Phase separation modulates membrane-associated receptor organization in dendritic spines

点击翻译按钮获取中文摘要

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.