Blocking aSyn phosphorylation reduces membrane interactions and accelerates inclusion formation.

<p>(A) Time lapse confocal imaging comparing the distribution of WT or S129A aSyn-GFP at the indicated time points after induction of aSyn expression. Nuclei were visualized by co-expressing NLS-TagRFP657. (B) Percentage of WT and S129A aSyn-GFP expressing cells presenting inclusions over time. n_wt = 48 cells, n_S129A = 55 cells. Inclusions were recognized by measuring GFP intensity.</p

S129A aSyn is more toxic for yeast cells than the WT aSyn.

<p>(A) Growth curve based on culture OD_600nm of yeast cells expressing WT (□), S129A (▴) or S129E (○) aSyn-GFP, compared to cells that are not expressing the human protein (◊). Cells used as inoculum were exponential-phase cells cultivated in raffinose medium that at time zero were transferred to galactose medium to induce aSyn-GFP expression. (B) aSyn-GFP versus Propidium Iodide (PI) fluorescence and Frequency of PI and GFP positive cells assessed by flow cytometry, in the indicated yeast cells, after 6 hours of aSyn expression induction (***p<0.001; one way ANOVA with Bonferroni's multiple comparison test). A representative result is shown from at least five independent experiments. Values represent the mean ± SD.</p

Inclusions formed by S129A aSyn-GFP are more heterogeneous than the ones formed by WT aSyn-GFP.

<p>(A) FRAP recovery curves for WT and S129A aSyn-GFP inclusions on yeast cells expressing aSyn forms at the indicated time points. For each strain, at least 15 inclusions in different cells were analyzed. Each plot represents mean ± SD for each time point for all FRAP experiments (for the WT aSyn-GFP expressing cells) or groups of FRAP experiments organized by recovery profiles (for the S129A aSyn-GFP expressing cells). (B) FRAP time lapse recording of a representative inclusion of each group (C) Area (pixels) and fluorescence intensity mean (in arbitrary units, a.u.) of the inclusions analyzed (***p<0.001 and ** p<0.01; one way ANOVA with Bonferroni's multiple comparison test). A representative result is shown from at least three independent experiments. Values represent the mean ± SD.</p

The percentage of Triton-X insoluble S129A aSyn increases during clearance.

<p>(A) WT and S129A aSyn-GFP protein levels in yeast cells assessed by western blot analysis of total protein extracts at the indicated time points of aSyn-GFP clearance (left panel). Densitometric analysis of the immunodetection of aSyn relative to the intensity obtained for GAPDH, used as loading control, presented in arbitrary units (a.u.) (right panel) (***p<0.001, *p<0.05; one way ANOVA with Bonferroni's multiple comparison test). (B) Triton-X soluble (TS) and Triton-X insoluble (TI) fractions of aSyn WT or S129A assessed by western blot analysis (upper panel) and determination by densitometric analysis of the percentage of TI aSyn, at the indicated time points of aSyn clearance (lower panel) (*p<0.05; Mann-Whitney test). GAPDH was used as an internal control for the experiment. A representative result is shown from at least four independent experiments. Values represent the mean ± SD.</p

S129A aSyn forms more inclusions and oligomeric species with higher molecular weight than the WT form.

<p>(A) Intracellular localization of the WT, S129A or S129E aSyn-GFP (left panel) and percentage of yeast cells containing aSyn-GFP inclusions (right panel), assessed by fluorescence microscopy at the indicated time points after aSyn-GFP expression induction (***p<0.001; one way ANOVA with Bonferroni's multiple comparison test). (B) WT, S129A or S129E aSyn-GFP expression and pS129 levels in yeast cells assessed by western blot analysis of total protein extracts, at the indicated time points after aSyn-GFP expression induction (left panel). Densitometric analysis of the immunodetection of aSyn-GFP relative to the intensity obtained for GAPDH, used as loading control, presented in arbitrary units (a.u.) (right panel). Results shown are from one representative experiment from a total of three independent experiments. (C) The aggregated species formed in yeast cells by the WT and S129A aSyn-GFP were resolved using sucrose gradients after 6 hour of aSyn-GFP expression induction. The resulting fractions were separated on an SDS-page gel followed by immunoblotting with an antibody against aSyn (left panel). The same amount of WT and S129A aSyn-GFP protein was applied to the sucrose gradients, as confirmed by western blot (right panel). (D) The aggregated species formed by WT and S129A aSyn-GFP were also resolved using size exclusion-chromatography (SEC) after 6 hours of aSyn-GFP expression induction. Fractions were collected and separated on SDS-PAGE followed by western blot analyses with an antibody against aSyn or pS129 aSyn. The molecular weights indicated correspond to the calibration curve performed in the SEC, as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004302#pgen.1004302.s003" target="_blank">Figure S3</a>. A representative result is shown from at least three independent experiments. Values represent the mean ± SD.</p

Modulation of ER-to-Golgi trafficking alters S129A aSyn inclusion formation.

