Regionalization of plasma membrane-bound flavoproteins of cerebellar granule neurons in culture by fluorescence energy transfer imaging

Abstract

Flavoproteins are components of plasma membrane redox chains, which have been suggested to play major roles in neuronal activity and survival. We found that the red/orange autofluorescence of mature primary cultures of cerebellar granule neurons (8-9 days in vitro) was largely quenched by millimolar concentrations of dithionite added to the extracellular medium, and pointed out that nearly 50% of this autofluorescence was due to plasma membrane-bound flavoproteins. We report in this work that the lipophilic neuronal plasma membrane markers N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)-pyridinium dibromide (RH-414) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64) can form fluorescence energy transfer donor-acceptor pairs with flavoproteins with calculated R (0) values between 3.7 and 4.2 nm. The quantification of the efficiency of fluorescence energy transfer with different concentrations of acceptor dyes has been worked out with re-suspended neurons. Using quantitative images of the neurons in culture, acquired with a CCD camera attached to an epifluorescence microscope, regionalization of the plasma membrane-bound flavoproteins of cerebellar granule neurons has been achieved from the quenching by dithionite of the fluorescence of the acceptor dye. The results unraveled that plasma membrane-bound flavoproteins are largely enriched in interneuronal contact sites forming clusters of 0.5-1 microm diameter size, which appears largely regionalized in the neuron's cell body.