Shirasu M, Morihara T, Okajima S, Tamai K, Mizoguchi A, Ide C, Hirasawa Y
Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Japan.
J Orthop Res. 1998 Nov;16(6):690-6. doi: 10.1002/jor.1100160610.
The present study, using confocal laser scanning microscopy and immunoelectron microscopy, examined the intracellular localization of tyrosine-phosphorylated proteins in cultured mouse dorsal root ganglion neurons with special reference to their growth cones. The growth cone is the specialized structure formed at the growing tip of the axon; characteristically highly motile with filopodia on the surface, it is responsible for the extension and guidance of the neurites to the appropriate targets during nerve regeneration. It has been suggested that protein-tyrosine phosphorylation plays an important role in the intracellular signal transduction that regulates the extension and motility of growth cones. By fluorescence immunocytochemistry, phosphotyrosine immunoreactivity was found in the growth cones and neurites. Some of the filopodia exhibited strong immunoreactivity at their tips. By immunoelectron microscopy, a large number of immunogold particles (gold particles conjugated to the secondary antibody) were seen to be distributed in the cytoplasm and some were observed on the plasma membrane in the growth cones, whereas in the neurites the density of immunogold particles was the same in the axoplasm as on the plasma membranes. These findings suggest that in the growth cones phosphotyrosines might mainly be involved in intracellular signaling for maintaining their high motility whereas in the neurites they might mostly be associated with the receptor proteins at the plasma membrane for adhesion as well as for growth of neurites. Thus, tyrosine phosphorylation might contribute to different functions for growth cones and neurites.
本研究运用共聚焦激光扫描显微镜和免疫电子显微镜，特别针对培养的小鼠背根神经节神经元生长锥，检测酪氨酸磷酸化蛋白的细胞内定位。生长锥是在轴突生长末端形成的特殊结构；其特点是表面有丝状伪足，高度活跃，在神经再生过程中负责将神经突延伸并引导至合适的靶点。有人提出，蛋白酪氨酸磷酸化在调节生长锥延伸和运动的细胞内信号转导中起重要作用。通过荧光免疫细胞化学法，在生长锥和神经突中发现了磷酸酪氨酸免疫反应性。一些丝状伪足的尖端呈现出强烈的免疫反应性。通过免疫电子显微镜观察，在生长锥中可见大量免疫金颗粒（与二抗结合的金颗粒）分布于细胞质中，部分也见于质膜上，而在神经突中，轴浆内免疫金颗粒的密度与质膜上相同。这些发现表明，在生长锥中，磷酸酪氨酸可能主要参与维持其高运动性的细胞内信号传导，而在神经突中，它们可能主要与质膜上用于神经突黏附及生长的受体蛋白相关。因此，酪氨酸磷酸化可能对生长锥和神经突的不同功能有贡献。
