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Defining endogenous TACC3–chTOG–clathrin–GTSE1 interactions at the mitotic spindle using induced relocalization

By Ellis L. Ryan, James Shelford, Teresa Massam-Wu, Richard Bayliss, Stephen J Royle

Posted 01 Jul 2020
bioRxiv DOI: 10.1101/2020.07.01.181818

A multiprotein complex containing TACC3, clathrin, and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand the composition of the complex and the interactions between complex members and with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein-protein interactions and additionally monitoring where and when these interactions occur. We used CRISPR/Cas9 gene-editing to add tandem FKBP-GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG and GTSE1 are ancillary to the complex, respectively binding to TACC3 and clathrin, but not each other. PIK3C2A, a membrane trafficking protein that binds clathrin, was previously proposed to also bind TACC3 and stabilize the TACC3-chTOG-clathrin-GTSE1 complex during mitosis. We show that PIK3C2A is not on the mitotic spindle and that knockout of this gene had no effect on the localization of the complex. We therefore conclude that PIK3C2A is not a member of the TACC3-chTOG-clathrin-GTSE1 complex. This work establishes that targeting the TACC3-clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex. ### Competing Interest Statement The authors have declared no competing interest.

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