Kanie Tomoharu, Liu Beibei, Love Julia F, Fisher Saxton D, Gustavsson Anna-Karin, Jackson Peter K
Baxter Laboratory, Department of Microbiology & Immunology and Department of Pathology, Stanford University, Stanford, United States.
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, United States.
Elife. 2025 Jan 30;14:e85999. doi: 10.7554/eLife.85999.
Distal appendages are ninefold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for the formation of the primary cilium, by regulating at least four critical steps: preciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here, we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in the RAB34 vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
远端附属物是附着于母中心粒远端的九重对称叶片状结构。这些结构对于初级纤毛的形成至关重要，通过调节至少四个关键步骤：纤毛前囊泡募集、鞭毛内运输（IFT）的募集和起始，以及CP110的去除。虽然已经鉴定出定位于远端附属物的特定蛋白质，但每种蛋白质究竟如何发挥作用以实现远端附属物的多种功能却知之甚少。在这里，我们全面分析了已知的和新发现的远端附属物蛋白（CEP83、SCLT1、CEP164、TTBK2、FBF1、CEP89、KIZ、ANKRD26、PIDD1、LRRC45、NCS1、CEP15）的精确定位、募集顺序及其在纤毛形成各步骤中的作用。使用CRISPR-Cas9基因敲除技术，我们表明远端附属物蛋白的募集顺序高度相互关联且层次更为复杂。我们的分析突出了两个蛋白质模块，即CEP83-SCLT1和CEP164-TTBK2，对远端附属物的结构组装至关重要。功能分析显示，CEP89在RAB34囊泡募集中具有选择性功能，而缺失整合成分CEP83-SCLT1-CEP164-TTBK2会严重损害纤毛形成的所有四个步骤。总的来说，我们的分析为远端附属物的组织和功能提供了更全面的视角，为纤毛组装的分子理解铺平了道路。
