Figure 4

Complementation assays and growth analysis of mutants with decreased inclusion formation capacity.

(a) Images of selected mutants with a decreased number of cells showing synphilin-1 (SY1) inclusions and the corresponding control mutant strains with only dsRed expression. Left panel, bright field; right panel, dsRed-synphilin-1 or dsRed. Scale bar, 5 μm. (b) Quantification of the percentage of cells with synphilin-1 inclusions in WT and mutants. Values are normalized such that the percentage of cells with inclusions in WT cells is set to 50%. A value of 0% means that there are no cells of that mutant with synphilin-1 inclusions. Error bars represent standard deviation from triplicates each containing about 200 cells. (c) Western blot analysis of the WT (his3Δ) strain and mutants expressing synphilin-1. Immunodetection was performed using primary antibodies directed against synphilin-1 or Adh2, which served as internal control protein. The quantification of synphilin-1 expression levels is shown. Error bars represent standard deviation from triplicates samples. Asterisks denote significant differences between WT and mutants (Student’s t-test): *P, 0.05; **P, 0.01; ***P, 0.001. (d) Complementation assays further confirmed that the reduced inclusion formation capacity of the mutants is solely due to the lack of function caused by the corresponding deletion. The complementation assays were performed with MoBY plasmids and the empty vector control (p5586). Error bars represent standard deviation from triplicates containing about 200 cells. (e) An increased synphilin-1 cytotoxicity effect (prolonged generation time) was observed from the selected mutants. The generation times of mutants expressing dsRed-synphilin-1 and solely dsRed (control) were measured at 30 °C. The differences of generation time increase between WT and mutants are shown. Data are represented as mean ± standard deviation SD. Asterisks denote significant differences between samples (Student’s t-test): *P, 0.05; **P, 0.01; ***P, 0.001.