A chemical genetic approach to probe the function of PINK1 in regulating mitochondrial dynamics

PINK1 and Parkin have been implicated in mitochondrial quality control, mitochondrial fusion/fission dynamics and axonal mitochondrial mobility in neurons^1,2. The PINK1-Parkin pathway is very dynamic and the biological responses mediated by the PINK1-Parkin pathway differ spatially and kinetically in different cell types². Therefore, tools to dynamically probe the function of the PINK1-Parkin pathway are highly desirable. Here we report a chemical genetic approach to probe the function of PINK1 in mitophagy and mitochondrial mobility. Our results validate the roles of PINK1 in recruitment of Parkin to mitochondria, mitochondrial mobility in neurons, and mitochondrial fusion/fission dynamics. In addition, we reveal that PINK1 is required for triggering Parkin translocation to mitochondria, but is dispensable for sustaining Parkin accumulation and consequent mitophagy.

CCCP or valinomycin induced Venus-Parkin translocation to mitochondria within 1 h with similar kinetics (Supplementary information, Figure S1B). We examined the ability of various PP1 analogs to block PINK1-dependent Parkin translocation in MEF cells expressing PINK1^AS or PINK1^WT (Figure 1D and Supplementary information, Figure S1C). The mitochondrial translocation of Venus-Parkin was quantified 1 h after CCCP treatment. 1-NA-PP1 showed a nanomolar IC₅₀ value (165.4 ± 6.5 nM). 1-NM-PP1, 3-MB-PP1 and 2,3-DMB-PP1 showed low micromolar IC₅₀ values (8.3 ± 0.4 μM, 10.2 ± 0.3 μM and 20.2 ± 0.3 μM, respectively). In contrast, 3-IB-PP1 showed no inhibition when tested at concentrations up to 40 μM. Importantly, all five bulky PP1 analogs showed no inhibition of wild-type PINK1 at concentrations up to 40 μM. These results demonstrate the high specificity of PINK1^AS and selectivity of PP1 analogs 1-NA-PP1 and 3-MB-PP1 as orthogonal inhibitors of PINK1 activity.

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