Okabe S, Hirokawa N
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
J Cell Biol. 1988 Aug;107(2):651-64. doi: 10.1083/jcb.107.2.651.
To study microtubule (MT) dynamics in nerve cells, we microinjected biotin-labeled tubulin into the cell body of chemically fused and differentiated PC12 cells and performed the immunofluorescence or immunogold procedure using an anti-biotin antibody followed by secondary antibodies coupled to fluorescent dye or colloidal gold. Incorporation of labeled subunits into the cytoskeleton of neurites was observed within minutes after microinjection. Serial electron microscopic reconstruction revealed that existing MTs in PC12 neurites incorporated labeled subunits mainly at their distal ends and the elongation rate of labeled segments was estimated to be less than 0.3 micron/min. Overall organization of MTs in the nerve cells was different from that in undifferentiated cells such as fibroblasts. Namely, we have not identified any MT-organizing centers from which labeled MTs are emanating in the cell bodies of the injected cells. Stereo electron microscopy revealed that some fully labeled segments seemed to start in the close vicinity of electron dense material within the neurites. This suggests new nucleation off some structures in the neurites. We have also studied the overall pattern of the incorporation of labeled subunits which extended progressively from the proximal part of the neurites toward their tips. To characterize the mechanism of tubulin incorporation, we have measured mean density of gold labeling per unit length of labeled segments at different parts of the neurites. The results indicate access of free tubulin subunits into the neurites and local incorporation into the neurite cytoskeleton. Our results lead to the conclusion that MTs are not static polymers but dynamic structures that continue to elongate even within the differentiated nerve cell processes.
为了研究神经细胞中的微管（MT）动力学，我们将生物素标记的微管蛋白显微注射到化学融合并分化的PC12细胞的细胞体中，并使用抗生物素抗体，随后是偶联荧光染料或胶体金的二抗，进行免疫荧光或免疫金程序。显微注射后几分钟内即可观察到标记的亚基掺入神经突的细胞骨架中。连续电子显微镜重建显示，PC12神经突中现有的微管主要在其远端掺入标记的亚基，标记段的伸长率估计小于0.3微米/分钟。神经细胞中微管的整体组织与成纤维细胞等未分化细胞中的不同。也就是说，我们在注射细胞的细胞体中未发现任何发出标记微管的微管组织中心。立体电子显微镜显示，一些完全标记的段似乎始于神经突内电子致密物质的附近。这表明在神经突中的某些结构上有新的成核作用。我们还研究了标记亚基掺入的整体模式，其从神经突的近端部分逐渐向其尖端延伸。为了表征微管蛋白掺入的机制，我们测量了神经突不同部位标记段每单位长度的金标记平均密度。结果表明游离微管蛋白亚基进入神经突并局部掺入神经突细胞骨架。我们的结果得出结论，微管不是静态聚合物，而是动态结构，即使在分化的神经细胞过程中也会继续伸长。
