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

NSF/BIO-DFG：ARL 13B 在控制睫状 cAMP 信号传导中的作用

• 批准号: 2329634
• 负责人: Tamara Caspary
• 金额: $ 72.44万
• 依托单位: Emory University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2329634&HistoricalAwards=false
• 关键词: NSF; BIO; DFG; role; ARL

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.

细胞是组织的组成部分，为了组织和生物体的形成和发挥功能，细胞必须有效地进行通讯。对细胞通讯至关重要的脊椎动物细胞的一个区室是初级纤毛，它是几乎所有细胞上的细长突起，起着蜂窝天线的作用。虽然初级纤毛对于细胞通讯至关重要，但纤毛如何参与细胞通讯尚不清楚。纤毛的完全丧失导致所有纤毛信号的完全丧失。纤毛中富含的蛋白质的损失会导致该蛋白质的细胞和纤毛库的损失，因此，解释纤毛与细胞体内发生的情况尚不清楚，卡斯帕里实验室开发了专门分离一种纤毛蛋白质功能的遗传工具。 Mick 和 Wachten 实验室分别在定义纤毛内的蛋白质组成和专门操纵纤毛内的信号传导方面拥有互补的专业知识，这项合作研究将结合三个实验室的技能。在埃默里大学，该项目将培训和交换学生研究纤毛内的信号传导，让学生参与基于队列的结构化研究经验，这些经验将培训学生的研究以及道德和科学演示，该项目将通过提供真诚的内容造福社会。向广大学生介绍研究经验，并通过交流让他们接触到各种技术来解决纤毛如何介导细胞通讯的基本问题。环磷酸腺苷 (cAMP) 信号传导是纤毛信号传导的核心信使。营信号级联在初级纤毛中富集，产生功能上独立的 cAMP 区室，最近的报告表明，纤毛 cAMP 信号成分的异常定位会改变纤毛信号传导，然而，细胞如何区分纤毛和细胞质 cAMP 信号输出以及哪些信号传导途径和细胞线索。 ARL13B 是一种在纤毛中高度富集的调节性 GTP 酶，目前尚不清楚。在这项研究中，Caspary、Mick 和 Wachten 实验室将研究初级纤毛中 ARL13B 的缺失是否会控制纤毛 cAMP 信号传导。目标 1 将揭示仅表达细胞中的睫状 cAMP“特征”。排除纤毛的 ARL13B。目标 2 将通过光遗传学刺激正常纤毛和排除 ARL13B 的纤毛内的 cAMP 信号传导，以定义常见的 RNA 和蛋白质组成。目标 3 将通过使用 3D 研究纤毛 cAMP 信号传导在重塑缺乏纤毛 ARL13B 的细胞形态中的作用。最后，目标 4 将操纵睫状膜的磷脂成分以确定其是否调节睫状体。通过这项研究，将以前所未有的分辨率破译初级纤毛内的 cAMP/ARL13B 信号传导。该美国/德国合作项目得到了美国国家科学基金会和德国研究协会 (DFG) 的支持。授予 NSF 的法定使命，并通过评估反映使用基金会的智力优点和更广泛的影响审查标准，被认为值得支持。