CAREER: Mechanistic studies of cilium proteins in cell signaling

该奖项的全部或部分资金来自《2021年美国救援计划法案》(公法117-2)。我们体内的大多数细胞都有初级纤毛(细胞表面的单个毛发状突起)，使它们能够通过化学信号相互作用。这个古老而神秘的细胞器就像一个无线电天线，允许细胞接收不同的信息，这取决于它们的天线包含的受体类型。影响纤毛信号的基因突变与许多疾病有关，这些疾病导致多个器官的畸形和功能障碍。这个项目关注的是发育中的大脑中的初级纤毛。在大脑形成过程中，产生神经细胞(神经元)的祖细胞相互协调，以确保在正确的时间在正确的位置产生特定类型的神经元。令人惊讶的是，关于它们的主纤毛中的哪些受体可以进行有效的交流，人们知之甚少。该项目将使用新技术记录位于神经前体细胞初级纤毛中的完整受体目录，该技术用名为生物素的特殊标签选择性地标记纤毛蛋白质，然后使用质谱仪鉴定这些标记的蛋白质。它还将破译其中一些蛋白质影响发育决定的机制。试点研究已经发现了以前被证明控制大脑发育的蛋白质，这些蛋白质出人意料地在纤毛中运作。总体而言，该项目的结果将为神经细胞前体如何协调构建大脑提供新的见解。它们促进了我们对大脑发育如何工作的理解，也将帮助我们更好地理解大脑发育障碍。此外，该项目将让当地的K-12学生和高中教师参与实验室研究，旨在激发当地青年对生物科学的持续兴趣，并实施一项综合教育计划，以促进来自代表性不足的少数族裔的学生参与和留住生物研究。这些活动将极大地改善加州中央山谷的生命科学教育。发育中的大脑中的神经前体细胞(也称为放射状胶质细胞，RG)以特定时间和空间的方式产生大脑的所有神经元。不同类型的神经元在不同的发育阶段和不同的大脑区域产生。这一过程是通过RG初级纤毛感应的细胞信号高度协调的。纤毛功能缺陷会导致纤毛疾病，这是一种广泛的疾病，通常涉及大脑结构缺陷。然而，对RG纤毛信号通路缺乏系统的了解。该项目将利用一种新的邻近标记工具(TurboID)和一种新的转基因小鼠模型(其中TurboID在RG细胞的纤毛中选择性表达)来识别RG纤毛中的新信号蛋白。具有严格对照的定量蛋白质组学研究将揭示真正的纤毛蛋白质在不同的大脑区域和不同的发育阶段中发挥作用。以纤毛为靶标的TurboID的初步研究令人惊讶地揭示了纤毛定位，这是一种先前报道的通过未知机制调节神经发生的蛋白质。初步数据显示，这种蛋白在纤毛中起作用，调节Hedgehog信号，这是纤毛中描述得最好的途径。研究这种新的纤毛蛋白与Hedgehog信号之间的相互作用将揭示胚胎神经发生的新调控机制，并展示纤毛蛋白质组学如何帮助解决大脑发育中长期存在的问题。通过系统地揭示RG纤毛所使用的时空分辨率的信号通路，该项目还将为未来的神经发育研究绘制新的方向。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。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).Most cells in our body have a primary cilium (a single hair-like projection on their cell surface) that allows them to interact with each other via chemical signals. This ancient and mysterious organelle acts like a radio antenna, allowing cells to receive different information, depending on which kinds of receptors their antenna contains. Gene mutations affecting ciliary signaling have been implicated in a host of diseases that result in the malformation and dysfunction of multiple organs. This project focuses on primary cilia in the developing brain. During brain formation, the progenitor cells that give rise to nerve cells (neurons) coordinate with each other to make sure that specific type of neurons are produced in the correct location at the right time. Surprisingly little is known about which receptors in their primary cilia allow effective communication. This project will document the full catalog of receptors situated in the primary cilia of neural progenitor cells using novel techniques that selectively label cilium proteins with a special tag named biotin, and then identify these marked proteins using mass spectrometry. It will also decipher the mechanisms by which some of these proteins influence developmental decisions. Pilot studies have already identified proteins previously shown to control brain development that are unexpectedly operating in the cilium. Overall, the results of this project will provide novel insights into how neural cell progenitors coordinate to build the brain. They advance our understanding of how brain development works and will also help us to better understand brain developmental disorders. In addition, this project will engage local K-12 students and high school teachers in laboratory research, aiming to inspire a sustained interest in the biological sciences among local youth, as well as implementing an integrated educational program to promote participation and retention of students from underrepresented minorities in biological research. These activities will substantially improve education in life science in the socioeconomically disadvantaged California Central Valley.Neural progenitors in the developing brain (also known as Radial Glia, RG) produce all of the brain’s neurons in a time- and space-specific manner. Different neuronal types are generated at distinct developmental stages and in discrete brain regions. This process is highly coordinated via cell signaling sensed by the RG primary cilia. Defects in cilium function lead to ciliopathies, a wide-ranging spectrum of disorders that usually involve brain structural defects. Yet a systematic understanding of RG cilium signaling pathways is lacking. This project will identify new signaling proteins in RG cilia by leveraging a new proximity labeling tool (TurboID) and a novel transgenic mouse model in which TurboID is selectively expressed in the cilium of RG cells. Quantitative proteomic studies with rigorous controls will reveal bona fide cilium proteins operating in discrete brain regions and across different developmental stages. Pilot studies with cilium-targeted TurboID have surprisingly revealed cilium localization of a protein previously reported to regulate neurogenesis through undetermined mechanisms. Preliminary data show that this protein operates in the cilium to regulate Hedgehog signaling, the best-described pathway in the cilium. Investigating the interaction between this new cilium protein and Hedgehog signaling will reveal new regulatory mechanisms in embryonic neurogenesis, and demonstrate how cilium proteomics can help solve standing long-questions in brain development. By systemically unveiling the signaling pathways used by RG cilia with spatiotemporal resolution, this project will also chart new directions for future neurodevelopmental studies.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.