BPS2026 – Modeling-informed experiments capture Arc-lipid interactions critical to intercellular communication

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) has been previously implicated to have a phospholipid-binding affinity to PI (3)P at the early endosome. Through the endosome-multivesicular body (MVB) pathway, Arc can form viral-like capsids encapsulating mRNA and are secreted as extracellular vesicles (EVs). While Arc has been shown to be a key regulator for synaptic plasticity, Arc’s ability to traffic important RNA cargo has not been clearly defined in the context of endosomal membranes. Furthermore, the mechanism of Arc’s binding to the membrane interface has been poorly defined, especially in the context of protein oligomerization and capsid assembly. Using all-atom molecular dynamics simulations and weight ensemble enhanced sampling, we predicted that Arc requires lipid binding to carry out its functional role of capsid formation and secretion. Following with site-directed mutagenesis, we validated Arc’s electrostatic interactions are primarily responsible for membrane association by creating a synthetic construct of mutated arginine and lysine residues along the matrix domain called KRRK-Arc. Using confocal microscopy, we found KRRK-Arc formed less puncta and was not compartmentalized within the MVB-endosome compartments. We perform modeling-informed in vitro binding experiments using nanodiscs to obtain binding kinetics between wild-type/KRRK-Arc and PI(3)P. To access the role of Arc-PI(3)P binding in the regulation of Arc oligomerization in cells, we use MINFLUX imaging to resolve Arc oligomer sizes. Our work summarizes key protein-lipid interactions that are necessary for downstream Arc interactions and will guide future studies for Arc intercellular communication.