Phase separation modulates membrane-associated receptor organization in dendritic spines

相分离调节树突棘中膜相关受体组织

• 批准号: RGPIN-2022-03274
• 负责人: Ditlev, Jonathon
• 金额: $ 2.26万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744128
• 关键词: Phase; separation; modulates; membrane; associated

BACKGROUND: The long-term goal of my research program is to understand how neuronal proteins organize themselves into membraneless structures through a process called phase separation, and how phase separation controls cellular responses to stimuli. Communication between neurons plays a central role in learning, memory, thought processing, and sensory functions, such as seeing or hearing, within the brain. Neuronal signal transmission is controlled by glutamate receptors on the membrane of neurons. These receptors are organized by binding partners to promote efficient signal transmission. Liquid-liquid phase separation of receptors and binding partners is a key mechanism by which receptors can be organized on membranes. Intrinsically disordered regions (IDRs) in binding partners have been shown to regulate phase separation to promote the organization of receptors on membranes. Modification of IDRs in these proteins can also modulate their phase separation. On the membrane of dendritic spines, Membrane Associated Guanylate Kinases (MAGUKs) and glutamate receptors interact with one another and form distinct clusters that are required for neuronal signal transmission. Both MAGUKs and glutamate receptors contain modifiable IDRs that have been implicated in the control of receptor cluster formation. We hypothesize that phase separation controls receptor organization on membranes. OBJECTIVES: We will biochemically reconstitute glutamate receptor and MAGUK protein clusters on model membranes and use total internal reflection fluorescence microscopy to evaluate receptor organization. MAGUKs will be either non-phosphorylated or phosphorylated wild-type IDRs or IDRs that are predicted to assume collapsed, intermediate, or extended conformations. We will use these MAGUKs to determine how the conformation of the IDR regulates glutamate receptor cluster formation. The aims of our research program are: 1)Characterize the biophysical properties of MAGUK IDRs and their contribution to phase separation. 2)Elucidate the role of IDR phosphorylation in MAGUK-dependent phase separation. 3)Investigate MAGUK IDR-regulated changes in receptor organization on cell membranes. SIGNIFICANCE: These studies will provide a fundamental understanding of the biophysical mechanisms by which MAGUK family proteins regulate the organization of glutamate receptors on the membranes of dendritic spines. The results from the research program will also provide insight into the general principles that govern how IDRs found between structured domains regulate the formation of other phase separated clusters on membranes found in many cell types. Highly qualified personnel trained within my research program will acquire expertise in biochemical studies, biophysical analysis, cell biology, and advanced light microscopy techniques which will position them well to be future leaders of the Canadian knowledge-based economy.

背景技术背景：我的研究计划的长期目标是了解神经元蛋白质如何通过称为相分离的过程将自己组织成无膜结构，以及相分离如何控制细胞对刺激的反应。神经元之间的通信在大脑内的学习、记忆、思维处理和感觉功能（如视觉或听觉）中起着核心作用。神经元的信号传递受神经元膜上的谷氨酸受体控制。这些受体由结合伴侣组织，以促进有效的信号传递。受体和结合伴侣的液-液相分离是受体可以在膜上组织的关键机制。结合伴侣中的内含子无序区（IDR）已被证明可以调节相分离，以促进膜上受体的组织化。这些蛋白质中IDR的修饰也可以调节它们的相分离。在树突棘的膜上，膜相关鸟苷酸激酶（MAGUKs）和谷氨酸受体彼此相互作用并形成神经元信号传递所需的不同簇。MAGUK和谷氨酸受体都含有可修饰的IDR，其涉及受体簇形成的控制。我们假设相分离控制膜上的受体组织。 目的：我们将生化重建谷氨酸受体和MAGUK蛋白质簇模型膜，并使用全内反射荧光显微镜来评估受体组织。MAGUK将是非磷酸化或磷酸化的野生型IDR或预测呈现折叠、中间或延伸构象的IDR。我们将使用这些MAGUKs来确定IDR的构象如何调节谷氨酸受体簇的形成。我们的研究计划的目的是：1）表征MAGUK IDRs的生物物理性质及其对相分离的贡献。 2)阐明IDR磷酸化在MAGUK依赖性相分离中的作用。3)研究MAGUK IDR调节的细胞膜受体组织变化。重要性：这些研究将提供MAGUK家族蛋白调节树突棘膜上谷氨酸受体组织的生物物理机制的基本理解。研究计划的结果还将提供对结构化结构域之间发现的IDR如何调节许多细胞类型中发现的膜上其他相分离簇的形成的一般原则的见解。在我的研究计划中培训的高素质人员将获得生物化学研究，生物物理分析，细胞生物学和先进的光学显微镜技术方面的专业知识，这将使他们成为加拿大知识经济的未来领导者。