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Investigating the Role of Lipid Anchors in Live Cell Membrane Organization Using Fluorescence Fluctuation Spectroscopy

Abstract

The cellular plasma membrane is a heterogeneous and dynamic two-dimensional fluid. It has long been considered the necessary boundary between the internal components of a cell and the surrounding environment. The organization of membrane proteins, originally thought to freely and randomly diffuse throughout the plasma membrane, has been recognized as essential to the proper functioning of a cell. Evidence from detergent extraction of membranes, model membranes, and nanometer resolution microscopy techniques requiring cell fixation have supported the idea that the membrane, through lipid-lipid and lipid-protein interactions, plays an active role in membrane protein organization. Advanced fluorescence microscopy and spectroscopy techniques offering subdiffraction resolution have added to the evidence and knowledge of heterogeneity in the membrane lipid bilayer of live cells, but have led to mixed results on the exact role that lipid-lipid and lipid-protein interactions play in lateral membrane organization.

Here we employ pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to study the co-diffusion of lipid anchors in live cell membranes. Lipid anchors are lipid moieties often found attached to membrane proteins. They function to tether proteins to membranes, and we ask if they also play a larger role in lateral membrane organization. We use fusions of fluorescent proteins and lipid anchors that participate in only lipid-lipid and lipid-peptide interactions without any direct protein-protein interactions. PIE-FCCS allows us to observe the co-diffusion of two different colored species while completely removing cross-talk due to spectral bleed-through and simultaneously measuring the fluorescence lifetimes to monitor energy transfer. We find a highly specific organizing scheme wherein different anchors partition into distinct domains, and we see no evidence of Förster resonance energy transfer through fluorescence lifetimes indicating the absence of tight anchor interactions.

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