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

Abstract

<jats:p>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.</jats:p>

chTOG is a conserved mitotic error correction factor

“…Previous microscopy studies suggested that chTOG localizes to kinetochores ( Campbell et al, 2019 ; Gutiérrez-Caballero et al, 2015 ; Ryan et al, 2020 ), similar to the budding yeast ortholog ( Miller et al, 2016 ; Miller et al, 2019 ); however, it was unclear if this population was simply bound to microtubule tips. To address this, we used engineered HCT116 cells where the endogenous chTOG genes were epitope tagged with EGFP ( Cherry et al, 2019 ) to determine whether chTOG specifically localizes to kinetochores throughout mitosis ( Figure 1a ).…”

Phase Separation during Germline Development

“…Endogenous TACC3 does not appear to phase-separate in mitotic cells, regardless of the presence or absence of centrosomes [98,103,104]. The LISD is thus likely a meiosis-specific mechanism for enriching microtubule regulatory factors within the large cytoplasmic volume of oocytes.…”

High Expression of TACC3 Is Associated with the Poor Prognosis and Immune Infiltration in Lung Adenocarcinoma Patients

“…This research was centered on TACC3, which is a well-recognized member of the TACC family and a centrosome and microtubuleassociated protein [29]. Whereas the function of TACC3 is yet to be comprehensively clarified, cumulative proof from research reports indicates that TACC3 is necessary for attachment of kinetochore-microtubule, assembly of centrosome dependent microtubule, and the alignment of spindledependent chromosome in the process of mitosis [30,31].…”