Concurrent diffusion of nicotinic acetylcholine receptors and fluorescent cholesterol disclosed by two-colour sub-millisecond MINFLUX-based single-molecule tracking

Nicotinic acetylcholine receptors (nAChRs) are ubiquitous neurotransmitter receptors predominantly located at the cell-surface of neurons and muscle cells. Their dynamics affect synaptogenesis at neurodevelopmental stages and the efficacy of synaptic transmission in the adult synapse. Here we exploit the enhanced capabilities of superresolution fluorescence MINFLUX microscopy to track for minute-long periods with nanometric precision and sub-millisecond time resolution the 2D translational dynamics of the bungarotoxin-labelled adult muscle-type nAChR in tandem with a fluorescent cholesterol analogue. To this end, we implemented a multiplexing procedure in continuous MINFLUX microscopy that enabled the simultaneous excitation of the two molecules using a single wavelength, followed by discrimination of their emissions via differential ratiometric recording. Single-molecule trajectories displayed a heterogeneous spectrum of diffusive behaviours (subdiffusive, Brownian and superdiffusive), with a predominance of the subdiffusive component, which became less pronounced upon cholesterol depletion. nAChRs spent most of their lifetime in confined areas of characteristic size (~ 0.005 µm ² ) lasting for ~ 100 ms. Further, MINFLUX captured regions where nAChR and fluorescent cholesterol moved jointly, both in confinement sojourns and along the free Brownian walks, which strongly indicated mutual interactions between the receptor macromolecule and the neutral lipid. To the best of our knowledge, this study constitutes the first series of experiments showing the diffusion dynamics of a transmembrane protein -a functionally important neurotransmitter receptor- together with a key membrane lipid in the native plasma membrane of a live cell at such high detail, thanks to the MINFLUX-based recordings.