S129A aSyn inclusions clearance is compromised.
(A) Schematic representation of the treatments performed: aSyn expression was induced for 6 hours in galactose medium (Gal) and then yeast cells were transferred to glucose medium (Glu) to repress aSyn expression for 6 hours. (B) Intracellular localization of WT or S129A aSyn-GFP and (C) percentage of yeast cells containing aSyn-GFP inclusions, assessed by fluorescence microscopy at the indicated time points of aSyn clearance (***p<0.001, **p<0.01; two-tailed unpaired t-test with Welch's correction). (D) Percentage of cells presenting 3 or less inclusions, or 4 or more inclusions per cell (**p<0.01, *p<0.05; Unpaired two-tailed t-test), and (E) inclusions size (µm2) at 0 or 6 hours of aSyn clearance. (F) SSC (side scatter) and aSyn-GFP fluorescence in the indicated yeast cells, assessed by flow cytometry, at 0 and 6 hours after aSyn clearance. A sub-population of yeast cells presenting higher levels of GFP fluorescence is distinguishable after 6 hours of WT or S129A aSyn clearance (left panel). The frequency of cells of this sub-population is represented the in right upper panel. In the right lower panel a fluorescence microscopy picture exemplifies the different GFP intensities observed in aSyn yeast cells after 6 hours of clearance [(a) indicates cells with lower levels of GFP fluorescence and (b) sub-population of yeast cells presenting higher levels of GFP fluorescence]. A representative result is shown from at least three independent experiments. Values represent the mean ± SD.