Ahmad F J, Pienkowski T P, Baas P W
Department of Anatomy, University of Wisconsin Medical School, Madison 53706.
J Neurosci. 1993 Feb;13(2):856-66. doi: 10.1523/JNEUROSCI.13-02-00856.1993.
We have used an indirect method to compare the dynamic properties of microtubules (MTs) in the main shaft and distal regions of the axon. Individual MTs are staggered along the length of the axon and consist of a labile domain situated at the plus end of a stable domain (Baas and Black, 1990). As a result of this organization and the plus-end-distal orientation of axonal MTs, the most distal region of the axon consists entirely of labile domains, while the main shaft consists of a mixture of labile and stable domains. In this study, we wished to determine whether the labile domains extending into the distal axon differ in their dynamic properties from the labile domains terminating in the main shaft. To address this issue, we used immunoelectron microscopy to compare the tyrosination state of the labile domains terminating in these 2 axon regions. Because detyrosination is a polymerspecific modification of alpha-tubulin that accumulates with time, the levels of tyrosinated alpha-tubulin will be a reflection of the age, and hence dynamic properties, of the polymer. To maximize our chances of visualizing potential differences, we varied the concentration of the primary antibody in these experiments. Our studies indicate that the stable domains are generally deficient in tyrosinated alpha-tubulin, while the labile domains contain clearly detectable levels. Within the labile domain, the subsection closer to the plus end of the MT contains relatively higher levels of tyrosinated alpha-tubulin than does the subsection farther from the plus end, suggesting that the levels of tyrosinated alpha-tubulin in the labile domain may gradually increase as one moves away from the stable domain toward the plus end of the MT. Although these observations apply to the labile domains in both regions of the axon, the labile domains extending into the distal region contain comparatively higher levels of tyrosinated alpha-tubulin than do the labile domains terminating in the main shaft. These results are consistent with the view that highly dynamic MT polymer is present throughout the axon, but that the polymer nearest the advancing growth cone is particularly dynamic.
我们采用了一种间接方法来比较轴突主轴和远端区域中微管（MTs）的动态特性。单个微管沿轴突长度交错排列，由位于稳定结构域正端的不稳定结构域组成（巴斯和布莱克，1990年）。由于这种结构以及轴突微管的正端向远侧的取向，轴突的最远端区域完全由不稳定结构域组成，而主轴则由不稳定和稳定结构域的混合物组成。在本研究中，我们希望确定延伸至轴突远端的不稳定结构域在动态特性上是否与终止于主轴的不稳定结构域不同。为了解决这个问题，我们使用免疫电子显微镜来比较终止于这两个轴突区域的不稳定结构域的酪氨酸化状态。由于去酪氨酸化是α-微管蛋白的一种聚合物特异性修饰，会随时间积累，酪氨酸化α-微管蛋白的水平将反映聚合物的年龄，从而反映其动态特性。为了最大程度地增加观察到潜在差异的机会，我们在这些实验中改变了一抗的浓度。我们的研究表明，稳定结构域通常缺乏酪氨酸化α-微管蛋白，而不稳定结构域含有明显可检测到的水平。在不稳定结构域内，靠近微管正端的子部分比远离正端的子部分含有相对较高水平的酪氨酸化α-微管蛋白，这表明随着从稳定结构域向微管正端移动，不稳定结构域中酪氨酸化α-微管蛋白的水平可能会逐渐增加。尽管这些观察结果适用于轴突两个区域的不稳定结构域，但延伸至远端区域的不稳定结构域比终止于主轴的不稳定结构域含有相对较高水平的酪氨酸化α-微管蛋白。这些结果与以下观点一致，即高动态微管聚合物存在于整个轴突中，但最靠近前进生长锥处的聚合物特别具有动态性。
