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.

Caveolin-rich lipid rafts of the plasma membrane of mature cerebellar granule neurons are microcompartments for calcium/reactive oxygen and nitrogen species cross-talk signaling

“…A c c e p t e d M a n u s c r i p t 8 microscope, and quantitative analysis of the average fluorescence intensity of the frames and of selected neuronal soma was done with the HCImage software using the region of interest (ROI) tool of this software, as in previous works [27,33,38,39,46]. Only fields devoid of large aggregates of neurons forming granules or small grain-like structures were selected for image acquisition, to minimize distortion of images by the sum of the fluorescence contributions of juxtaposed layers of neurons.…”

Cytosolic Calcium Homeostasis in Neurons — Control Systems, Modulation by Reactive Oxygen and Nitrogen Species, and Space and Time Fluctuations

“…Experimental evidences have pointed out that there is a large mesh/network of lipid raftsassociated nanodomains in the plasma membrane of the soma of primary cultures of cerebellar granule neurons, where they are particularly enriched in neuron/neuron contact areas [130], and microscopy images have also shown a distribution map that closely overlap with the distribution map of flavoproteins bound to the plasma membrane [130,261], consistent with the association of the flavoproteins nNOS and cytochrome b 5 reductase with these nanodomains. Because of the strong impairment of the activity of calcium transport systems present in these nanodomains by many ROS/RNS that can be generated in the neuronal cytoplasm under a variety of cellular stress conditions, it should be expected that even exposure of neurons to a relatively mild oxidative stress should elicit a partial failure of the control of calcium homeostasis within these neurons.…”

Are There Lipid Membrane-Domain Subtypes in Neurons with Different Roles in Calcium Signaling?

“…In this case, modulation by superoxide anion might be associated with superoxide anion producing enzymes of very specific sources, also clustering within lipid membrane domains [32]. Flavoproteins, such as the enzyme cytochrome b 5 reductase (Cb 5 R), have been established to form complexes within plasma membrane lipid membrane domains of cerebellar granule neurons, as those described by our laboratory [28]. Cb 5 R is one of the major sources of superoxide anion in the plasma membrane lipid membrane domains of cerebellar granule neurons [29,31].…”

“…Methods for lipid membrane-domain isolation based on differential gradient centrifugation cannot discern the existence of lipid membrane nanodomain subtypes. Particularly, cytochemical and histochemical studies combined with physicochemical techniques based on quantitative fluorescence energy transfer (FRET) techniques, as those conducted by the research group led by Prof. Gutierrez-Merino, have provided insights into this matter by identification of proteins in clusters complexing with protein markers of lipid membrane domains (caveolin and flotillin isoforms) at a distance <100 nm in studies performed in neurons and brain tissue using the appropriate secondary fluorescent antibodies against the primary antibody of the selected lipid membrane-domain marker (Box 1) [28][29][30][31][32][33][34][35][36]. As discussed in these articles, this is a particular case of FRET from one donor to multiple acceptors, a situation in which the maximum range of FRET distance is significantly expanded, as analyzed in detail in former studies with purified biological membranes [23,[37][38][39].…”