Fig. 2. Quantification of nucleocytoplasmic partitioning during early development.

a Assay to quantify nucleocytoplasmic partitioning via multiplexed proteomics. Embryonic lysate, developed at 16 °C, was collected at various stages: nuclei were enriched by filtration through membranes of defined pore sizes. Shown are example epifluorescence images of lysate, supernatant, and flow-through stained with Hoechst (blue) and MitoTracker (red). Supernatant and flow-through fractions were digested into tryptic peptides, labeled with isobaric tags, and subjected to accurate multiplexed proteomic analysis. Xenopus illustrations © Natalya Zahn (2022)^86,95. b Nucleocytoplasmic partitioning quantification for three examples. Dots indicate measured fraction of each protein in nuclei. Solid lines indicate fit sigmoids. Blue dashed line indicates median fit from all nuclear proteins. c Quantification of time-dependent nuclear fraction for ~2k nuclear proteins and ~600 transcription factors (TFs)^20,71,96. Solid line indicate the median and shaded area 50% spread. d, e Measurements for multisubunit protein complexes. Solid line indicates the median and shaded area 50% spread. d DNA repair complexes enter embryonic nuclei at various times, yet their subunits enter simultaneously. Shown are nuclear entry quantifications for homologous recombination (HR), the nonhomologous DNA end-joining repair (NHEJ), and the Fanconi anaemia (FA). An apparent time delay of nuclear entry for some DNA repair complexes explains previous observations that early embryos bypass DNA repair to accommodate fast cell divisions^30–32. Oocyte nuclear fractions are shown as standard box plots. e Change in nucleocytoplasmic partitioning for nuclear pore complex (NPC) subunits. Top: Our nuclear fraction measurement for NPC proteins shows rapid incorporation onto embryo’s exponentially increasing nuclear surface. Middle: Quantification of NPC nuclear fraction via MS agrees well with immunofluorescence-quantified nuclear surface change. The x-axis is the relative nuclear surface measurement to that of embryos at 22.5 h post-fertilization. Bars indicate standard errors. Bottom: Illustration of dynamic localization of NPC proteins in the oocyte versus the embryo. Oocyte stores NPC subunits in endoplasmic reticulum membranes embedded with pore complexes, called annulate lamellae^34,35.