Figure 1. Quantification of input-to-output responses for a minimal PINK1/Parkin synthetic circuit.

(A) Cartoon of PINK1/Parkin positive feedback loop. Yellow circles: phosphorylation sites. Conformational changes of activated Parkin’s UBL (ubiquitin-like) and catalytic RING2 domains are shown.

(B) Hypothetical input-response relationship (curve) illustrating a PINK1 input threshold for circuit activation (vertical dashed line). Inputs: discrete mitochondrial PINK1 concentrations, held stable over time.

(C and D) PINK1/Parkin synthetic circuit using rapalog-induced $\text{PINK}{\text{1}}^{\text{mito}}$ recruitment. Mitochondrial targeting sequence (MTS) of PINK1, amino acid 1–109, removed. T82L: mutation required for rapalog binding.

(E) Live-cell imaging approach. Rapalog treatment: 200 nM. SNAP-647: fluorescent SNAP ligand for far-red imaging of MtTether (STAR Methods). Bar length not to scale.

(F and G) Representative time-lapse images of cells with (F) or without (G) Parkin^mito recruitment in response to induced $\text{PINK}{\text{1}}^{\text{mito}}$ recruitment. Scale bars: 10 μm. Relative intensity visualization range noted in (F).

(H and I) Quantification of circuit input (mean $\text{PINK}{\text{1}}^{\text{mito}}$ concentration), circuit response (max Parkin^mito recruitment), and the time of Parkin^mito recruitment for cells in (F) and (G). AFU: arbitrary fluorescence units. Co-localization: intensity correlation (Pearson, STAR Methods). Black points: time points in (F) and (G). See also Figures S1 and S2.