Funakoshi T, Takeda S, Hirokawa N
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo, Japan.
J Cell Biol. 1996 Jun;133(6):1347-53. doi: 10.1083/jcb.133.6.1347.
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.
为了确定在轴突中运输的微管蛋白分子是聚合物还是寡聚体，我们对光激活后微管蛋白分子的运动进行了电子显微镜分析。尽管先前在光漂白或光激活后的光学显微镜分析表明大多数轴突微管是静止的，但它们的灵敏度不足以检测到预期仅占轴突中微管蛋白分子总数一小部分的主动运输的微管蛋白分子。此外，最近一些使用间接方法的研究表明，主动聚合物运输是微管蛋白运输的一种机制（巴斯，P.W.，F.J.艾哈迈德。1993。《细胞生物学杂志》120:1427 - 1437；于，W.，V.E.森通泽，F.J.艾哈迈德，和P.W.巴斯，1993，《细胞生物学杂志》122:349 - 359；艾哈迈德，F.J.，和P.W.巴斯。1995。《细胞科学杂志》108:2761 - 2769）。因此，运输的微管蛋白分子是否为聚合物仍有待确定。为了弄清楚这个问题，我们使用光激活技术在轴突中的微管蛋白分子上做了荧光标记，并使用抗荧光素抗体进行电子显微镜免疫细胞化学分析。使用这种新方法，我们在检测运输的微管蛋白分子方面实现了高分辨率和高灵敏度。在通透后固定的细胞中，我们没有发现易位的微管。在未通透固定的细胞中，除了聚合物外还保留了寡聚体和异二聚体，我们发现在光激活区域远端的区域比近端区域有更多的标记。这些数据表明，微管蛋白分子不是作为聚合物运输，而是作为异二聚体或寡聚体通过主动机制而非扩散进行运输。
