Figure 1

Heterologous expression of αSyn elevates basal [Ca²⁺]_cyt and amplifies transient [Ca²⁺]_cyt responses in yeast. (a) Determination of basal cytosolic Ca²⁺ levels using aequorin-based luminescence measurement of WT yeast cells expressing human αSyn for indicated time or harbouring the empty vector (Ctrl.). Mean±S.E.M., n=8; ***P<0.001 and **P<0.01. (b) Flow cytometric quantification of oxidative stress indicated by the superoxide-driven conversion of non-fluorescent dihydroethidium to fluorescent ethidium (DHE→Eth.) of WT yeast cells expressing human αSyn for indicated time or harbouring the empty vector (Ctrl.). Mean±S.E.M., n=8; ***P<0.001. (c) Survival determined by clonogenicity of WT yeast cells expressing human αSyn or harbouring the empty vector control for 24 h and 48 h. Cells were plated on YEPD agar plates. Mean±S.E.M., n=10; ***P<0.001. (d) Aequorin-equipped yeast cells harbouring the vector control or expressing αSyn for 16 h were challenged with 150 mM CaCl₂ and transient [Ca²⁺]_cyt responses were observed for 70 s. Data was normalized to maximum peak amplitude of control cells. Mean±S.E.M., n=6. (e) Western blot analysis of αSyn expression in WT cells. Cells were harvested at indicated time points after induction of galactose-driven expression. Blots were probed with antibodies directed against FLAG-epitope to detect FLAG-tagged αSyn and against glyceraldehyd-3-phosphate dehydrogenase (GAPDH) as loading control. (f) Fluorescence microscopic analysis of WT cells expressing GFP-tagged αSyn (αSyn^GFP) or harbouring the corresponding vector control (Ctrl.^GFP) at indicated time points. (g) Determination of basal cytosolic Ca²⁺ levels using aequorin-based luminescence measurement of yeast cells expressing human αSyn or harbouring the empty vector (Ctrl.) after growth for 12 h, 20 h or 24 h on galactose media (promoter induction) supplemented or not with 2 mM ethylene glycol tetraacetic acid (EGTA). Data has been normalized to equally treated vector control cells. Mean±S.E.M., n=6; **P<0.01 and *P<0.05. (h) Flow cytometric quantification of oxidative stress by assessing the ROS-driven conversion of dihydroethidium to ethidium (DHE→Eth) upon expression of αSyn for indicated time and supplementation of media with 2 mM EGTA. Mean±S.E.M., n=4–8; ***P<0.001 and **P<0.01. (i) Survival determined by clonogenicity of yeast cells expressing αSyn or harbouring the empty vector control for 48 h and supplementation of galactose medium with 2 mM EGTA. Cells were plated on YEPD agar plates. Mean±S.E.M., n=12; ***P<0.001