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 later al distribution of pyrene-labeled lipids in the membranes through visu alization of the excimer/monomer (E/M) intensity ratio, has been set u p, studying the association of a fluorescent derivative of GM1 ganglio side (pyreneGM1) to rat cerebellar granule cells in culture. The imagi ng results show that the mean E/M ratio value, under experimental cond itions leading to the association of pyreneGM1 with the plasma membran e, is significantly higher in neuritic processes than in cell bodies a nd, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylamino-diphenylhexa triene) shows the presence of domains having different fluidity and th at the average fluidity is higher in cell bodies than in neuritic proc esses. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in th e two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more cluster ed state at the level of neuritic processes than in cell bodies and lo cally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation i maging technique to study membrane behavior and dynamics of pyrene-lab eled lipids, with a particular insight into their aggregative properti es.