Active transport of photoactivated tubulin molecules in growing axons revealed by a new electron microscopic analysis
Active transport of photoactivated tubulin molecules in growing axons revealed by a new electron microscopic analysis
复制标题
DOI：
10.1083/jcb.133.6.1347
复制
发表时间：
1996-06-01
影响因子：
7.8
通讯作者：
Hirokawa, N
中科院分区：
文献类型：
--
作者：
Funakoshi, T;Takeda, S;Hirokawa, N
为了确定是否微管蛋白分子在轴突中运输的聚合物或寡聚体，我们进行了光活化后的微管蛋白分子的运动的电子显微镜分析。虽然以前的光学显微镜分析后，光漂白或光活化表明，大多数的轴突微管是固定的，他们没有足够的灵敏度，以允许检测积极运输的微管蛋白分子，预计只有一小部分的总微管蛋白分子的轴突。此外，最近使用间接方法的一些研究表明主动聚合物转运是微管蛋白转运的机制（Baas，P.W.，F. J.艾哈迈德1993. 120：1427-1437; Yu，W.，V.E. Centonze，F.J. Ahmad和P.W.低音. 1993. 122：349-359; Ahmad，F.J.，和P.W.低音. 1995. J. Cell Sci 108：2761-2769）。因此，运输的微管蛋白分子是否是聚合物仍有待确定。为了澄清这个问题，我们使用光活化技术在轴突中的微管蛋白分子上进行荧光标记，并使用抗荧光素抗体进行电子显微镜免疫细胞化学。利用这种新的方法，我们实现了高分辨率和高灵敏度的检测微管蛋白分子的运输。在透化后固定的细胞中，我们没有发现易位的微管。在那些固定而不透化，其中除了聚合物的低聚物和异二聚体被保存，我们发现更多的标签在该地区的远端的光活化区域比在近端区域。这些数据表明，微管蛋白分子的运输不是作为聚合物，而是作为异源二聚体或寡聚体的主动机制，而不是通过扩散。
To determine whether tubulin molecules transported in axons are polymers or oligomers, we carried out electron microscopic analysis of the movement of the tubulin molecules after photoactivation. Although previous optical microscopic analyses after photobleaching or photoactivation had suggested that most of the axonal microtubules were stationary, they were not sufficiently sensitive to allow detection of actively transported tubulin molecules which were expected to be only a small fraction of total tubulin molecules in axons. In addition, some recent studies using indirect approaches suggested active polymer transport as a mechanism for tubulin transport (Baas, P.W., F.J. Ahmad. 1993. J. Cell Biol. 120:1427-1437; Yu, W., V.E. Centonze, F.J. Ahmad, and P.W. Bass. 1993. J. Cell Biol. 122:349-359; Ahmad, F.J., and P.W. Bass. 1995. J. Cell Sci 108:2761-2769). So, whether transported tubulin molecules are polymers or not remain to be determined. To clear up this issue, we made fluorescent marks on the tubulin molecules in the axons using a photoactivation technique and performed electron microscopic immunocytochemistry using anti-fluorescein antibody. Using this new method we achieved high resolution and high sensitivity for detecting the transported tubulin molecules. In cells fixed after permeabilization, we found no translocated microtubules. In those fixed without permeabilization, in which oligomers and heterodimers in addition to polymers were preserved, we found much more label in the regions distal to the photoactivated regions than in the proximal regions. These data indicated that tubulin molecules are transported not as polymers but as heterodimers or oligomers by an active mechanism rather than by diffusion.