Department of Cell Biology, M. Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
Cytoskeleton (Hoboken). 2010 Mar;67(3):161-71. doi: 10.1002/cm.20433.
Basal bodies which nucleate cilia and flagella, and centrioles which organize centrosomes share the same architecture characterized by the ninefold symmetry of their microtubular shaft. Among the conserved proteins involved in the biogenesis of the canonical 9-triplet centriolar structures, Sas-6 and Bld10 proteins have been shown to play central roles in the early steps of assembly and in establishment/stabilization of the ninefold symmetry. Using fluorescent tagged proteins and RNAi to study the localization and function of these two proteins in Paramecium, we focused on the early effects of their depletion, the consequences of their overexpression and their functional interdependence. We find that both genes are essential and their depletion affects cartwheel assembly and hence basal body duplication. We also show that, contrary to Sas6p, Bld10p is not directly responsible for the establishment of the ninefold symmetry, but is required not only for new basal body assembly and stability but also for Sas6p maintenance at mature basal bodies. Finally, ultrastructural analysis of cells overexpressing either protein revealed two types of early assembly intermediates, hub-like structures and generative discs, suggesting a conserved scaffolding process.
基体是纤毛和鞭毛的核，中心粒组织中心体，它们具有相同的结构，其微管轴具有九倍对称性。在参与经典的三联中心粒结构生物发生的保守蛋白中，Sas-6 和 Bld10 蛋白已被证明在组装的早期步骤以及在九倍对称性的建立/稳定中发挥核心作用。我们使用荧光标记蛋白和 RNAi 研究了这两种蛋白在草履虫中的定位和功能，重点研究了它们缺失的早期影响、过表达的后果以及它们的功能依赖性。我们发现这两个基因都是必需的，它们的缺失会影响车轮辐条的组装，从而影响基体的复制。我们还表明，与 Sas6p 相反，Bld10p 不是建立九倍对称性的直接原因，而是不仅需要新的基体组装和稳定性，而且需要 Sas6p 在成熟基体上的维持。最后，过表达任一蛋白的细胞的超微结构分析显示出两种类型的早期组装中间体，轮毂状结构和生殖盘，表明存在保守的支架过程。
