Intracellular distribution of glutathionylated proteins in cultured dermal fibroblasts by immunofluorescence.

Abstract

S-glutathionylation is a mechanism of signal transduction by which cells respond effectively and reversibly to redox inputs. The glutathionylation regulates most cellular pathways. It is involved in oxidative cellular response to insult by modulating the transcription factor Nrf2 and inducing the expression of antioxidant genes (ARE); it contributes to cell survival through nuclear translocation of NFkB and activation of survival genes, and to cell death by modulating the activity of caspase 3. It is involved in mitotic spindle formation during cell division by binding cytoskeletal proteins thus contributing to cell proliferation and differentiation. Glutathionylation also interfaces with the mechanism of phosphorylation by modulating several kinases (PKA, CK) and phosphatases (PP2A, PTEN), thus allowing a cross talk between the two processes of signal transduction. Glutathionylation of proteins may also act on cell metabolism by modulating enzymes involved in glycosylation, in the Krebs cycle and in mitochondrial oxidative phosphorylation. Perturbations in protein glutathionylation status may contribute to the etiology of many diseases, thus it is clear the importance to visualize the distribution of glutathionylated proteins in subcellular compartments. This chapter describes the immunofluorescence technique that permits simultaneous detection of glutathionylated proteins and their localization in cellular compartments, using multiple stained cell samples. By confocal laser microscopy analysis of the immunofluorescent cells it is possible to obtain detailed information of submicroscopic structures inside cells and tissues, and to perform correct co-localization analysis between two proteins. The association between glutathione, nuclear lamina, and cytoskeleton has been investigated by employing a helpful assay consisting on the in situ extraction of the cellular matrix from cultured dermal fibroblasts followed by multiple stainings with several primary antibodies. This protocol can be used for the detection of the intracellular distribution and expression of interest proteins and can be customized for a large variety of cells and tissues.