Table S2 from CEP19 cooperates with FOP and CEP350 to drive early steps in the ciliogenesis programme

Primary cilia are microtubule-based sensory organelles necessary for efficient transduction of extracellular cues. To initiate cilia formation, ciliary vesicles (CVs) are transported to the vicinity of the centrosome where they dock to the distal end of the mother centriole and fuse to initiate cilium assembly. However, to this date, the early steps in cilia formation remain incompletely understood. Here, we demonstrate functional interplay between CEP19, FOP and CEP350 in ciliogenesis. Using three-dimensional structured-illumination microscopy (3D-SIM) imaging, we mapped the relative spatial distribution of these proteins at the distal end of the mother centriole and show that CEP350/FOP act upstream of CEP19 in their recruitment hierarchy. We demonstrate that CEP19 CRISPR KO cells are severely impaired in their ability to form cilia, analogous to the loss of function of CEP19 binding partners FOP and CEP350. Using GFP-tagged deletion constructs of CEP19, we show that the C-terminus of CEP19 is required for both its localization to centrioles and for its function in ciliogenesis. Critically, this region also mediates the interaction between CEP19 and FOP/CEP350. Interestingly, a morbid obesity-associated R82* truncated mutant of CEP19 cannot ciliate nor interact with FOP and CEP350, indicative of a putative role for CEP19 in ciliopathies. Finally, analysis of CEP19 KO cells using thin-section electron microscopy revealed marked defects in the docking of CVs to the distal end of the mother centrioles. Together, these data demonstrate a role for the CEP19, FOP and CEP350 module in ciliogenesis and the possible effect of disrupting their functions in ciliopathies.

初级纤毛（Primary cilia）是基于微管的感觉细胞器，对于高效转导细胞外信号至关重要。为启动纤毛形成，纤毛囊泡（ciliary vesicles, CVs）会被转运至中心体附近，在此停靠于母中心粒的远端并发生融合，以启动纤毛组装。然而截至目前，纤毛形成的早期步骤仍未被完全阐明。本研究揭示了CEP19、FOP与CEP350在纤毛发生过程中的功能互作关系。借助三维结构照明显微镜（three-dimensional structured-illumination microscopy, 3D-SIM）成像技术，我们绘制了这些蛋白在母中心粒远端的相对空间分布图谱，并证实CEP350与FOP在蛋白招募层级中位于CEP19的上游。本研究证实，CEP19的CRISPR敲除细胞的纤毛形成能力受到严重损害，这与CEP19的结合伴侣FOP和CEP350的功能缺失表型一致。通过使用带有GFP标签的CEP19缺失构建体，我们发现CEP19的C端结构域不仅是其定位至中心粒所必需的，同时也是其在纤毛发生中发挥功能所不可或缺的。至关重要的是，该结构域同时介导了CEP19与FOP/CEP350之间的相互作用。有趣的是，一种与病态肥胖相关的CEP19 R82*截短突变体既无法介导纤毛形成，也不能与FOP及CEP350发生相互作用，这提示CEP19可能在纤毛病（ciliopathies）中发挥关键作用。最后，通过超薄切片电子显微镜对CEP19敲除细胞进行分析，本研究发现纤毛囊泡向母中心粒远端的停靠过程存在显著缺陷。综上，本研究的数据证实了CEP19、FOP与CEP350复合体在纤毛发生中的功能，同时也揭示了其功能失调可能与纤毛病的发生相关。