Mechanism of microtubule stabilization in neurites : a novel approach capable of directly observing and testing microtubule stability.

在神经突的生长和伸长过程中，微管（MT）不仅数量增加，而且逐渐变得更加稳定。为了阐明稳定的分子机制，我们开发了一种新的方法，能够通过视频增强微分干涉对比（DIC）显微镜结合流动池技术直接观察神经炎MT的稳定性。我们发现，培养超过5天的背根神经节细胞的已建立的突起含有MT，其在细胞外持续时间更长，30分钟以上（Tashiro等，J.Neurosci.Res.，50，81 - 93，1997）。为了进一步研究微束激光照射后微束的形态变化，我们采用视频增强DIC显微镜观察微束激光照射后微束的长度变化。MT片段开始缩短两侧的横切网站，更迅速地从新产生的正端比从负端。缩短的速率和模式与膜去除后的持续时间相关;稳定的MT随着间歇性停顿缓慢缩短，而更不稳定的MT以更高的速率持续缩短。横切还揭示了稳定的MT锚定到基底或解体暂时停止的特定点的存在（Kurachi等人，从纯化的微管蛋白和MT相关蛋白tau重新组装的MT对通过灌注的稀释没有抗性。我们还发现天然tau的去磷酸化导致其MT成核活性的选择性降低（Morita-Fujimura et al.，生物化学、生物物理学、研究和通信，225，462 - 468，1996）。这些结果表明，除了通过MT相关蛋白沿着长度的稳定化之外，还必须考虑MT末端和特定点的保护。

During outgrowth and elongation of neurites, microtubules (MTs) not only increase in quantity but also become progressively more stabilized. To elucidate the molecular mechanism of stabilization, we developed a novel approach capable of directly observing the stability of neuritic MTs by video-enhanced differential interference contrast (DIC) microscopy combined with a flow cell technique.By dissolving the membrane with detergent perfusion, we found that the established neurites of dorsal root ganglion cells cultured for more than 5 days contained MTs which persisted outside the cell for more than 30 minutes (Tashiro et al.J.Neurosci.Res., 50,81-93,1997). These stable MTs were usually single and floating above the substratum, with only point attachments along the length.For further characterization, we transected the exposed MTs by laser microbeam irradiation and observed their length changes with video-enhanced DIC microscopy. MT fragments started to shorten on both sides of the transection site, more rapidly from the newly generated plus ends than from the minus ends. The rate and pattern of shortening correlated with the persistence time after membrane removal ; stable MTs shortened slowly with intermittent pauses, while the more labile MTs shortened continuously at higher rates. Transection also revealed the presence of specific points where stable MTs are anchored to the substratum or disassembly is transiently halted (Kurachi et al., submitted).MTs reassembled from purified tubulin and MT-associated protein tau were not resistant to dilution by perfusion. We also found that dephosphorylation of the native tau resulted in the selective decrease in its MT-nucleation activity (Morita-Fujimura et al., Biochem.Biophys.Res.Comm., 225,462-468,1996).These results show that protection at MT ends and at specific points must be considered in addition to stabilization along the length by MT-associated proteins.