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Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

Masako Okumura, Toyoaki Natsume, Masato T. Kanemaki, Tomomi Kiyomitsu
doi: https://doi.org/10.1101/277202
Masako Okumura
1Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
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Toyoaki Natsume
3Division of Molecular Cell Engineering, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111, Mishima, Shizuoka 411-8540, Japan
4Department of Genetics, SOKENDAI, Yata 1111, Mishima, Shizuoka 411-8540, Japan
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Masato T. Kanemaki
2Precursory Research for Embryonic Science and Technology (PRESTO) Program, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
3Division of Molecular Cell Engineering, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111, Mishima, Shizuoka 411-8540, Japan
4Department of Genetics, SOKENDAI, Yata 1111, Mishima, Shizuoka 411-8540, Japan
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Tomomi Kiyomitsu
1Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
2Precursory Research for Embryonic Science and Technology (PRESTO) Program, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
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  • For correspondence: kiyomitsu@bio.nagoya-u.ac.jp
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Abstract

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment/activation by NuMA’s N-terminal long arm, and NuMA’s direct microtubule-binding activities to achieve a multiplicity of microtubule interactions. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

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Posted March 06, 2018.
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Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble
Masako Okumura, Toyoaki Natsume, Masato T. Kanemaki, Tomomi Kiyomitsu
bioRxiv 277202; doi: https://doi.org/10.1101/277202
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Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble
Masako Okumura, Toyoaki Natsume, Masato T. Kanemaki, Tomomi Kiyomitsu
bioRxiv 277202; doi: https://doi.org/10.1101/277202

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