Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy.

Astrid Gesper,Ingela Parmryd,Patrick Happel,Sven-Göran Eriksson,Philipp Hagemann,Stefan Wennmalm

doi:10.3389/fcell.2020.00767

Abstract

Fluorescence correlation spectroscopy (FCS) is frequently used to study diffusion in cell membranes, primarily the plasma membrane. The diffusion coefficients reported in the plasma membrane of the same cell type and even within single cells typically display a large spread. We have investigated whether this spread can be explained by variations in membrane topography throughout the cell surface, that changes the amount of membrane in the FCS focal volume at different locations. Using FCS, we found that diffusion of the membrane dye DiI in the apical plasma membrane was consistently faster above the nucleus than above the cytoplasm. Using live cell scanning ion conductance microscopy (SICM) to obtain a topography map of the cell surface, we demonstrate that cell surface roughness is unevenly distributed with the plasma membrane above the nucleus being the smoothest, suggesting that the difference in diffusion observed in FCS is related to membrane topography. FCS modeled on simulated diffusion in cell surfaces obtained by SICM was consistent with the FCS data from live cells and demonstrated that topography variations can cause the appearance of anomalous diffusion in FCS measurements. Furthermore, we found that variations in the amount of the membrane marker DiD, a proxy for the membrane, but not the transmembrane protein TCRζ or the lipid-anchored protein Lck, in the FCS focal volume were related to variations in diffusion times at different positions in the plasma membrane. This relationship was seen at different positions both at the apical cell and basal cell sides. We conclude that it is crucial to consider variations in topography in the interpretation of FCS results from membranes.

Highlights

In diffusion studies of membrane molecules with optical techniques, a common assumption is that the membrane is both flat and aligned with the imaging plane
In the analysis of data from widespread methods employed for diffusion studies of membrane components like fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and single particle tracking (SPT) it is still assumed that the membrane is locally flat and aligned with the imaging plane
While it is possible that membrane organization might correspond to these models, non-flat surfaces alone can create the appearance of anomalous diffusion – something we have previously demonstrated for SPT-analysis (Adler et al, 2010, 2019)

Summary

Introduction

In diffusion studies of membrane molecules with optical techniques, a common assumption is that the membrane is both flat and aligned with the imaging plane. In the analysis of data from widespread methods employed for diffusion studies of membrane components like fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and single particle tracking (SPT) it is still assumed that the membrane is locally flat and aligned with the imaging plane. While it is possible that membrane organization might correspond to these models, non-flat surfaces alone can create the appearance of anomalous diffusion – something we have previously demonstrated for SPT-analysis (Adler et al, 2010, 2019)

Methods

Results

Conclusion