Long-Term Live Observation of Membrane Protein Interaction with Lipid Nanodomains Show Dependence on Cell Cycle and Time After Transfection

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The study of cholesterol-stabilized nanodomains in the cell membrane, aka lipid rafts, is complicated by differences in techniques, cell type, physiological parameters and possible changes of cell and membrane composition during the cell cycle and after transient transfection. We recently developed a non-destructive and non-perturbing technique combining fluorescence correlation spectroscopy (FCS) with spatially-resolved camera TIRF imaging, bimFCS. This method provides a quantification of the strength of the membrane protein - lipid nanodomain interaction, the membrane protein concentration and its diffusion coefficient. As an optical method it can be combined with other imaging methods such as we record changes in cytoskeleton structure simultaneously. bimFCS data was acquired with hourly intervals for periods longer than one day while keeping the cells growing at physiological temperature on the microscope stage. Transiently transfecting PtK2 or CHO cells with GPI-anchored membrane proteins associated with cholesterol-stabilized nanodomains, we observed a higher association with such domains in earlier stages of protein expression and slower diffusion compared to later times. Using synchronized cell cultures, we also quantify the membrane protein interaction with lipid nanodomains and record the images of membrane cytoskeletal actin bundles as function of the cell cycle. To discriminate between possible membrane ultrastructure models, we combine the experimental results with diffusion simulations of molecules interacting with such domains in the presence and absence of linear actin meshwork acting as pinpoints for the domains.