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

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.

Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells

“…Unfortunately, we could not directly follow the molecular events during the very early stage of T‐cell activation, as the required temporal resolution at single‐molecule sensitivity is unattainable to the currently available live‐cell microspectroscopy techniques. Furthermore, we conducted our measurements only 5–10 min after the cell‐surface contact to avoid potential bias of the readouts owing to nonplanar topography of the initial contact sites [68], during which cell activation could have induced additional aggregation of TCR. However, as simulations predict that the local lipid composition and order respond to immobilisation of a membrane protein within microseconds [42], this lipid–protein interplay may represent the very first subtle, though likely not decisive, membrane response in sensing of external binding.…”

Adaptive Lipid Immiscibility and Membrane Remodeling Are Active Functional Determinants of Primary Ciliogenesis

“…Interestingly, this issue has recently been hinted to be a possible source in misinterpreting molecular diffusion values in cells, for instance, when using single particle tracking and FCS techniques. [ 46 ] More so, considering that cellular morphology is not planar, and that irregular 3D topology can macro and microscopically change continuously over a short period of time, such as in cell filopodia or cellular protrusions. [ 46,47 ] On the other hand, RICS‐based techniques like LICSR, can minimize bleaching artefacts coming from illuminating highly static components compared to FCS‐based techniques.…”

“…[ 46 ] More so, considering that cellular morphology is not planar, and that irregular 3D topology can macro and microscopically change continuously over a short period of time, such as in cell filopodia or cellular protrusions. [ 46,47 ] On the other hand, RICS‐based techniques like LICSR, can minimize bleaching artefacts coming from illuminating highly static components compared to FCS‐based techniques. This is because the stack of images can be processed applying a moving average filter to subtract an immobile fraction or very slow‐moving cellular feature.…”

“…Additionally, RICS‐based methods as LICSR, advantageously can be employed to measure diffusion coefficients at the plasma membrane, where morphology is not planar and irregular 3D topology change over a short period of time continuously in comparison to artefact‐prone single and scanning‐FCS methods. [ 46,47 ] To this extend, RICS, as a non‐single molecule technique would, in principle, be less influenced by out‐of‐focus intensity fluctuations. However, this cannot be completely discarded.…”

Aggregation and immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells

“…Unfortunately, we could not directly follow the molecular events during the very early stage of T cell activation, as the required temporal resolution at single-molecule sensitivity is unattainable to the currently available live-cell microspectroscopy techniques. Furthermore, we conducted our measurements only 5-10 min after the cell-surface contact to avoid potential bias of the readouts owing to non-planar topography of the initial contact sites (65), during which cell activation could have induced additional aggregation of TCR. However, as simulations predict that the local lipid composition and order respond to immobilisation of a membrane protein within microseconds (42), this lipid-protein interplay may represent the very first subtle, though likely not decisive, membrane response in sensing of external binding.…”