Saengsawang Witchuda, Rasenick Mark M
Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Methods Cell Biol. 2013;115:173-89. doi: 10.1016/B978-0-12-407757-7.00012-8.
Microtubules, major components of the cytoskeleton, play important roles in a variety of cellular functions including mitosis, intracellular transport, and the modulation of cell morphology. Several studies have demonstrated that specific G-protein alpha subunits bind to tubulin with a high affinity (~130 nM) and elicit various functional effects on tubulin and microtubules. In this chapter, we present a description of the protocols for several methods that are used to determine the interaction between heterotrimeric G proteins and tubulin, as well as functional consequences of the interactions including protocols for protein purification, binding assays, tubulin GTPase assays, microtubule dynamics assays, and assays for cytoskeletal consequences of G-protein-coupled receptor signaling.
微管是细胞骨架的主要成分，在包括有丝分裂、细胞内运输和细胞形态调节在内的多种细胞功能中发挥重要作用。多项研究表明，特定的G蛋白α亚基以高亲和力（约130 nM）与微管蛋白结合，并对微管蛋白和微管产生各种功能影响。在本章中，我们描述了几种用于确定异源三聚体G蛋白与微管蛋白之间相互作用的方法的实验方案，以及这些相互作用的功能后果，包括蛋白质纯化、结合测定、微管蛋白GTP酶测定、微管动力学测定以及G蛋白偶联受体信号传导的细胞骨架后果测定的实验方案。
