Phase separation modulates membrane-associated receptor organization in dendritic spines

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。神经元之间的交流在大脑的学习、记忆、思维处理和感觉功能（如视觉或听觉）中起着核心作用。神经元信号传递是由神经元膜上的谷氨酸受体控制的。这些受体通过结合伙伴组织以促进有效的信号传递。受体与结合伙伴的液液相分离是受体在膜上组织的关键机制。结合伙伴中的内在无序区（IDRs）已被证明可以调节相分离以促进膜上受体的组织。修饰这些蛋白质中的idr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）和谷氨酸受体相互作用并形成神经元信号传递所需的不同簇。MAGUKs和谷氨酸受体都含有可改变的idr，这些idr与受体簇形成的控制有关。我们假设相分离控制着膜上受体的组织。目的：我们将在模型膜上生化重建谷氨酸受体和MAGUK蛋白簇，并使用全内反射荧光显微镜评估受体的组织。maguk要么是非磷酸化的，要么是磷酸化的野生型idr，要么是预测为塌陷、中间或扩展构象的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 idr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调控细胞膜受体组织的变化。意义：这些研究将为了解MAGUK家族蛋白调控树突棘膜上谷氨酸受体组织的生物物理机制提供基础。该研究项目的结果还将提供对控制结构域之间发现的idr如何调节许多细胞类型中膜上其他相分离簇的形成的一般原理的见解。在我的研究项目中训练的高素质人才将获得生化研究、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，这些将使他们成为加拿大知识经济的未来领导者。

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.