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Abstract：
Primary cilia are finger-like sensory organelles that extend from the bodies of most cell types and have a distinct lipid and protein composition from the plasma membrane. This partitioning is maintained by a diffusion barrier that restricts the entry of non-ciliary proteins, and allows the selective entry of proteins harboring a ciliary targeting sequence (CTS). However, CTSs are not stereotyped and previously reported sequences are insufficient to drive efficient ciliary localisation across diverse cell types. Here, we describe a short peptide sequence that efficiently targets transmembrane proteins to primary cilia in all tested cell types, including human neurons. We generate human-induced pluripotent stem cell (hiPSC) lines stably expressing a transmembrane construct bearing an extracellular HaloTag and intracellular fluorescent protein, which enables the bright, specific labeling of primary cilia in neurons and other cell types to facilitate studies of cilia in health and disease. We demonstrate the utility of this resource by developing an image analysis pipeline for the automated measurement of primary cilia to detect changes in their length associated with altered signaling or disease state.
摘要：
初级纤毛是指状感觉器官，从大多数细胞类型的身体延伸出来，并具有与质膜不同的脂质和蛋白质组成。这种分配是由限制非纤毛蛋白进入的扩散屏障维持的，并允许选择性进入具有睫状靶向序列（CTS）的蛋白质。然而,CTSs不是定型的,并且先前报道的序列不足以驱动跨不同细胞类型的有效纤毛定位。这里,我们描述了一个短的肽序列，有效地将跨膜蛋白靶向所有测试细胞类型的初级纤毛，包括人类神经元。我们产生了稳定表达跨膜构建体的人诱导多能干细胞（hiPSC）系，该构建体带有细胞外HaloTag和胞内荧光蛋白，这使得光明，神经元和其他细胞类型中初级纤毛的特异性标记，以促进纤毛在健康和疾病中的研究。我们通过开发用于自动测量初级纤毛的图像分析管道来检测与信号传导或疾病状态改变相关的长度变化，从而证明了该资源的实用性。
