Abstract
Faithful genome partition during cell division relies on proper congression of chromosomes to the spindle equator before sister chromatid segregation. Here we uncover a kinesin-7 motor, kinetochore-associated kinesin 1 (KAK1), that is required for mitotic chromosome congression in Arabidopsis. KAK1 associates dynamically with kinetochores throughout mitosis. Loss of KAK1 results in severe defects in chromosome congression at metaphase, yet segregation errors at anaphase are rarely observed. KAK1 specifically interacts with the spindle assembly checkpoint protein BUB3.3 and both proteins show interdependent kinetochore localization. Chromosome misalignment in BUB3.3-depleted plants can be rescued by artificial tethering of KAK1 to kinetochores but not vice versa, demonstrating that KAK1 acts downstream of BUB3.3 to orchestrate microtubule-based chromosome transport at kinetochores. Moreover, we show that KAK1’s motor activity is essential for driving chromosome congression to the metaphase plate. Thus, our findings reveal that plants have repurposed BUB3.3 to interface with a specialized kinesin adapted to integrate proper chromosome congression and checkpoint control through a distinct kinetochore design.
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All data of this study are available in the main text or the Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We thank A. Schnittger and S. Komaki for sharing the SAC plasmids and T. Nakagawa for providing pGWB vectors. This study was supported by the National Natural Science Foundation of China (grant no. 32270354), Natural Science Foundation of Sichuan Province (grant no. 2022NSFSC1651) and Sichuan Forage Innovation Team Program (grant no. SCCXTD-2024-16).
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X.D. conceived and supervised the project. X.D., X.T., Y.T. and Y.H. performed most of the experiments. X.D., X.T. and Y.T. analysed the data. X.D. wrote the paper. K.C., H.L. and B.L. read and revised the paper. All authors approved the paper submission.
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Extended data
Extended Data Fig. 1 KAK1 belongs to the kinesin-7 group.
The phylogenetic tree shows the evolutionary relationship of the KAK1 protein from the plant species A. thaliana to other members of the kinesin-7 subfamily, including CENP-E from H. sapiens and KIP2 from S. cerevisiae. Amino acid sequences are aligned using the MUSCLE method. The tree is constructed using the neighbor-joining method within MEGA software, with gaps automatically removed using the complete-deletion option. The estimated evolutionary distances are indicated by the numbers above each branch, representing amino acid substitutions per site. The reliability of each branch is shown by the score next to each node, with the highest value being 100.
Extended Data Fig. 2 Defective chromosome congression in the kak1 mutant.
a, Chromosomes congression and segregation in WT and kak1 plants with or without oryzalin treatment. The fluorescent signals are detected by immunofluorescence, with MTs detected by the anti-tubulin antibody shown in green, and DNA detected by DAPI shown in magenta. Scale bars, 5 μm. b, Quantitative assessment of cells exhibiting misaligned chromosomes at metaphase and anaphase.
Extended Data Fig. 3 Pull-down assays of recombinant MBP fusions of KAK1 variants with GST- BUB3.3 immobilized beads.
The additional bands in the KAK1(1-600) sample line likely correspond to degradation products of the KAK1 N-terminal fragment during the co-purification process. The experiment was repeated three times with similar results.
Extended Data Fig. 4 Impact of SAC mutants on chromosome congression.
a-f, Representative immunofluorescence images of mitotic cells from different SAC mutant backgrounds, including bub3.3 (a), mps1 (b), bmf1 (c), bmf2 bmf3 (d), mad1 (e), and mad2 (f). The MTs are visualized in green using an anti-tubulin antibody, and DNA is stained with DAPI (magenta). Scale bars, 5 μm. g-h, Quantitative assessment of cells exhibiting misaligned chromosomes at metaphase (g) and anaphase (h) in different SAC mutant backgrounds.
Extended Data Fig. 5 Loss of KAK1 in mad1 and bub3.3 mutants does not affect plant growth.
Representative image of growth phenotypes of 3-week-old WT, kak1, mad1, kak1 mad1, bub3.3, and kak1 bub3.3 plants. Scale bars, 1 cm.
