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.
Highlights
Many forms of cell membrane bound signaling require the interaction of diffusing membrane proteins, such as dimerization of or kinase activity on a receptor
For data taken from the same bilayer, normalized fluorescence correlation spectroscopy (FCS) curves of different binned-pixels show that the autocorrelation time increases with increasing bin size, as expected for a diffusion-dominated process (Fig. 1a)
Two simulations of two-dimensional free diffusion analyzed by binned-imaging FCS (bimFCS) were overlaid on the experimental data (Fig. 1b): a super-resolution simulation using a δ-function as point-spread function (PSF) and ω2 set as equal to the pixel area divided by π, and a simulation using the point spread function (PSF) of the microscope and computed equivalent ω ‘s for the binned pixels
Summary
Many forms of cell membrane bound signaling require the interaction of diffusing membrane proteins, such as dimerization of or kinase activity on a receptor. Some diffusing proteins are corralled between “fences” created by cytoskeleton-anchored membrane-associated proteins[8]; other diffusing proteins are transiently captured or trapped in either protein
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