Stractural and molecular biological study of the ciliary basal apparatus during ciliogenesis

1.我们获得了6个单克隆抗体标记与基体延伸纤毛的横纹根。这些抗体通过免疫印迹鉴定205-215 kDa的蛋白质。使用克隆R67，我们揭示了复杂的形态与单纤毛的条纹小根。205-215抗原是细根暗条纹的组成成分。纹状根在人下颌下腺所有类型的细胞中均呈阳性染色。提示横纹小根与中心粒的定位有关，在正常生理条件下，横纹小根可能与中心粒结合，而不形成孤立纤毛.秋水仙胺处理导致PtK_2细胞中心粒相关小根的延长和/或结构复杂化，此外，胞质内游离小根的组织化。这些变化是可逆的，在药物去除后2小时几乎消失。这些观察结果可能表明细胞质微管和纹状小根之间的功能关系，以及除了中心粒外，细胞质中还存在小根成核因子.应用免疫荧光和免疫电镜技术研究了γ-微管蛋白在睫状体基底器中的定位。除了基底体外，在外侧基底足，特别是基底足帽中还发现了γ-微管蛋白，证实了以前的观察结果，即基底足是微管组织中心.在单纤毛延伸的细胞中，基体和单个中心粒由一束或两束带有少量周期性条纹的细丝相互连接。我们把这些纤维束称为条纹连接器。连接器可以发挥重要作用，稳定在顶端细胞质中的基体。

1. We obtained six monoclonal antibodies that labeled striated rootlets associated with basal bodies extending cilia. These antibodies identified proteins of 205-215 kDa by immunoblotting. Using a clone R67, we revealed the complicated morphology of striated rootlets associated with solitary cilia. The 205-215 antigens were constitutive components of dark striations of the rootlet. Striated rootlets were positively stained in all cell types in the human submandibular glands. It is suggested that striated rootlets are concerned with the positioning of centrioles and may be associated with centrioles under normal physiological conditions, without formation of solitary cilia.2. Treatment with colcemid resulted in the elongation and/or structural complication of the centriole-associated rootlets in PtK_2 cells and additionally, organization of intracytoplasmic free rootlets. The changes were reversible and almost disappeared 2 hours after removal of the drug. These observations may indicate functional relationships between cytoplasmic microtubules and striated rootlets, and also the existence of rootlet-nucleating factors in the cytoplasm in addition to centrioles.3. The localization of gamma-tubulin in the ciliary basal apparatus was studied by fluorescence immunohistochemistry and immunoelectron microscopy. Ip addition to basal bodies, gamma-tubulin was identified in the lateral basal foot, especially the basal foot cap, confirming the previous observations that the basal foot serves as the microtubule organizing center.4. In the cells extending solitary cilia, the basal body and the single centriole were interconnected by one or two bundles of thin filaments with a few periodic striations. We have called these bundles the striated connector. The connector could play an important role for stabilising the basal body in the apical cytoplasm.