Institute for Molecular and Cell Physiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
J Biol Chem. 2012 Nov 9;287(46):38559-68. doi: 10.1074/jbc.M112.369785. Epub 2012 Sep 27.
Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.
目前，关于 Tau 蛋白在细胞内的转运模型表明，Tau 蛋白与微管片段一起通过马达蛋白共转运，并且在细胞质中扩散，而 Tau 蛋白与微管结合时被认为是静止的，与细胞质中游离扩散处于平衡状态。观察到微管依赖性驱动蛋白家族的成员在微管上表现出布朗运动，这使我们假设 Tau 蛋白的扩散也可能与微管有关。我们使用单分子全内反射荧光显微镜来探测单个荧光标记的 Tau 分子在微管上的扩散。这使我们能够避免由于溶液中标记的 Tau 过量而掩盖微管依赖性扩散的问题，这种情况可能会在体内过表达实验中发生。我们发现，大约一半的单独检测到的 Tau 分子能够在微管上双向移动，距离可达数微米。扩散参数，如扩散系数、相互作用时间和扫描微管长度，与 Tau 浓度无关。然而，Tau 与微管晶格的结合和扩散对离子强度和 pH 敏感，并且在微管蛋白的带负电荷的 C 末端被酶去除后会大大减少。我们提出 Tau 在微管晶格的引导下进行一维扩散，这是 Tau 分布的另一种可能的机制。通过这种一维微管晶格扩散，Tau 可以被引导到微管的两端，即在微管聚合过程中需要 Tau 的部位，而不依赖于定向马达蛋白依赖性运输。在微管沿微管的主动运输可能受到损害的情况下，这可能很重要。