<p>(A) Intracellular localization of the WT or S129A aSyn-GFP (left panel) and percentage of yeast cells containing aSyn-GFP inclusions (right panel) in the indicated yeast strains, assessed by fluorescence microscopy after 6 hours of aSyn-GFP expression induction (*** p<0.001 <i>vs</i> WT; ^###<0.001 <i>vs</i> S129A; one way ANOVA with Bonferroni's multiple comparison test). (B) WT or S129A aSyn-GFP expression and pS129 levels in the indicated yeast strains assessed by western blot analysis of total protein extracts 6 hours after aSyn-GFP expression induction. A representative result is shown from at least three independent experiments. Values represent the mean ± SD.</p

S129A aSyn impairs <i>ATG8</i> induction.

<p>(A) Autophagy evaluated by mCherry-Atg8 processing assay in yeast cells expressing mCherry-Atg8 under the regulation of the endogenous promoter alone (control) or together with either WT or S129A aSyn, at the indicated time points, assessed by western blot (upper panel) (hi:hours of induction; hc: hours of clearance). <i>ATG8</i> induction quantified by the total mCherry signal (mCherry-Atg8 and free mCherry signal, detected with anti-mCherry) (lower left panel); autophagic flux quantified by measuring the vacuolar degradation of the Atg8 domain reporter (ratio of free mCherry to total mCherry signal) (lower right panel). (***p-value<0.001; one way ANOVA with Bonferroni's multiple comparison test). Results shown are from one representative experiment from a total of five independent experiments. (B) aSyn-GFP and mCherry-Atg8 fluorescence intensity in yeast cells expressing mCherry-Atg8 alone (control) or together with either WT or S129A aSyn, at the indicated time points, assessed by flow cytometry (upper panel). mCherry-Atg8 MFI from all cells (lower left panel) and from the indicated sub-population (lower right panel) (***p<0.001, one way ANOVA, with Bonferroni's multiple comparison test). Yeast cells not expressing aSyn-GFP or mCherry-Atg8 were also used as an additional control (empty). A representative result is shown from at least four independent experiments. Values represent the mean ± SD.</p

Plasma membrane to vacuole endocytic trafficking of WT and S129A aSyn.

<p>(A) aSyn inclusions localize to membrane and endosomal compartments. Cells coexpressing Jen1-mCherry and WT or S129A aSyn-GFP were grown at 30°C in glucose and shifted to galactose for 6 hours. Jen1-mCherry is constitutively synthesized, trafficked to the membrane, and re-absorbed through the endocytic pathway, eventually ending up in the vacuole. Since mCherry is pH stable it is visible in the vacuole. aSyn-GFP inclusions colocalize with Jen1-mCherry in endosome compartments throughout the entire endocytic pathway. (B) aSyn foci cluster with vesicles. Images shown are still frames from a time-lapse series showing internalization of aSyn through the endocytic pathway. In some cases of low level of aSyn aggregation enters the vacuole. As the aggregation level increases, however, aSyn-cotaining vesicles are trapped, likely in late endosomes. (C) aSyn inclusions are present as vesicles or vesicle clusters (arrows). Expression of S129A aSyn-GFP was induced for 8 hours. 3D reconstruction and single z sections (2D) are shown. Each z series was acquired with 0.4 micron step size and 21 total steps.</p

ER-to-Golgi trafficking genes are modifiers of S129A aSyn toxicity.

<p>(A) aSyn-GFP versus Propidium Iodide (PI) fluorescence and Frequency of PI positive cells assessed by flow cytometry 6 hours after aSyn expression induction (^§§§p<0.001 <i>vs</i> empty strain; *** p<0.001 <i>vs</i> WT; ^###<0.001 or ^#p<0.05 <i>vs</i> S129A; one way ANOVA with Bonferroni's multiple comparison test). Results shown are from one representative experiment from three independent experiments. Values represent the mean ± SD. (B) Spotting assay of the indicated yeast cells. Cell suspensions were adjusted to the same OD_600nm, serially diluted and spotted onto the surface of the solid medium containing either glucose (control) or galactose (induced aSyn expression) as carbon source. A representative result is shown from at least three independent experiments.</p

Strains used in this study.

<p>Strains used in this study.</p