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The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles

Y. Rose Citron, Carey J. Fagerstrom, Bettina Keszthelyi, Bo Huang, Nasser M Rusan, Mark J S Kelly, View ORCID ProfileDavid A. Agard
doi: https://doi.org/10.1101/200204
Y. Rose Citron
1University of California, San Francisco
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Carey J. Fagerstrom
2National Heart, Lung, and Blood Institute, National Institutes of Health
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Bettina Keszthelyi
1University of California, San Francisco
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Bo Huang
3University of California, San Francisco & Chan Zuckerberg Biohub
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Nasser M Rusan
2National Heart, Lung, and Blood Institute, National Institutes of Health
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Mark J S Kelly
1University of California, San Francisco
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David A. Agard
4University of California, San Francisco & Howard Hughes Medical Institute
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  • ORCID record for David A. Agard
  • For correspondence: agard@msg.ucsf.edu citron@msg.ucsf.edu
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Abstract

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes a significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution interactions are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results we suggest a model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.

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Posted October 10, 2017.
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The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles
Y. Rose Citron, Carey J. Fagerstrom, Bettina Keszthelyi, Bo Huang, Nasser M Rusan, Mark J S Kelly, David A. Agard
bioRxiv 200204; doi: https://doi.org/10.1101/200204
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The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles
Y. Rose Citron, Carey J. Fagerstrom, Bettina Keszthelyi, Bo Huang, Nasser M Rusan, Mark J S Kelly, David A. Agard
bioRxiv 200204; doi: https://doi.org/10.1101/200204

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