Exogenous tau interacts with Parkin, trapping it in the cytosol.Figure 5A Co-immunoprecipitations of V5-tagged tau and GFP-Parkin. The V5 tag was used to pull down tau and the membrane was probed for V5 (using an antibody raised in a different species) and GFP. IP = immunoprecipitation, Flow = flow-through lysate following removal of beads. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. B Maximum projection images of proximity ligation assays (PLAs) for tau and endogenous Parkin in cells expressing hTau-GFP, hP301L-GFP or GFP. Inset = negative control (antibodies to tau and histone H3, which are not known binding partners). C Quantification of PLA signals in maximum projections normalised to the GFP signal. Results were analysed with a Kruskal-Wallis test, χ2 (2) = 27.2, p < 0.0001, followed by Dunn's post hoc analysis to test for differences from the GFP control, n = 17 cells in GFP condition, 14 cells in hTau condition and 27 cells in hP301L condition. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. D PLA for Parkin and hP301L in an aged pR5 mice and a littermate control. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. E Schematic of tau constructs used in PLA experiments. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. F, G PLA for Parkin and ∆Tau, revealing interactions via the projection domain of tau. Data were analysed with a Mann-Whitney test (U = 185, p = 0.9224, n = 21, 18 cells/group for hTau and ∆Tau, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. H, I PLA for Parkin and the microtubule-binding domain construct (MTBD). Data were analysed with a t-test (t = 5.226, p < 0.0001, n = 16, 17 cells/group for hTau and MTBD, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. J, K Cells expressing tau constructs together with GFP-Parkin (24 h) with and without CCCP treatment. Parkin clusters were counted, illustrating that Parkin translocation was impaired by ∆Tau, but not MTBD. Data were analysed with a Kruskal-Wallis test χ2 (2) = 10.4, p = 0.0056, followed by Dunn's post-hoc, n = 67, 42, 53 cells/ group for for hTau, ∆Tau, and MTBD, respectively. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. L The V5 fluorescence intensity in each cell analysed in (K) (Mann-Whitney test, U = 953, p = 0.2332, n = 42, 53 cells/group for ∆Tau and MTBD, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. M Cells were transfected with GFP-Parkin and hTau-V5, hP301L-V5 or an empty vector control, and treated with CCCP. PLAs were performed for tau and Parkin. Notably, at Parkin clusters (arrow), no interactions between tau and Parkin were evident. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001. N 3D reconstructions in IMARIS, illustrating a lack of PLA signals in Parkin clusters. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.