The biophysical interaction network that couples zona occludens-1 to membrane proteins at tight junctions

Tight junctions are dynamic membrane-bound protein assemblies that maintain barrier function for tissues as cells divide, grow, and move through development and throughout life. Maintenance of tissue barriers requires spatially separated intracellular and extracellular signaling pathways to be linked through novel and dynamic protein interaction networks. Tight junction intracellular signaling is governed by zona occludens-1 (ZO-1), a Membrane-Associated GUanylate Kinase (MAGUK) family protein that recruits and assembles transmembrane proteins and scaffolds them to the actin cytoskeleton through its own oligomerization, localization to membranes, and phase separation. Here we report comprehensive quantitative studies that nearly completely maps the intracellular interaction network between ZO-1 and tight junction transmembrane proteins that elucidates the biophysical principles governing their binding and a mechanism for how stimuli transmit through membranes. We establish where, how, and with whom each protein interacts, and identify how membrane proteins enable ZO-1 and greater tight junction function through specific interactions or by phase separation. Our findings reveal the intracellular interactome that triggers and regulates tight junction assembly, the active and direct roles each membrane protein plays in these processes, and provide a mechanism for how intra/extracellular signaling is coupled to transmit mechanochemical signals between cells via tight junctions. These insights provide a prospective blueprint for how other MAGUK/membrane protein interaction networks form and function across biology.