Department of Materials, University of California, Santa Barbara, CA, USA.
Neuroscience Research Institute, University of California, Santa Barbara, CA, USA Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA.
J Alzheimers Dis. 2014;39(2):301-14. doi: 10.3233/JAD-131274.
We tested the hypothesis that mutant tau proteins that cause neurodegeneration and dementia differentially alter kinesin translocation along microtubules (MTs) relative to normal tau in vitro. We employed complementary in vitro motility assays using purified recombinant kinesin, purified recombinant tau, and purified bovine brain α:β tubulin to isolate interactions among these components without any contribution by cellular regulatory mechanisms. We found that kinesin translocates slower along MTs assembled by any of three independent tau mutants (4-repeat P301L tau, 4-repeat ΔN296 tau, and 4-repeat R406W tau) relative to its translocation rate along MTs assembled by normal, 4-repeat wild type (WT) tau. Moreover, the R406W mutation exhibited isoform specific effects; while kinesin translocation along 4-repeat R406W tau assembled MTs is slower than along MTs assembled by 4-repeat WT tau, the R406W mutation had no effect in the 3-repeat tau context. These data provide strong support for the notion that aberrant modulation of kinesin translocation is a component of tau-mediated neuronal cell death and dementia. Finally, we showed that assembling MTs with taxol before coating them with mutant tau obscured effects of the mutant tau that were readily apparent using more physiologically relevant MTs assembled with tau alone, raising important issues regarding the use of taxol as an experimental reagent and novel insights into therapeutic mechanisms of taxol action.
我们检验了这样一个假设，即引起神经退行性变和痴呆的突变 tau 蛋白与正常 tau 相比，会在体外不同地改变驱动蛋白沿微管（MTs）的易位。我们采用了互补的体外运动分析，使用纯化的重组驱动蛋白、纯化的重组 tau 蛋白和纯化的牛脑α：β微管蛋白，以分离这些成分之间的相互作用，而不受任何细胞调节机制的影响。我们发现，与正常的 4 重复 WT tau 组装的 MT 相比，任何三种独立的 tau 突变体（4 重复 P301L tau、4 重复ΔN296 tau 和 4 重复 R406W tau）组装的 MT 上，驱动蛋白的易位速度较慢。此外，R406W 突变表现出同工型特异性效应；虽然驱动蛋白沿 4 重复 R406W tau 组装的 MT 的易位速度比沿 4 重复 WT tau 组装的 MT 慢，但该突变在 3 重复 tau 背景中没有影响。这些数据为 tau 介导的神经元细胞死亡和痴呆是异常调节驱动蛋白易位的一个组成部分的观点提供了有力支持。最后，我们表明，在用突变 tau 涂覆之前用紫杉醇组装 MT 会掩盖突变 tau 的作用，而用单独的 tau 组装的更具生理相关性的 MT 则很容易观察到这些作用，这就提出了关于紫杉醇作为实验试剂的使用以及紫杉醇作用的治疗机制的新见解的重要问题。
