Data_Sheet_1_Synchronizing Protein Traffic to the Primary Cilium.docx

The primary cilium is able to maintain a specific protein composition, which is critical for its function as a signaling organelle. Here we introduce a system to synchronize biosynthetic trafficking of ciliary proteins that is based on conditional aggregation domains (CADs). This approach enables to create a wave of ciliary proteins that are transported together, which opens novel avenues for visualizing and studying ciliary import mechanisms. By using somatostatin receptor 3 (SSTR3) as model protein we studied intracellular transport and ciliary import with high temporal and spatial resolution in epithelial Madin-Darby canine kidney (MDCK) cells. This yielded the interesting discovery that SSTR3, besides being transported to the primary cilium, is also targeted to the basolateral plasma membrane. In addition, we found a similar behavior for another ciliary protein, nephrocystin-3 (NPHP3), thus suggesting a potential correlation between ciliary and basolateral trafficking. Furthermore, our CAD-based system allowed assembling a large dataset in which apical and basolateral surface SSTR3 signals could be compared to ciliary SSTR3 signals on a single cell level. This enabled to generate novel complementary evidence for the previously proposed lateral import mechanism of SSTR3 into the cilium along the plasma membrane.

初级纤毛（primary cilium）能够维持特定的蛋白质组成，这对于其作为信号细胞器的功能至关重要。本文介绍一种基于条件性聚集结构域（conditional aggregation domains, CADs）的系统，可同步调控纤毛蛋白质的生物合成转运过程。该方法可同步产生一批共同转运的纤毛蛋白质，为可视化研究纤毛蛋白导入机制开辟了全新途径。本研究以生长抑素受体3（somatostatin receptor 3, SSTR3）作为模型蛋白，在上皮性Madin-Darby犬肾（Madin-Darby canine kidney, MDCK）细胞中以高时空分辨率解析了其胞内转运与纤毛导入过程。研究得到了一项有趣的发现：SSTR3除被转运至初级纤毛外，还可被靶向定位至基底外侧质膜。此外，我们在另一款纤毛蛋白——肾囊性蛋白3（nephrocystin-3, NPHP3）中发现了类似的靶向行为，这提示纤毛转运与基底外侧转运之间可能存在潜在关联。此外，基于CAD的实验系统可构建大规模数据集，实现在单细胞水平上比较顶侧与基底外侧质膜表面的SSTR3信号与纤毛内SSTR3信号。这为此前提出的"SSTR3沿质膜侧向导入纤毛"的机制提供了全新的互补性实验证据。