Multi-body Fluctuation-Induced Forces Between Membrane Proteins: Insights from Mesoscale Simulations
The spatial organization of membrane-associated proteins is essential for a wide range of cellular processes, including signal transduction, endocytosis, and cell adhesion. While protein clustering can be driven by direct short-range forces, indirect interactions mediated by the membrane itself, particularly those arising from thermal shape fluctuations, are potentially sufficient to drive clustering in the absence of direct binding. In this study, we investigate how fluctuation-induced interactions contribute to the lateral organization of membrane inclusions using mesoscale simulations. Our approach is based on dynamically triangulated surfaces and is parameterized by three mesoscale quantities that capture local membrane rigidification and curvature induction. We show that local membrane rigidification drives the non-random organization of membrane inclusions and, above a critical concentration threshold, induces a fully segregated state. This threshold depends strongly on the magnitude of the induced rigidification. We further demonstrate that membrane tension only weakly affects lateral organization away from the threshold but has a pronounced effect near it. Extending our analysis to spherical geometries, we obtain similar behavior relevant to experiments on small unilamellar vesicles. In mixed systems containing two types of stiff inclusions, we find that stiffer proteins act as nucleation centers for softer proteins. Finally, we show that protein-induced curvature, combined with fluctuation-mediated clustering, can drive membrane shape remodeling. Our results are consistent with previous findings while additionally extending the characterization across the full parameter space and to new conditions of direct biological relevance. Overall, our findings suggest that local suppression of membrane-shape fluctuations by proteins generates effective attractive forces capable of driving protein reorganization on membranes, with broad implications for cell biology and the design of membrane-associated nanoparticles. STATEMENT OF SIGNIFICANCEThis study demonstrates that membrane protein lateral organization can emerge solely from membrane-mediated interactions driven by thermal shape fluctuations, independent of direct protein-protein interactions. Although theoretical models have long predicted such forces, their non-additive nature and apparent weakness left it unclear whether they could overcome mixing entropy. Here, we show that these forces can indeed be sufficient to drive clustering and, crucially, that they promote the formation of heterogeneous protein assemblies. These findings suggest that protein accumulations observed in experimental membrane-imaging data may arise from membrane fluctuations rather than direct affinities, highlighting the need for careful consideration when interpreting membrane-imaging experiments.