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

细胞是组织的基石，必须有效地沟通，以使组织和生物体的形成和功能。 脊椎动物细胞中对细胞通讯至关重要的一个区室是初级纤毛，几乎所有细胞上的细长突起，用作细胞天线。 虽然很明显，初级纤毛是至关重要的细胞通信，纤毛如何参与细胞通信还没有得到很好的理解。 纤毛在功能上或物理上分离具有挑战性。 纤毛的完全丧失导致所有纤毛信号传导的丧失;纤毛中富集的蛋白质的完全丧失导致该蛋白质的细胞和纤毛池的丧失。 因此，要破译纤毛和细胞体内发生的事情是很模糊的。 卡斯帕里实验室开发了遗传工具来分离纤毛内特定纤毛蛋白的功能;米克和瓦赫滕实验室分别在定义纤毛内的蛋白质组成和操纵纤毛内特定信号传导方面具有互补的专业知识。这项合作研究将结合联合收割机的技能，培训和交换学生在调查纤毛内的信号。 在埃默里大学，这个项目将让学生参与以队列为基础的结构化研究体验，这将训练学生沿着道德和科学演示进行研究。 本项目将通过为广大学生提供真实的研究经验，并通过交流，使他们接触到各种技术，以解决纤毛如何介导细胞通讯的基本问题，从而造福社会。环磷酸腺苷（cAMP）信号是纤毛信号的中心信使。 cAMP信号级联的组分在初级纤毛中富集，产生功能上分离的cAMP隔室。 最近的报告表明，纤毛cAMP信号成分的异常定位改变纤毛信号。 然而，细胞如何区分纤毛和细胞质cAMP信号传导输出，以及哪些信号传导途径和细胞线索被纤毛与细胞质cAMP信号传导特异性地参与尚不清楚。 ARL 13 B是纤毛中高度富集的调节性GT3。 卡斯帕里实验室设计了一种排除纤毛的ARL 13 B变体，并表明它保留了其已知的生物化学活性，从而能够分离纤毛ARL 13 B功能。 在这项研究中，Caspary，Mick和Wachten实验室将研究初级纤毛中ARL 13 B的缺失是否控制肾上皮细胞系中的纤毛cAMP信号传导。 目的1将阐明仅表达纤毛排除的ARL 13 B的细胞中的纤毛cAMP“签名”。 目的2将在正常和Ar 113 B排除的纤毛内光遗传学地刺激cAMP信号传导，以确定共同的RNA和蛋白质组成。 目的3将通过使用3D培养来研究纤毛cAMP信号传导在缺乏纤毛ARL 13 B的细胞的形态重塑中的作用。 最后，目标4将操纵睫状膜的磷脂组成，以确定它是否调节纤毛内的纤毛cAMP/ARL 13 B信号传导。 通过这项研究，初级纤毛中的信号作用将以前所未有的分辨率被破译。这项美国/德国合作项目得到了美国国家科学基金会和德国研究共同体（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价值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.