Fluorescence excimer formation imaging: a new technique to investigate association to cells and membrane behavior of glycolipids

A new fluorescence ratio imaging technique aiming to monitor the lateral distribution of pyrene-labeled lipids in the membranes through visualization of the excimer/monomer (E/M) intensity ratio, has been set up, studying the association of a fluorescent derivative of GM1 ganglioside (pyreneGM1) to rat cerebellar granule cells in culture. The imaging results show that the mean E/M ratio value, under experimental conditions leading to the association of pyreneGM1 with the plasma membrane, is significantly higher in neuritic processes than in cell bodies and, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylaminodiphenylhexatriene) shows the presence of domains having different fluidity and that the average fluidity is higher in cell bodies than in neuritic processes. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in the two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more clustered state at the level of neuritic processes than in cell bodies and locally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation imaging technique to study membrane behavior and dynamics of pyrene-labeled lipids, with a particular insight into their aggregative properties.