Familial Parkinson's Disease Mutant E46K α-Synuclein Localizes to Membranous Structures, Forms Aggregates, and Induces Toxicity in Yeast Models
Figure 4
E46K α-synuclein aggregates in a time- and concentration-dependent manner in fission yeast. (a) Time course: Left—TCP1 cells expressing WT, A53T, or E46K α-synuclein (grown on media without thiamine) visualized using a fluorescent microscope over a 48-hour time period. Right—Quantification of the number of α-synuclein aggregates in each cell plotted as a percent of total cells that fluoresced . E46K and A53T α-synuclein aggregated more intensely and quickly than WT. (b) Concentration: Left—TCP1 cells expressing WT, A53T, or E46K α-synuclein were grown in decreasing concentrations of thiamine (10, 1.0, 0.1, and 0.0 μM) to induce increasing expression of α-synuclein and cells were visualized using a fluorescent microscope at 24 hours. Right—Quantification of number of aggregates in each cell plotted as a percent of total cells that fluoresced . All three α-synuclein variants aggregated in a concentration-dependent manner but only at 0.1 and 0.0 μM thiamine. Middle—Western blot of TCP1 cells expressing WT or E46K α-synuclein grown in 0 μM or 10 μM thiamine concentration indicates a low level of expression even in strongly repressing media (10 μM thiamine).
