Kohli Priyanka, Höhne Martin, Jüngst Christian, Bertsch Sabine, Ebert Lena K, Schauss Astrid C, Benzing Thomas, Rinschen Markus M, Schermer Bernhard
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
EMBO Rep. 2017 Sep;18(9):1521-1535. doi: 10.15252/embr.201643846. Epub 2017 Jul 14.
Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane-associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high-stoichiometric presence of actin-binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F-actin causes further enrichment of the actin-binding and actin-related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin-binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.
初级纤毛是存在于多种细胞类型表面的感觉性、触角样细胞器。它们与统称为纤毛病的多种疾病有关。由于纤毛是细胞信号传导的重要调节因子，纤毛膜的组成需要严格调控。为了了解纤毛膜的调控过程，我们报道了一种基因工程酶特异性靶向纤毛膜，以实现生物素化并鉴定膜相关蛋白质组。对综合数据集的生物信息学分析揭示了纤毛内部肌动蛋白结合蛋白的高化学计量存在。免疫荧光染色和互补相互作用蛋白质组分析证实了这些发现。分支F-肌动蛋白的解聚导致纤毛中肌动蛋白结合蛋白和肌动蛋白相关蛋白进一步富集，包括肌球蛋白5a（Myo5a）。有趣的是，Myo5a基因敲除会降低纤毛形成，而在诱导纤毛解体后，纤毛中观察到Myo5a水平升高。总之，我们提出了一种新方法来研究哺乳动物细胞中纤毛膜蛋白质组的动态变化，并将肌动蛋白结合蛋白鉴定为纤毛的机械敏感成分，其可能在纤毛膜动态变化中具有重要功能。
