Abstract
The nuclear pore complex (NPC) is a cornerstone of eukaryotic cell functionality, orchestrating the nucleocytoplasmic shuttling of macromolecules. Here we report that Plant Nuclear Envelope Transmembrane 1 (PNET1), a transmembrane nucleoporin, is an adaptable NPC component that is mainly expressed in actively dividing cells. PNET1’s selective incorporation into the NPC is required for rapid cell growth in highly proliferative meristem and callus tissues in Arabidopsis. We demonstrate that the cell cycle-dependent phosphorylation of PNET1 coordinates mitotic disassembly and post-mitotic reassembly of NPCs during the cell cycle. PNET1 hyperphosphorylation disrupts its interaction with the NPC scaffold, facilitating NPC dismantling and nuclear membrane breakdown to trigger mitosis. In contrast, nascent, unphosphorylated PNET1 is incorporated into the nuclear pore membrane in the daughter cells, where it restores interactions with scaffolding nucleoporins for NPC reassembly. The expression of the human PNET1 homologue is required for and markedly upregulated during cancer cell growth, suggesting that PNET1 plays a conserved role in facilitating rapid cell division during open mitosis in highly proliferative tissues.
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Data availability
The raw data files for the RNA-seq analysis have been deposited in the NCBI GEO under accession number GSE273725 and are publicly available. The raw data files for all mass spectrometry analyses have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository (identifiers PXD054475 and PXD054530) and are publicly available. Source data are provided with this paper.
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Acknowledgements
We thank C. Gutierrez at Centro de Biologia Molecular Severo Ochoa and D. Bergmann at Stanford University for sharing PlaCCI seeds and B. Liu at UC Davis for sharing the CYCD3;1–Myc construct and GFP–TUA6 N. benthamiana transgenic seeds. We also thank J. Shen from UC Berkeley for assistance with the callus induction assay, M. Nomoto and Y. Tada from Nagoya University for providing reagents for cell-free protein synthesis, L. Xu from Chinese Academy of Sciences for sharing the detached leaf regeneration scRNA-seq data35 and D. Schichnes from the Rausser College of Natural Resources Biological Imaging Facility at UC Berkeley for assistance with fluorescence imaging. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (grant no. R35GM154623-01 to Y.G., grant nos R01GM135706 and S10OD030441 to S.X. and a diversity supplement to A.V.R.), the National Institute of Food and Agriculture (HATCH project nos CA-B-PLB-0243-H to Y.G. and CA-D-PLB-2850-H to X.X.), the Carnegie Endowment Fund to the Carnegie Mass Spectrometry Facility, and the US National Science Foundation (grant no. 2049931 to Y.G.).
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Y.F., Y.T. and Y.G. conceived the project. Y.F. and P.X. imaged and quantified the root phenotypes. Y.F. and K.H. performed the callus induction assay. Y.F. and H.N. performed the GUS staining assay. M.J. contributed to RNA extraction and qPCR. A.V.R. and S.X. performed the phosphorylation site identification through mass spectrometry. Y.F., Y.L. and X.X. performed scRNA-seq reanalysis on shoot inflorescence and regeneration tissue from the detached leaf data. Y.F. conducted all the rest of the experiments and data analyses. Y.F. and Y.G. wrote the paper with input from all authors.
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Extended data
Extended Data Fig. 1 PNET1 is a conserved cell cycle regulator.
a, AlphaFold predictions of human TMEM209 and Arabidopsis PNET1 structures (https://alphafold.ebi.ac.uk/). pLDDT score shows the confidence of the prediction. b, The pnet1 T-DNA and CRISPR mutant alleles. c, Root length measurement of 6-day-old seedlings (n = 30 for each background, two-tailed t-tests). The lower and upper whiskers indicate the minimum and maximum values, respectively. The upper boundary, middle line, and the lower boundary of the box indicate the 75th percentile, medium, and 25th percentile, respectively. Scale bar, 5 mm. d, Fluorescence imaging of the RAM in Dex::PNET1–GFP / pnet1-1 seedling with dex treatment. Scale bar, 20 μm. e, Quantification of the percentage of cells at different cell cycle stages within the epidermal layer of the RAM under homeostatic conditions (n = 10 seedlings for each background, two-tailed t-tests). Data are presented as mean values ± SD.
Extended Data Fig. 2 PNET1 is specifically and highly expressed in actively dividing cells.
a, GUS staining of 14-day-old proPNET1::GUS and WT non-transgenic seedlings. No specific and consistent staining pattern was observed. Scale bars, 5 mm. b, PNET1 expression pattern in de novo root tissues regenerated from detached Arabidopsis leave. Data was obtained by reanalyzing scRNA-seq data published previously35. H3.1 and CDKB2;1 are cell cycle markers for the S and G2/M phase, respectively. c, GUS staining of WT and proPNET1::GUS plants at 0 and 4 days past shoot removal (dpsr). Scale bars, 0.1 mm. Panels a and c represent results from two independent experiments conducted with separate reporter lines, yielding similar outcomes.