Extended Data Fig. 6 The kak1 mad1 double mutant exhibits increased sensitivity to oryzalin treatment.
a, Representative images of 10-day-old seedlings of the WT, kak1, mad1, and kak1 mad1 genotypes, grown either with or without 75 nM oryzalin. Scale bars, 1 cm. b, Quantification of root lengths in the seedlings with and without oryzalin treatment. Graph bars represent means ± SD of six seedlings per genotype. The statistical significance (***P < 10-6) was determined by one-way ANOVA followed by Tukey test. The experiment was repeated three times with similar results.
Extended Data Fig. 7 The kak1 mad1 double mutant generates aneuploid daughter cells.
a, Representative immunofluorescence images of WT cells (n = 52) at the end of mitosis, where the forming daughter cells consistently exhibit 10 kinetochore signals, as detected by the anti-CENH3 antibody (green). The MTs (magenta) and DNA (blue) are also visualized. b, Representative immunofluorescence images of kak1 mad1 mutant cells (n = 55) displaying aberrant chromosome segregation patterns. In the double mutant cells, the daughter cell pairs exhibit an unequal distribution of kinetochore signals, with pairs having 9 + 11 or 8 + 12 CENH3 signals. The immunofluorescence images are obtained through confocal-based z-stack projections of cells undergoing cytokinesis, allowing the visualization of all kinetochore signals in the forming daughter cells. Scale bars, 5 μm.
Extended Data Fig. 8 KAK1 is not essential for kinetochore localization of core SAC proteins.
a-b, GFP-MAD1 is detected at kinetochores upon expression in mad1 mutant (a) and kak1 mutant (b) backgrounds in representative cells at prophase (top row) and metaphase (bottom row). c-d, BMF3-GFP is detected at kinetochores upon expression in bmf3 mutant (c) and kak1 (d) mutant backgrounds. The fluorescent signals are detected by immunofluorescence and merged images have GFP-tagged proteins detected by anti-GFP shown in green, MTs detected by anti-tubulin shown in magenta, and DNA detected by DAPI shown in blue. Micrographs are representative of more than 60 cells from three independent lines with similar results. Scale bars, 5 μm.
Supplementary information
Supplementary Information
Supplementary Figs. 1–6 and Table 1.
Supplementary Video 1
Live-cell imaging of WT cells expressing GFP–TUB6 and histone H1.2–RFP. Images were acquired every 20 s. Scale bars, 5 μm.
Supplementary Video 2
Live-cell imaging of kak1 mutant cells expressing GFP–TUB6 and histone H1.2–RFP. Images were acquired every 20 s. Scale bars, 5 μm.
Supplementary Video 3
Live-cell imaging of mad1 mutant cells expressing GFP–TUB6 and histone H1.2–RFP. Images were acquired every 20 s. Scale bars, 5 μm.
Supplementary Video 4
Live-cell imaging of kak1 mad1 double-mutant cells expressing GFP–TUB6 and histone H1.2–RFP. Images were acquired every 20 s. Scale bars, 5 μm.
Supplementary Video 5
Live-cell imaging of bub3.3 mutant cells expressing GFP–TUB6 and histone H1.2–RFP. Images were acquired every 20 s. Scale bars, 5 μm.
Supplementary Video 6
Live-cell imaging of kak1 bub3.3 double mutant cells expressing GFP–TUB6 and histone H1.2–RFP. Images were acquired every 20 s. Scale bars, 5 μm.
Supplementary Data 1
Co-immunoprecipitation and mass spectrometry results for GFP–BUB3.3 as a bait.
Supplementary Data 2
Co-immunoprecipitation and mass spectrometry results for KAK1–GFP as a bait.
Source data
Source Data Fig. 1
Statistical source data.
Source Data Fig. 6
Statistical source data.
Source Data Fig. 7
Statistical source data.
Source Data Extended Data Fig. 3
Unprocessed western blots.
Source Data Extended Data Fig. 6
Statistical source data.
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Tang, X., He, Y., Tang, Y. et al. A kinetochore-associated kinesin-7 motor cooperates with BUB3.3 to regulate mitotic chromosome congression in Arabidopsis thaliana. Nat. Plants 10, 1724–1736 (2024). https://doi.org/10.1038/s41477-024-01824-7
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DOI: https://doi.org/10.1038/s41477-024-01824-7
