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

项目摘要 这个项目的目标是确定与睫毛病变相关的突变对睫毛的影响。 病理生理学。纤毛疾病是一种根源于睫毛功能障碍的疾病，临床表现重叠 特征，包括发育迟缓、智力残疾、多指、视网膜营养不良和进行性 肾、肝受累。虽然个别罕见，但纤毛疾病合并影响1/500人和 大约30种不同的纤毛疾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， 尽管确切的细胞机制仍然难以捉摸。初级纤毛为触角状突起。 几乎存在于每一个细胞上；它从细胞体延伸出来，在那里它接收和解释信号，从而允许 细胞对它们的环境做出反应。纤毛由过渡区从细胞质中分离出来。 它管理着蛋白质的交易。一种专用的主动转运系统，鞭毛内转运，运输蛋白质 穿过这一屏障进入纤毛，并结合多种方法保留蛋白质和 有选择性的出口。参与这种选择性蛋白质运输的蛋白质涉及一系列 纤毛病变，表明纤毛蛋白含量的异常可能与这些疾病的病因有关。 我将使用我的新人类纤毛分离方案和最先进的质谱学方法来 评估全球纤毛蛋白组成，确定对照组和纤毛病变细胞之间的差异- 相关的亚形突变。这项工作将提供一个全面的，不偏不倚的目录错误本地化 Joubert(K99)和Bardet-Biedl(R00)综合征中的蛋白质，从而为未来的工作提供了丰富的资源 剖析纤毛疾病中涉及的蛋白质网络。在一个补充方法中，我将确定如何 纤毛病变相关突变通过内源性标记键影响蛋白质运输的动力学 纤毛病变蛋白，并使用活细胞显微镜跟踪它们的运动。这项工作将回答关键问题 关于纤毛病变相关突变对纤毛进入、保留和退出的影响的问题。 这项申请提出了创新的技术，可以很容易地扩展到其他蛋白质/纤毛疾病，以及 重要的是，在人类疾病背景下研究蛋白质含量和动态，而不是在零的情况下 动物模型中的突变。总之，这项工作将阐明纤毛疾病的病因，并催化 未来疗法的发展。

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.