Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells
Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells
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从哺乳动物细胞中分离功能性微管蛋白二聚体和微管蛋白相关蛋白
DOI：
10.1016/j.cub.2016.04.069
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发表时间：
2016
期刊：
影响因子：
9.2
通讯作者：
N. Galjart
中科院分区：
文献类型：
--
作者：
Nuo Yu;L. Signorile;Sreya Basu;Sophie Ottema;J. Lebbink;K. Leslie;Ihor Smal;D. Dekkers;Jeroen A. A. Demmers;N. Galjart
微管（MT）细胞骨架形成一个动态的丝状网络，对许多过程至关重要，包括有丝分裂、细胞极性和形状、神经突生长和迁移以及纤毛发生[1,2]。MTs由α/β-微管蛋白异源二聚体组成，其动态行为部分受微管蛋白相关蛋白（TAPs）调控。在这里，我们描述了一个新的系统来研究哺乳动物微管蛋白和tap。我们使用2A“自切割”肽共表达等量的三标记α-微管蛋白和β-微管蛋白，并通过快速两步法从转染的HEK293T细胞中分离出功能性荧光微管蛋白二聚体。我们还制造了两种突变的小管蛋白，它们在小管病患者中引起脑畸形。然后，我们应用基于配对质谱的方法鉴定HEK293T细胞中的微管蛋白结合蛋白，并描述了新的和已知的tap。我们发现CKAP5和具有TOG(L)结构域[4]的MT +末端跟踪蛋白CLASPs可以有效地结合微管蛋白，高尔基蛋白相关蛋白GCC185也可以有效地与CLASPs[5]相互作用。CLASP1的n端TOGL结构域有助于微管蛋白结合，并允许CLASP1作为自主的mt生长促进因子发挥作用。有趣的是，突变型微管蛋白与许多tap（包括CLASPs和GCC185）结合较差，并且与细胞mt结合效率较低。此外，这些突变体在细胞中的表达会损害涉及tap的几种mt生长相关过程。因此，稳定的微管蛋白- tap相互作用调节MT在细胞中的成核和生长。总之，我们的研究结果为研究微管蛋白的相互作用和功能提供了资源，并扩大了微管蛋白相关疾病机制的范围。
The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated by tubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cells and describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains [4], bind tubulin efficiently, as does the Golgi-associated protein GCC185, which interacts with the CLASPs [5]. The N-terminal TOGL domain of CLASP1 contributes to tubulin binding and allows CLASP1 to function as an autonomous MT-growth-promoting factor. Interestingly, mutant tubulins bind less well to a number of TAPs, including CLASPs and GCC185, and incorporate less efficiently into cellular MTs. Moreover, expression of these mutants in cells impairs several MT-growth-related processes involving TAPs. Thus, stable tubulin-TAP interactions regulate MT nucleation and growth in cells. Combined, our results provide a resource for investigating tubulin interactions and functions and widen the spectrum of tubulin-related disease mechanisms.
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影响因子：
11.8
作者：
Al-Bassam, Jawdat;Kim, Hwajin;Brouhard, Gary;van Oijen, Antoine;Harrison, Stephen C.;Chang, Fred
通讯作者：
Chang, Fred
CLASP2 具有两个不同的 TOG 结构域，对微管动力学的贡献不同。
影响因子：
5.6
作者：
Maki,Takahisa;Grimaldi,AshleyD;Fuchigami,Sotaro;Kaverina,Irina;Hayashi,Ikuko
通讯作者：
Hayashi,Ikuko
影响因子：
64.5
作者：
Tischfield MA;Baris HN;Wu C;Rudolph G;Van Maldergem L;He W;Chan WM;Andrews C;Demer JL;Robertson RL;Mackey DA;Ruddle JB;Bird TD;Gottlob I;Pieh C;Traboulsi EI;Pomeroy SL;Hunter DG;Soul JS;Newlin A;Sabol LJ;Doherty EJ;de Uzcátegui CE;de Uzcátegui N;Collins ML;Sener EC;Wabbels B;Hellebrand H;Meitinger T;de Berardinis T;Magli A;Schiavi C;Pastore-Trossello M;Koc F;Wong AM;Levin AV;Geraghty MT;Descartes M;Flaherty M;Jamieson RV;Møller HU;Meuthen I;Callen DF;Kerwin J;Lindsay S;Meindl A;Gupta ML Jr;Pellman D;Engle EC
通讯作者：
Engle EC
影响因子：
3.5
作者：
Tian, Guoling;Jaglin, Xavier H.;Cowan, Nicholas J.
通讯作者：
Cowan, Nicholas J.
影响因子：
11.8
作者：
Efimov, Andrey;Kharitonov, Alexey;Kaverina, Irina
通讯作者：
Kaverina, Irina