Integrating functional proteomics, genome engineering, and live-cell microscopy to the study of ciliopathies

项目摘要 该项目的目标是确定纤毛病变相关的突变如何有助于睫状体 病理生理学睫状体病是根源于睫状体功能障碍的疾病， 特征，包括发育迟缓、智力残疾、多指（趾）畸形、视网膜营养不良和进行性 肾脏和肝脏受累。虽然个别罕见，但纤毛病变合并影响1/500的个体， 约30种不同的纤毛病中的每一种都是由与纤毛相关的特定蛋白质网络的功能障碍引起的， 尽管精确的细胞机制仍然难以捉摸。初级纤毛是触角状突起 几乎在每一个细胞上都可以找到;它从细胞体延伸出来，在那里它接收和解释信号，从而允许 细胞对环境做出反应。纤毛通过过渡带与细胞质分隔开 调节蛋白质运输的基因一个专门的主动运输系统，鞭毛内运输， 结合纤毛穿过这一屏障，并与多种蛋白质保留方法结合使用， 选择性出口参与这种选择性蛋白质转运的蛋白质涉及一系列的蛋白质转运。 纤毛病变，表明异常的纤毛蛋白含量可能有助于这些疾病的病因。 我将使用我的新的人类纤毛分离方案和最先进的质谱方法， 评估总体纤毛蛋白组成，确定对照组和携带纤毛病变的细胞之间的差异- 相关的亚晶突变。这项工作将提供一个全面的，公正的目录， Joubert（K99）和Bardet-Biedl（R 00）综合征中的蛋白质，从而为未来的工作提供了丰富的资源， 剖析纤毛病变中的蛋白质网络。作为补充，我将确定如何 睫状体病相关突变通过内源性标记关键影响蛋白质运输的动力学 纤毛病蛋白质，并使用活细胞显微镜观察它们的运动。这项工作将回答关键 关于纤毛病相关突变对纤毛进入、保留和退出的影响的问题。 本申请提出了创新的技术，很容易扩展到其他蛋白质/纤毛病变， 重要的是，研究蛋白质含量和动力学在人类疾病的背景下，而不是与空 动物模型中的突变。总之，这项工作将揭示纤毛虫病的病因并催化 未来的治疗方法。

Project Summary The goal of this project is to determine how ciliopathy-related mutations contribute to ciliary pathophysiology. Ciliopathies are disorders rooted in ciliary dysfunction and exhibit overlapping clinical features, including developmental delay, intellectual disability, polydactyly, retinal dystrophy, and progressive involvement of the kidney and liver. While individually rare, ciliopathies combined affect 1/500 individuals and each of the ~30 distinct ciliopathies is caused by dysfunction of a specific protein network related to the cilium, although the precise cellular mechanisms remain elusive. The primary cilium is an antenna-like projection found on nearly every cell; it extends from the cell body, where it receives and interprets signals, thus allowing cells to respond to their environment. Cilia are partitioned from the cellular cytoplasm by the transition zone that regulates protein trafficking. A dedicated active transport system, intraflagellar transport, moves proteins bound for the cilium across this barrier and works in conjunction with multiple methods for protein retention and selective egress. The proteins involved in this selective protein transport are implicated in a range of ciliopathies, indicating that aberrant ciliary protein content likely contributes to the etiology of these disorders. I will use my novel human cilia isolation protocol and state-of-the-art mass spectrometry approach to assess global ciliary protein composition, defining differences between controls and cells harboring ciliopathy- associated hypomorphic mutations. This work will provide a comprehensive, unbiased catalog of mislocalized proteins in Joubert (K99) and Bardet-Biedl (R00) syndromes, thus providing a rich resource for future work to dissect the protein networks involved in ciliopathies. In a complementary approach, I will determine how ciliopathy-associated mutations affect the dynamics of protein trafficking by endogenously tagging key ciliopathy proteins and following their movement using live-cell microscopy. This work will answer critical questions about the impact of ciliopathy-associated mutations on entry into, retention within, and exit from cilia. This application proposes innovative techniques that are easily extendable to other proteins/ciliopathies, and importantly, investigates protein content and dynamics in the human disease context rather than with null mutations in animal models. Together, this work will shed light on the etiology of ciliopathies and catalyze the development of future therapies.