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 its composition and how it interacts with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein–protein interactions and, additionally, monitor 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 (also known as CKAP5) and GTSE1 are ancillary to the complex, binding respectively to TACC3 and clathrin, but not each other. We also show that PIK3C2A, a clathrin-binding protein that was proposed to stabilize the TACC3–chTOG–clathrin–GTSE1 complex during mitosis, is not a member of the 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.

含有 TACC3、网格蛋白和其他蛋白质的多蛋白复合物与有丝分裂纺锤体的稳定性有关。为了在抗癌背景下破坏这种复合物，我们需要了解它的组成以及它如何与微管相互作用。细胞中蛋白质的诱导重定位是分析蛋白质-蛋白质相互作用的有效方法，此外还可以监测这些相互作用发生的地点和时间。我们使用 CRISPR/Cas9 基因编辑为每个复杂成员添加串联 FKBP-GFP 标签。内源标记蛋白从有丝分裂纺锤体到线粒体的重新定位以及对其他蛋白质的影响的评估使我们能够确定 TACC3 和网格蛋白是核心复合体成员，并且 chTOG（也称为 CKAP5）和 GTSE1 是复合体的辅助成分，结合分别与 TACC3 和网格蛋白相关，但彼此不相关。我们还表明，PIK3C2A（一种网格蛋白结合蛋白，被提议在有丝分裂期间稳定 TACC3-chTOG-网格蛋白-GTSE1 复合物）并不是该复合物的成员。这项工作确定，靶向 TACC3-网格蛋白界面或其微管结合位点是最有可能破坏这种多蛋白复合物介导的纺锤体稳定性的两种策略。