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

Area of Science:

• Cell biology
• Biophysics
• Membrane dynamics

Background:

• Fluorescence correlation spectroscopy (FCS) is widely used to measure diffusion in cell membranes, particularly the plasma membrane.
• Observed diffusion coefficients in plasma membranes often show considerable variability within the same cell type or even within a single cell.

Purpose of the Study:

• To investigate if variations in cell membrane topography explain the spread in FCS diffusion measurements.
• To determine the influence of membrane surface roughness on diffusion dynamics.

Main Methods:

• Utilized fluorescence correlation spectroscopy (FCS) to measure the diffusion of the membrane dye DiI.
• Employed live-cell scanning ion conductance microscopy (SICM) to map cell surface topography.
• Performed FCS modeling with simulated diffusion on SICM-derived surfaces.

Main Results:

• Diffusion of DiI was faster in the apical plasma membrane above the nucleus compared to above the cytoplasm.
• SICM revealed that the plasma membrane above the nucleus is smoother than other areas, correlating with faster diffusion.
• FCS modeling confirmed that topography variations can lead to apparent anomalous diffusion.
• Variations in the membrane marker DiD within the FCS focal volume correlated with diffusion time differences, unlike transmembrane or lipid-anchored proteins.

Conclusions:

• Cell surface topography is a critical factor influencing FCS measurements of membrane diffusion.
• The uneven distribution of membrane roughness must be considered when interpreting FCS data from cellular membranes.
• Topographical variations can account for apparent anomalies in diffusion observed via FCS.