Ciliary control of Gli protein activity in Hedgehog pathway signaling

描述(申请人提供)：初级纤毛是细胞表面的微米级突起，允许细胞接收和处理来自外部环境的各种信号。Hedgehog途径是由初级纤毛调节的最具特征的信号级联之一，几乎该途径的所有组成部分都动态地定位于纤毛，并需要纤毛来发挥其功能。纤毛功能障碍与HH信号在发育模式和组织动态平衡中的关键作用一致，会导致出生缺陷和一组统称为纤毛疾病的儿科疾病。关键是， 虽然初级纤毛现在被认为在HH信号转导中具有重要作用，但HH通路组件进出纤毛并受纤毛调控的分子机制仍不清楚。Gli家族蛋白是HH途径的最终效应者，经历纤毛依赖的翻译后加工成转录激活和抑制形式。在这里，我将结合生化、活体成像和功能基因组学方法，通过解剖纤毛运输和Gli蛋白的加工来研究纤毛在HH途径中的作用。利用我在博士后研究中开发的新工具，我将：1)使用显微镜分析来监测Gli蛋白的纤毛进出，并确定这些事件是如何由HH信号调控的；2)剖析纤毛运输机制在Gli蛋白运输和调控中的功能角色；以及3)应用功能基因组学工具来确定将上游HH途径蛋白的活性与Gli蛋白运输和加工的动态变化联系起来的因子。该奖项的指导阶段将在斯坦福大学进行，由纤毛贩运专家Maxence Nachury博士和HH领域的领导者Matthew Scott博士指导。在此期间，我将使用成像分析来剖析Gli蛋白运输，并开发生化工具来评估纤毛运输成分对Gli3调节的贡献。此外，我将与功能基因组学专家迈克尔·巴西克博士合作，启动一项基于RNAi的筛查，旨在识别纤毛HH信号的新调节因子。在转到一个独立的职位后，我将进一步研究Gli蛋白交易和POST的机制 翻译法规。我还将继续我的功能基因组学研究，使用强大的遗传相互作用分析工具来验证和表征从我的主要屏幕上分离出来的HIT基因。综上所述，该项目将提高我们对初级纤毛中HH信号的理解，为基于纤毛的发育障碍提供新的见解，并为成功的独立研究计划提供基础。这一奖项将使我在HH途径的功能分析和基于RNAi的筛选技术方面获得所需的培训，从而确保拟议研究的成功，并加快我向独立的过渡。

DESCRIPTION (provided by applicant): The primary cilium is a micron-scale protrusion from the cell surface that allows cells to receive and process a variety of signals from the external environment. The Hedgehog pathway is one of the best-characterized signaling cascades regulated by the primary cilium, with virtually all components of the pathway found to dynamically localize to cilia and to require cilia for their function. Consistent with the key role of Hh signaling in developmental patterning and tissue homeostasis, cilium dysfunction leads to birth defects and to a group of pediatric disorders known collectively as ciliopathies. Critically, while primary cilia are now recognized to have an essential role in Hh signaling, the molecular mechanisms by which Hh pathway components traffic to and from cilia and are regulated by cilia remain unknown. Gli family proteins are the ultimate effectors of the Hh pathway, undergoing cilium-dependent post- translational processing into transcriptional activator and repressor forms. Here I will investigate the role of cilia in the Hh pathway by dissecting the ciliary trafficking and processing of Gli proteins through a combination of biochemical, live imaging and functional genomics approaches. Leveraging new tools developed in my post- doctoral studies, I will: 1) use microscopy assays to monitor the ciliary entry and exit of Gli proteins and determine how these events are regulated by Hh signaling; 2) dissect the functional role of the ciliary trafficking machinery in Gli protein trafficking and regulation; and3) apply functional genomics tools to identify factors that link the activity of upstream Hh pathway proteins to dynamic changes in Gli protein transport and processing. The mentored phase of this award will be carried out at Stanford University under the mentorship of Dr. Maxence Nachury, an expert in ciliary trafficking, and Dr. Matthew Scott, a leader in the Hh field. During this time, I will use imaging assays to dissect Gli protein trafficking and develop biochemical tools to assess the contribution of ciliary trafficking components to Gli3 regulation. Additionally I will work with Dr. Michael Bassik, an expert in functional genomics, to initiate an RNAi-based screen aimed at identifying new regulators of ciliary Hh signaling. After moving to an independent position, I will further investigate the mechanisms of Gli protein trafficking and post translational regulation. I will also continue my functional genomics studies, using powerful genetic interaction analysis tools to validate and characterize hit genes isolated from my primary screen. In summary, this project will improve our understanding of Hh signaling in primary cilia, give new insights into cilium-based developmental disorders, and provide the foundation for a successful independent research program. This award will allow me to gain needed training in functional analysis of the Hh pathway and in RNAi-based screening technologies, thereby ensuring the success of the proposed research and accelerating my transition to independence.