Mercken M, Fischer I, Kosik K S, Nixon R A
Laboratory for Molecular Neuroscience, McLean Hospital, Belmont, Massachusetts 02178, USA.
J Neurosci. 1995 Dec;15(12):8259-67. doi: 10.1523/JNEUROSCI.15-12-08259.1995.
Microtubule-associated proteins (MAPs), such as tau, modulate neuronal shape and process outgrowth by influencing the stability and organization of microtubules. The dynamic nature of MAP-microtubule interactions in vivo, however, is poorly understood. Here, we have assessed the stability of these interactions by investigating the synthesis and axoplasmic transport of tau in relation to that of tubulin and other MAPs within retinal ganglion cells of normal adult mice in vivo. Using immunoprecipitation and Western blot analysis with anti-tau monoclonal and polyclonal antibodies, we unequivocally identified in optic axons a family of 50-60 kDa tau isoforms and a second 90-95 KDa tau family, the members of which were shown to contain the domain of tau encoded by exon 4A. To measure the rates of translocation of tau proteins in vivo, we injected mice with 35S-methionine intravitreously and, after 6-30 d, quantitated the radiolabeled tau isoforms immunoprecipitated from eight consecutive 1.1 mm segments of the nerve and optic tract and separated by electrophoresis. Linear regression analysis of protein transport along optic axons showed that the tau isoforms advanced at a rate of 0.2-0.4 mm/d, and other radiolabeled MAPs, identified by their association with taxol-stabilized microtubules, moved three- to fivefold more rapidly. By contrast, tubulins advanced at 0.1-0.2 mm/d, significantly more slowly than tau or other MAPs. These studies establish that tau is not cotransported with tubulin or microtubules, indicating that associations of tau with microtubules within axons are not as stable as previously believed. Our findings also reveal differences among various MAPs in their interactions with microtubules and provide evidence that assembly and reorganization of the microtubule network is an active process even after axons establish connections and fully mature.
微管相关蛋白（MAPs），如tau蛋白，通过影响微管的稳定性和组织来调节神经元的形状和突起生长。然而，体内MAP-微管相互作用的动态特性仍知之甚少。在这里，我们通过研究正常成年小鼠视网膜神经节细胞内tau蛋白与微管蛋白及其他MAPs的合成和轴浆运输，评估了这些相互作用的稳定性。利用抗tau单克隆和多克隆抗体进行免疫沉淀和蛋白质印迹分析，我们在视神经轴突中明确鉴定出一个50 - 60 kDa的tau蛋白异构体家族和第二个90 - 95 kDa的tau蛋白家族，其成员显示含有由外显子4A编码的tau结构域。为了测量体内tau蛋白的转运速率，我们向小鼠玻璃体内注射35S-甲硫氨酸，6 - 30天后，对从神经和视束连续的8个1.1 mm节段中免疫沉淀并经电泳分离的放射性标记tau蛋白异构体进行定量。沿视神经轴突的蛋白质运输的线性回归分析表明，tau蛋白异构体以0.2 - 0.4 mm/d的速率前进，而通过与紫杉醇稳定的微管结合鉴定的其他放射性标记MAPs移动速度快三到五倍。相比之下，微管蛋白以0.1 - 0.2 mm/d的速率前进，明显慢于tau蛋白或其他MAPs。这些研究表明，tau蛋白不与微管蛋白或微管共同运输，这表明轴突内tau蛋白与微管的结合不如先前认为的那么稳定。我们的研究结果还揭示了各种MAPs与微管相互作用的差异，并提供证据表明即使在轴突建立连接并完全成熟后，微管网络的组装和重组也是一个活跃的过程。