Extended Data Fig. 3 PNET1 regulates cell proliferation rate.
a, Relative expression levels of PNET1 in WT and two independent PNET1 overexpression lines. RT-qPCR was performed using 6-day-old seedlings (n = 3 biological replicates, two-sided t-tests). Data are presented as mean values ± SD. b, Representative images of calli derived from hypocotyl explants after growing on CIM2 for 4 days and then SIM for 12 days. Arrowheads indicate regenerated shoots. Scale bars, 1 mm. c, The number of regenerated shoots per callus was measured 12 days after growing on SIM (n = 40 for each genetic background). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison tests (P < 0.05). Exact P values are provided in the Source Data. The lower and upper whiskers indicate the minimum and maximum values, respectively. The upper boundary, middle line, and the lower boundary of the box indicate the 75th percentile, medium, and 25th percentile, respectively. d, Leaves of GFP–TUA6 transgenic N. benthamiana were co-infiltrated with Agrobacteria carrying 35S::PNET1–CFP and Dex::CYCD3;1–mCherry constructs. Representative images of epidermal cells undergoing cell division at 48 hours after dex treatment. Arrows label dividing cells with observable mitotic spindle structures. The experiment was repeated three times with similar results. Scale bar, 50 µm.
Extended Data Fig. 4 Identification of PNET1 interactors using proximity labeling and Alphafold prediction.
a, Total protein extracts from 35S::PNET1–3xHA–TurboID seedlings treated with biotin were immunoblotted with streptavidin and anti-HA antibody. Samples from WT seedlings were used as control. b, Predicted protein-protein interaction between PNET1 and Nup43 using Alphafold2. The interaction surface details are shown in the lower panel.
Extended Data Fig. 5 Phosphorylation of PNET1 affects its interaction with Nup43 and the cell cycle rate.
a, Y2H analysis with Nup43 as prey and PNET1D and PNET1A as bait. Diploid yeasts were grown on DDO and TDO supplemented with 0.5 mM 3-AT media. EV, empty vector. The experiment was repeated twice with similar results. b, Localization of PNET1A–CFP, GFP–TUA6, and CYCD3;1–mCherry in N. benthamiana epidermal cells. Scale bar, 10 µm. c, The dex-inducible CYCD3;1–mCherry was coexpressed with PNET–CFP, PNET1A–CFP, or free CFP in N. benthamiana epidermal cells. Percentage of dividing cells after 24, 36, or 48 hours of dex treatment was quantified. Two hundred cells were examined per replicate (n = 10 replicates, two-tailed t-tests). Data are presented as mean values ± SD. d, Measurements of PNET1D–mCherry or PNET1A–mCherry signal intensity on the NE using the same microscope settings (n = 20 nuclei). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison tests. Exact P values are provided in the Source Data. e, Multiple sequence alignment of PNET1 and its homologs in indicated eukaryotic species using ClustalW. Positions of detected phosphorylation sites on AtPNET1 are outlined by red boxes, and conserved phosphorylation sites are colored with an orange background. The design of partial phospho-mimic PNET1 mutant variants (PNET1Da–e) is labeled below the sequence alignment. f, Transient coexpression of PNET1 and PNET1 phospho-mimic/deficient variants fused to mCherry with Nup160–GFP in N. benthamiana. The experiment was repeated twice with similar results. Scale bars, 10 μm. g and h, Representative images of 6-day-old WT and 35S::PNET1D–YFP transgenic seedlings in WT (g) and pnet1 mutant (h) background, along with measurements of their root length (n = 40 for each line, two-tailed t-tests). Scale bars, 1 cm. For all box plots, the lower and upper whiskers indicate the minimum and maximum values, respectively. The upper boundary, middle line, and the lower boundary of the box indicate the 75th percentile, medium, and 25th percentile, respectively.
Supplementary information
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Supplementary Tables 1–4.
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Statistical source data.
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Unprocessed western blots for Fig. 4d.
Source Data Fig. 5 (download PDF )
Unprocessed western blots for Fig. 5a,b,d.
Source Data Extended Data Fig. 4 (download PDF )
Unprocessed western blots for Extended Data Fig. 4a.
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Fang, Y., Tang, Y., Xie, P. et al. Nucleoporin PNET1 coordinates mitotic nuclear pore complex dynamics for rapid cell division. Nat. Plants 11, 295–308 (2025). https://doi.org/10.1038/s41477-025-01908-y
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DOI: https://doi.org/10.1038/s41477-025-01908-y
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