Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green

Disorganization of the cytoskeleton of neurons has major consequences on the transport of neuro-transmitters via the microtubule network. The interaction of cytoskeleton proteins (actin and tubulin) was studied in neuronal SK-N-BE cells treated with tetracosanoic acid (C24:0), which is cytotoxic and increased in Alzheimer's disease patients. When SK-N-BE cells were treated with C24:0, mitochondrial dysfunctions and a non-apoptotic mode of cell death were observed. Fluorescence microscopy revealed shrunken cells with perinuclear condensation of actin and tubulin. After staining with rhodamine-phalloidin and with an antibody raised against alpha-/beta-tubulin, modifications of F-actin and alpha-/beta-tubulin levels were detected by flow cytometry. Lower levels of alpha-tubulin were found by Western blotting. In C24:0-treated cells, spectral analysis and fluorescence recovery after photo-bleaching (FRAP) measured by confocal microscopy proved the existence of fluorescence resonance energy transfer (FRET) when actin and tubulin were stained with tubulin tracker and rhodamine-phalloidin demonstrating actin and tubulin co-localization/interaction. In control cells, no FRET was observed. Our data demonstrate quantitative changes in actin and tubulin, and modified interactions between actin and tubulin in SK-N-BE cells treated with C24:0. They also show that FRET confocal imaging microscopy is an interesting method for specifying the impact of cytotoxic compounds on cytoskeleton proteins.