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bioRxiv · 10.1101/2025.07.19.665708

CDK1-dependent N-terminal NuMA phosphorylation promotes dynein-dynactin-NuMA assembly for accurate chromosome segregation

van Toorn, M.·Shintomi, K.·Kiyomitsu, T.
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Abstract

The microtubule-based motor dynein and its cofactor dynactin fulfil essential functions throughout the cell cycle, including organelle transport and mitotic spindle assembly. To achieve these diverse functions, dynein-dynactin associates with different activating adaptors. Nuclear Mitotic Apparatus (NuMA) is a mitosis-specific adaptor that connects dynein-dynactin with microtubules to focus mitotic spindle poles. NuMAs C-terminal microtubule-binding activity is promoted by mitotic phosphorylation, but regulation of NuMAs N-terminal interaction with dynein-dynactin remains unclear. Here, we combine a membrane-tethering assay, quantitative proteomics, and live functional analyses in human cells to show that the interaction between NuMAs N-terminus and dynein-dynactin is cell cycle-regulated and driven by mitotic phosphorylation. We identify highly conserved CDK1 consensus sites proximal to NuMAs dynein heavy chain-binding site, which are phosphorylated by CDK1-Cyclin B1 in cells and in vitro. This CDK1-dependent phosphorylation, together with NuMAs Spindly-like motif, is crucial for stable dynein-dynactin-NuMA (DDN) complex formation. Replacement of endogenous NuMA with phosphorylation-deficient NuMA mutants leads to aberrant dynein distribution on mitotic spindles, resulting in chromosome mis-segregation and micronucleus formation. Together, our results highlight CDK1-dependent N-terminal NuMA phosphorylation as a crucial mitotic switch that constitutes a regulatable core of multivalent interactions with dynein-dynactin to assemble stable DDN complexes for accurate chromosome segregation.

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van Toorn, M., Shintomi, K., Kiyomitsu, T.. 2025-07-21. CDK1-dependent N-terminal NuMA phosphorylation promotes dynein-dynactin-NuMA assembly for accurate chromosome segregation. https://doi.org/10.1101/2025.07.19.665708

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