuclei contain nucleosomes comparable to the amount of natural maternal pronuclei (Figure 4). However, despite the presence of nucleosomes and nucleosome-binding protein RCC1, DNA-conjugated beads injected into interphase mouse fertilized-eggs failed to form functional nuclei with nuclear transport activity (Suzuki et al, 2019). This suggests that the presence of nucleosomes and RCC1 is essential, but not sufficient for the formation of functional nuclei.…”

“…mRNA probes for histone H2B-mCherry, RCC1-EGFP, and Nup153-EFGP were generated as previously (Suzuki et al, 2019), and that for superfolder GFP-nucleoplasmin (NPM)-NLS (sfGFP-NLS) was generated as previously described (Oda et al, 2023). Briefly, cDNA encoding a protein of interest fused with fluorescent protein was cloned into pcDNA3.1 containing a poly(A) tail sequence for mRNA synthesis.…”

“…To identify such factors required for functional pronuclear formation, we used reconstitution approaches in living mouse oocytes by combining in vitro approaches. In the previous report, we introduced DNA-coated beads into mouse fertilized-eggs, and found that DNA-coated beads had the ability to form the nucleus with NPCs in mouse eggs (Suzuki et al, 2019). However, this reconstructed nucleus lacked nuclear transport activity.…”

Transition to the structurally vulnerable nuclear state is an integral part of mouse embryonic development

“…Live cell imaging was performed using an inverted fluorescence microscope (Ti-U, Nikon) equipped with a motorized sample stage (MS-2000, Applied Scientific Instruments), a piezo-driven objective scanner (PIFOC, Physics Instruments) with a piezo controller (E-665, Physics Instruments), a stage-top incubator (TIZ, Tokai Hit), a spinning-disk confocal unit (CSU-X1, Yokogawa) with two excitation lasers (488 nm and 561 nm; OBIS LS, Coherent), a silicone-immersion objective lens (UPlanSApo 60× 1.30 NA, Olympus) and a sCMOS camera (Neo4.1, Andor). To begin with, the fertilized embryos prepared as above were injected with mRNA (20 ng/μl of histone H2B-mCherry or 300 ng/μl of emerin-EGFP) at 1–2 h post-insemination and then transferred to drops of KSOM medium (10 µl each), which was supplemented with 0.00025% of polyvinyl alcohol (PVA; P8136-250G, Sigma-Aldrich) ( 47 ) and placed on a glass-bottom culture dish (P35G-1.5-14-C, MatTek) filled with paraffin oil (26137-85, Nacalai Tesque). The embryos were cultured in a cell culture incubator before being transferred to the microscope’s stage-top incubator, which was maintained at 37°C and filled with humidified air containing 5% CO 2 .…”