Primary cilia are microtubule-based organelles that are important for signaling and sensing in eukaryotic cells. Unlike the thoroughly studied motile cilia, the three-dimensional architecture and molecular composition of primary cilia are largely unexplored. Yet, studying these aspects is necessary to understand how primary cilia function in health and disease. We developed an enabling method for investigating the structure of primary cilia isolated from MDCK-II cells at molecular resolution by cryo-electron tomography. We show that the textbook ‘9 + 0’ arrangement of microtubule doublets is only present at the primary cilium base. A few microns out, the architecture changes into an unstructured bundle of EB1-decorated microtubules and actin filaments, putting an end to a long debate on the presence or absence of actin filaments in primary cilia. Our work provides a plethora of insights into the molecular structure of primary cilia and offers a methodological framework to study these important organelles.

初级纤毛是基于微管的细胞器，对于真核细胞中的信号传导和传感非常重要。与彻底研究的运动纤毛不同，初级纤毛的三维结构和分子组成在很大程度上尚未被探索。然而，研究这些方面对于了解初级纤毛在健康和疾病中的功能是必要的。我们开发了一种通过冷冻电子断层扫描以分子分辨率研究从 MDCK-II 细胞分离的初级纤毛结构的可行方法。我们表明，教科书上的微管双联体“9 + 0”排列仅存在于初级纤毛基部。几微米外，该结构变成一束非结构化的 EB1 修饰的微管和肌动蛋白丝，结束了关于初级纤毛中是否存在肌动蛋白丝的长期争论。我们的工作提供了对初级纤毛分子结构的大量见解，并提供了研究这些重要细胞器的方法框架。