Confinement energy landscape classification reveals membrane receptor nano-organization mechanisms.

The cell membrane organization has an essential functional role through the control of membrane receptor confinement in micro- or nanodomains. Several mechanisms have been proposed to account for these properties, though some features have remained controversial, notably the nature, size, and stability of cholesterol- and sphingolipid-rich domains- or lipid rafts. Here, we probed the effective energy landscape actin on single nanoparticle-labeled membrane receptors confined in raft nanodomains - Epidermal Growth Factor, C. perfringens ε-toxin and C. Septicum α-toxin receptors (EGFR , CPεTR and CSαTR,) - and compared it with hop-diffusing transferrin receptors (TfR). By establishing a new analysis pipeline combining Bayesian inference, decision trees and clustering approaches, we systematically classified single protein trajectories according to the type of effective confining energy landscape. This revealed the existence of only two distinct organization modalities: (A) confinement in a quadratic energy landscape for EGF, CPεT and CSαT receptors and (B) free diffusion in confinement domains resulting from the steric hindrance due to F-actin barriers for TfR. The further characterization of effective confinement energy landscapes by Bayesian inference revealed the role of interactions with the domain environment in cholesterol- and sphingolipid-rich domains with (EGFR) or without (CPεTR and CSαTR) interactions with F-actin, to regulate the confinement energy depth. These two distinct mechanisms result in the same organization type (A). We revealed that the apparent domain sizes for these receptor trajectories resulted from Brownian exploration of the energy landscape in a steady-state like regime at a common effective temperature, independently of the underlying molecular mechanisms. These results highlight that confinement domains may be adequately described as interaction hotspots rather than rafts with abrupt domain boundaries. Altogether, these results support a new model for functional receptor confinement in membrane nanodomains and pave the way to the constitution of an atlas of membrane protein organization.

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