NSF/BIO-DFG: The role of ARL 13B in controlling ciliary cAMP signaling

细胞是组织的基石，必须进行有效的沟通，才能使组织和有机体形成和发挥作用。脊椎动物细胞的一个隔间是初级纤毛，它是几乎所有细胞上的细长突起，起着细胞天线的作用。虽然初级纤毛对细胞通信至关重要，但纤毛如何参与细胞通信还不是很清楚。纤毛在功能上或物理上难以隔离。纤毛的完全丧失会导致所有纤毛信号的丧失；纤毛中丰富的蛋白质的完全丧失会导致该蛋白质的细胞池和纤毛池的丧失。因此，破译纤毛和细胞体内发生的事情是不清楚的。卡斯帕里实验室开发了基因工具来分离纤毛内一种纤毛蛋白的功能；米克和瓦赫滕实验室在确定纤毛内的蛋白质成分和操纵纤毛内的特定信号方面分别拥有互补的专业知识。这项合作研究将结合3个实验室的技能，培训和交流研究纤毛内信号的学生。在埃默里，这个项目将让学生参与基于队列的结构化研究体验，培训学生进行研究以及伦理和科学陈述。这个项目将通过向广泛的学生提供真正的研究经验来造福社会，并通过交流，让他们接触到一系列不同的技术，以解决纤毛如何调节细胞通信的基本问题。环磷酸腺苷(CAMP)信号是纤毛信号的中心信使。CAMP信号级联的组成部分在初级纤毛中丰富，产生一个功能上独立的cAMP隔间。最近的报道表明，纤毛cAMP信号成分的异常定位改变了纤毛信号。然而，细胞如何区分纤毛和胞浆cAMP信号输出，以及纤毛和胞浆cAMP信号具体涉及哪些信号通路和细胞信号，目前尚不清楚。Arl13b是一种高度富含纤毛的调节性GTP酶。卡斯帕里实验室设计了Arl13b的纤毛排除变体，并表明它保留了已知的生化活性，从而能够分离纤毛Arl13b的功能。在这项研究中，Caspary、Mick和Wachten实验室将调查初级纤毛中Arl13b的缺失是否控制了肾上皮细胞系中的纤毛cAMP信号。目标1将在只表达纤毛排除的Arl13b的细胞中解开纤毛环“签名”。目的2将光遗传刺激正常纤毛和Arl13b排除的纤毛内的cAMP信号，以确定共同的RNA和蛋白质组成。目的通过三维培养，探讨纤毛cAMP信号在缺失纤毛Arl13b的细胞形态重塑中的作用。最后，目标4将操纵纤毛膜的磷脂组成，以确定它是否调节纤毛内的纤毛cAMP/Arl13b信号。通过这项研究，初级纤毛内的信号作用将以前所未有的分辨率得到破译。这个美国和德国的合作项目得到了美国国家科学基金会和德国科学基金会(DFG)的支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Cells are the building blocks of tissues and must communicate effectively in order for tissues and organisms to form and function. One compartment of vertebrate cells critical for cell communication is the primary cilium, a slender projection on almost all cells that functions as a cellular antenna. While it is clear that primary cilia are critical for cellular communication, how cilia participate in cellular communication is not well understood. The cilium is challenging to functionally or physically isolate. The complete loss of cilia results in a loss of all ciliary signaling; a complete loss of proteins enriched in cilia leads to a loss of the cellular and ciliary pools of that protein. Thus, deciphering what is happening within the cilium versus the cell body is murky. The Caspary Lab developed genetic tools to isolate function of one ciliary protein specifically within cilia; the Mick and Wachten labs have complementary expertise in defining the protein composition within the cilium and manipulating signaling specifically within cilia, respectively. This collaborative research will combine the skills of the 3 labs to train and exchange students in investigating the signaling within cilia. At Emory, this project will involve students in cohort-based, structured research experiences that will train the students in research along with ethics and scientific presentation. This project will benefit society by providing bona fide research experiences to a broad swath of students and, through exchange, expose them to a diverse array of techniques to address the fundamental question of how cilia mediate cell communication.Cyclic AMP (cAMP) signaling is a central messenger for ciliary signaling. Components of the cAMP signaling cascade are enriched in primary cilia, generating a functionally separate cAMP compartment. Recent reports demonstrate that aberrant localization of ciliary cAMP signaling components alters ciliary signaling. However, how the cell distinguishes between ciliary and cytoplasmic cAMP signaling outputs and which signaling pathways and cellular cues are specifically engaged by ciliary versus cytoplasmic cAMP signaling is not known. ARL13B is a regulatory GTPase highly enriched in cilia. The Caspary Lab engineered a cilia-excluded variant of ARL13B and showed that it retains its known biochemical activities to enable the isolation of ciliary ARL13B function. In this research, the Caspary, Mick, and Wachten labs will investigate whether loss of ARL13B in the primary cilium controls ciliary cAMP signaling in a kidney epithelial cell line. Aim 1 will unravel the ciliary cAMP “signature” in cells expressing only cilia-excluded ARL13B. Aim 2 will optogenetically stimulate cAMP signaling within normal and Arl13B-excluded cilia to define the common RNA and protein composition. Aim 3 will investigate the role of ciliary cAMP signaling in remodeling morphology of cells lacking ciliary ARL13B through use of 3D cultures. Finally, Aim 4 will manipulate the phospholipid composition of the ciliary membrane to determine whether it regulates ciliary cAMP/ARL13B-signaling within cilia. Through this research, the role of signaling within the primary cilium will be deciphered at unprecedented resolution.This collaborative US/Germany project is supported by the US National Science Foundation and the Deutsche Forschungsgemeinschaft (DFG).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.