Effect of Receptor Dimerization on Membrane Lipid Raft Structure Continuously Quantified on Single Cells by Camera Based Fluorescence Correlation Spectroscopy

Abstract

Membrane bound cell signaling is modulated by the membrane ultra-structure, which itself may be affected by signaling. However, measuring the interaction of membrane proteins with membrane structures in intact cells in real-time poses considerable challenges. In this paper we present a non-destructive fluorescence method that quantifies these interactions in single cells, and is able to monitor the same cell continuously to observe small changes. This approach combines total internal fluorescence microscopy with fluorescence correlation spectroscopy to measure the protein’s diffusion and molecular concentration in different sized areas simultaneously. It correctly differentiates proteins interacting with membrane fences from proteins interacting with cholesterol-stabilized domains, or lipid rafts. This method detects small perturbations of the membrane ultra-structure or of a protein’s tendency to dimerize. Through continuous monitoring of single cells, we demonstrate how dimerization of GPI-anchored proteins increases their association with the structural domains. Using a dual-color approach we study the effect of dimerization of one GPI-anchored protein on another type of GPI-anchored protein expressed in the same cell. Scans over the cell surface reveal a correlation between cholesterol stabilized domains and membrane cytoskeleton.

Caveolae and lipid sorting: Shaping the cellular response to stress

“…This is a challenge, as purification of caveolae with the neck subdomain intact would be difficult. However, modern methods of lipid analysis and lipid localization are approaching the required resolution/sensitivity to make these distinctions in cells (Contreras et al, 2012; Owen et al, 2012; Huang et al, 2015; Zhou et al, 2015b). Enrichment of specific acidic lipids such as phosphatidylglycerol and lysophosphatidylglycerol, and an increase in long-chain unsaturated fatty acids, was observed in a model bacterial system for caveola formation compared with the lipidome of the prokaryotic host (Walser et al, 2012).…”

Plasma Membrane Organization of Epidermal Growth Factor Receptor in Resting and Ligand-Bound States

“…Cells were incubated with 3 mM mbCD and D and t 0 of EGFR were monitored for up to 1 h. 3 mM mbCD is sufficient to deplete cholesterol-containing domains (37,38). Since in the ITIR-FCS diffusion law, multiple observation areas are measured in a single FCS experiment, one can monitor the time evolution of spatial heterogeneity of a tracer on the membrane under perturbing conditions (23,39).…”

Imaging FCS Delineates Subtle Heterogeneity in Plasma Membranes of Resting Mast Cells

“…Importantly, such actin-based compartmentalization imposes fundamental membrane organization by preventing Lo/Ld lipid phase separation (30)(31)(32)(33). Considerable evidence supports the view that nanoscale Lo-like channels align along the picket fences, either by the effects of critical behavior and pinned Lo-preferring components (30,31,34) or by stabilization with Lo-preferring protein pickets (35,36). In the hierarchical model, Lo-like nanodomains (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) nm) and protein complexes (3-10 nm) also exist within the corrals.…”