Mechanisms of Protein Self-Assembly Coupled to Membrane Mechanics in the Cell

Clathrin-mediated endocytosis (CME) is an essential pathway used by all eukaryotes for the transport of extracellular cargo into the cell. By controlling many of the signals that are transmitted between cells, CME is a key component in the development of organisms and neurotransmission. Although the basic mechanism of clathrin-coated vesicle formation is known, the process is sensitive to membrane composition and mechanics, and the concentrations, interactions, and chemical modifications of dozens of cytoplasmic proteins and cargo receptors. Thus key outstanding questions remain: how does cargo control the nucleation and growth of clathrin-coated structures, thus ensuring proper uptake in response to changing stimuli from the external environment? While formation of clathrin-coated structures in cells is clearly linked to cargo levels, this coupling is not retained in vitro, where structures assemble without any cargo present. Establishing the mechanisms and frequency whereby clathrin-coat remodeling can drive productive vesicle formation is critical to understanding when cargo is internalized in healthy or diseased cells. The problem is a natural target for biophysical modeling because the fundamental structure of the problem (the clathrin cage) is known, but predicting how cargo uptake depends on the lipid and cargo composition and the mechanical properties of the membrane is difficult because of the nonlinear coupling between protein assembly and mechanics. Our proposed work will determine when and how lipid and cargo binding will control nucleation and growth of clathrin coated structures. We will address an open question on how curved vesicles can emerge from initially flat lattices in time. Our simulations will predict responses and rescue from PIP2 and cargo inhibition, where clathrin-coat formation effectively terminates in cells, with experimental tests from our expert collaborators. We will quantify the role of clathrin polymerization forces and cooperativity in controlling nucleation on membranes, as well as how it responds to changes to membrane rigidity. We will work with our experimental collaborators to iteratively refine our model against new conditions. The impact of this proposal will be a validated model of CME that can be used to predict cargo receptor uptake in response to external stimuli and environmental changes that directly alter the plasma membrane. The methods and mechanistic insights from this work on CME will transfer to other pathways like cell division, intracellular trafficking, and viral budding, where proteins must similarly sense the membrane environment to ensure assembly at the right time and place. Project Number: 1R35GM161901-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Margaret Johnson | Institution: JOHNS HOPKINS UNIVERSITY, BALTIMORE, MD | Award Amount: $420,936 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-J (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11260873