Yu Ka Lou, Keijzer Nanda, Hoogenraad Casper C, Akhmanova Anna
Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands.
Methods Mol Biol. 2011;777:293-316. doi: 10.1007/978-1-61779-252-6_21.
Microtubule organization and dynamics are controlled by a large set of cellular factors. An important group of microtubule regulators is microtubule plus-end-tracking proteins (+TIPs), which accumulate specifically at the growing microtubule ends, affect different phases of dynamic instability, and link microtubules to various cellular structures. +TIPs include a very diverse set of proteins with widely different structural properties. One of the most conserved and ubiquitous +TIP families are end-binding (EB) proteins, which can track growing microtubule ends autonomously in the absence of any other factors. In contrast, the majority of other known +TIPs cannot recognize the growing microtubule plus ends on their own; instead, they "hitchhike" to the plus ends by interacting with one of the members of the EB family. Therefore, the association with EBs and the ability to track growing microtubule ends are tightly linked, and binding to the EBs can be used to identify new +TIPs. In this chapter, we describe two affinity purification techniques, glutathione S-transferase and biotinylation tag-based pull-down assays that proved to be very useful for the identification of new EB-interacting +TIPs and their binding partners by mass spectrometry. We also discuss cytological techniques that can be applied to confirm plus-end localization of newly identified proteins.
微管的组织和动力学受大量细胞因子控制。微管调节因子的一个重要群体是微管正端追踪蛋白（+TIPs），它们特异性地聚集在生长中的微管末端，影响动态不稳定性的不同阶段，并将微管与各种细胞结构相连。+TIPs包括一组结构特性差异很大的蛋白质。最保守且普遍存在的+TIP家族之一是末端结合（EB）蛋白，它们在没有任何其他因子的情况下能够自主追踪生长中的微管末端。相比之下，大多数其他已知的+TIPs自身无法识别生长中的微管正端；相反，它们通过与EB家族的成员之一相互作用“搭便车”到正端。因此，与EBs的结合以及追踪生长中微管末端的能力紧密相连，与EBs的结合可用于鉴定新的+TIPs。在本章中，我们描述了两种亲和纯化技术，即基于谷胱甘肽S - 转移酶和生物素化标签的下拉分析，这些技术已被证明对于通过质谱鉴定新的与EB相互作用的+TIPs及其结合伙伴非常有用。我们还讨论了可用于确认新鉴定蛋白质正端定位的细胞学技术。
