Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling
Fig 5
(A) Scheme of the lentivirus construct LVGFAPEGFP-mMetRSL274G driving the expression of EGFP-mMetRSL274G from the simplified GFAP promoter version gfaABC1D. (B) Primary neuron-glia cocultures (DIV 14), infected with the lentivirus LVGFAPEGFP-mMetRSL274G, expressed the enzyme cell type-specifically in astrocytes. Cultures were incubated with either 4 mM AHA, ANL or Met for 4 h in Met-free medium, and AHA or ANL incorporation was visualized using a TAMRA-alkyne tag in a subsequent ‘click-reaction’. Astrocytes are identified employing GFAP immunostaining. AHA labeling occurred in a non-cell-selective manner both in neurons and astrocytes. TAMRA-positive proteins are detected solely in GFAP-positive, EGFP-mMetRSL274G expressing astrocytes upon incubation with ANL. Comparable TAMRA signals are observable in ANL and AHA labeled cells with a high nuclear and cytoplasmatic intensity of tagged proteins (scale bar = 10 μm). (C) Primary neuron-glia cocultures were infected with LVGFAPEGFP-mMetRSL274G to express EGFP-mMetRSL274G in astrocytes. Cocultures (DIV 22) were subjected to either 4 mM AHA, ANL or Met in Met-free medium for 4 h. Live-staining with 2 μg/ml propidium iodide for dead or dying cells revealed a non-significant increase in the percentage of dead cells of the counted EGFP-mMetRSL274G-positive astrocyte population after ANL incubation. Numbers at the X-axis indicate the total number of EGFP-mMetRSL274G positive astrocytes counted (n = 5 independent experiments; represented data are mean +/- SEM; ONE-way ANOVA, p>0.05).