Heterogeneity of cell membrane structure studied by single molecule tracking.

Gregory I Mashanov,Tatiana A Nenasheva,Alla Mashanova,Lucia Sivilotti,Remigijus Lape,Nigel J M Birdsall,Justin E Molloy

doi:10.1039/d1fd00035g

Abstract

Heterogeneity in cell membrane structure, typified by microdomains with different biophysical and biochemical properties, is thought to impact on a variety of cell functions. Integral membrane proteins act as nanometre-sized probes of the lipid environment and their thermally-driven movements can be used to report local variations in membrane properties. In the current study, we have used total internal reflection fluorescence microscopy (TIRFM) combined with super-resolution tracking of multiple individual molecules, in order to create high-resolution maps of local membrane viscosity. We used a quadrat sampling method and show how statistical tests for membrane heterogeneity can be conducted by analysing the paths of many molecules that pass through the same unit area of membrane. We describe experiments performed on cultured primary cells, stable cell lines and ex vivo tissue slices using a variety of membrane proteins, under different imaging conditions. In some cell types, we find no evidence for heterogeneity in mobility across the plasma membrane, but in others we find statistically significant differences with some regions of membrane showing significantly higher viscosity than others.

Highlights

It is thought that structural heterogeneity of the plasma membrane plays a critical role in a variety of cell functions.[1,2] This contrasts with our classical view of the plasma membrane as a uid mosaic bilayer of phospholipids and other amphipathic molecules within which proteins can diffuse in an unhindered manner.[3]
We examined the mobility of a C-terminally-tagged M2-receptor eGFP-fusion protein where the uorophore is positioned on the intracellular-side of the molecule in contrast to the extrinsic, synthetic labelling with Cy3B-telenzepine used in the previous experiments
We found that the diffusive motion of M2 receptors at the plasma membrane of HL1 cells, primary cardiomyocytes and zebra sh cardiac tissue slices all gave pooled mean-squared displacement (MSD) vs. dT plots that were linear (Fig. S4†), with no obvious evidence of anomalous diffusion

Summary

Introduction

It is thought that structural heterogeneity of the plasma membrane plays a critical role in a variety of cell functions.[1,2] This contrasts with our classical view of the plasma membrane as a uid mosaic bilayer of phospholipids and other amphipathic molecules within which proteins can diffuse in an unhindered manner.[3]. Interaction of membrane proteins with the cortical cytoskeleton[9] or extracellular matrix can lead to obstructions to free-diffusion[10] (“picketfences”). Together these features contribute to anomalous diffusive behaviour[11,12] whereby MSD vs dT plots are non-linear and show distinct downward curvature. Tracking of single uorophores in live cells is technically challenging because the spatial resolution and temporal precision of tracking is limited by photon emission rate and photobleaching of the uorescent moiety. To measure small-scale, rapid movements, high laser power is required so that single uorophores are bright and can be tracked with high-precision at fast video frame rate, but the cost is they soon photobleach. At low laser power, uorophores can be tracked for longer but tracking resolution

Results

Discussion

Conclusion