Quantification of live cell membrane compartmentalization with high-speed single lipid tracking through interferometric Scattering Microscopy

Abstract

The lateral dynamics of lipids on the cellular membranes are one of the most challenging topics to study in membrane biophysics, needing simultaneously high spatial and temporal resolution. In this study, we have employed Interferometric scattering Microscopy (ISCAT) to explore the dynamics of a biotinylated lipid analogue labelled with streptavidin-coated gold nanoparticles (20 and 40nm in diameter) at 2kHz sampling rate. We developed a statistics-driven analysis pipeline to analyse both ensemble average and single trajectory Mean Squared Displacements from each dataset, and to discern the most likely diffusion mode. We found that the use of larger tags slows down the target motion without affecting the diffusion mode. Moreover, we determined from our statistical analysis that the prevalent diffusion mode of the tracked gold-labelled lipids is compartmentalized diffusion. This model describes the motion of particles diffusing on a corralled surface, with a certain probability of changing compartment. This is compatible with the picket-fence model of membrane structure, already observed by similar studies. Through our analysis, we could determine significant physical parameters, such as average compartment size, dynamic localization uncertainty, and the intra- and inter-compartmental diffusion rates. We then simulated diffusion in an environment compatible with the experimentally-derived parameters and model. The closeness of the results from the analysis of experimental and simulated trajectories validates our analysis and the proposed description of the cell membrane. Finally, we introduce the confinement strength metric to compare diffusivity measurements across techniques and experimental conditions, which we used to successfully compare the present results with other related studies.

Implications of different membrane compartmentalization models in particle-based in silico studies

“…Taylor et al [ 68 ] developed new imaging techniques that have overcome speckling issues associated with iSCAT, while Taylor & Sandoghdar [ 69 ] have given a review of iSCAT. Reina et al [ 31 ] have also used iSCAT to explore lateral membrane diffusion dynamics and to characterize different diffusion modes along with average compartment size, location uncertainty and diffusion rates.…”

“…The way the fences are modelled in these particle-based reaction–diffusion simulations varies. For example, Andrade et al [ 30 ] and colleagues [ 31 ] use infinite thin barriers that can be crossed with a certain hop probability. While these two studies used the approach in continuous space, this type of restriction can also be implemented in lattice-based simulations [ 32 ].…”

Quantitative analysis of peroxisome tracks using a Hidden Markov Model

“…However, accuracy is challenged by the sufficient spatial and temporal resolution of the observation technique, such as optical microscopy, and often, even more importantly, by data analysis. Directly recorded spatiotemporal diffusion tracks are usually compromised by noise and anomalous diffusion, such as local confinements or directed motions, which may be relatively rare [1][2][3][4][5] . This also holds for organelles in the cellular cytosol, such as endocytosed or transported vesicles or migration of peroxisomes.Peroxisomes are ubiquitous organelles in eukaryotic cells fulfilling many metabolic functions.…”