Figure 3.
Automated Microfluidics Feedback Control of α-Synuclein Expression at Nine Increasing Levels to Observe and Quantify α-Synuclein Aggregation Thresholds in Both WT α-Synuclein Strain and A53T Mutant Strain
(A–F) Six control experiments were performed to increase α-synuclein protein expression stepwise in both the WT α-synuclein strain (A–C) and mutant A53T α-synuclein strain (D–F). Population-averaged α-synuclein expression (blue with SD in gray) was tightly regulated to the target expression levels (yellow) by automatically switching between galactose and glucose (brown) as directed by the controller (STAR Methods). α-Synuclein-GFP fluorescence in single cells is normalized to mCherry fluorescence within each cell (STAR Methods) to enable comparison across cells, strains, and experiments. Examples of images below each time course show WT and A53T mutant α-synuclein-GFP at the indicated time points (A–F). Prior to the formation of inclusions, α-synuclein-GFP is mainly on the membrane, whereas inclusions appear as bright cytoplasmic spots.