Figure 7Confocal microscopy of HEK sensor cells treated with 24h-old Tau condensates (Tau:tRNA) shows different types of intracellular CFP/YFP-Tau accumulations: spherical intranuclear Tau (NUC), bright cytoplasmic accumulations (CYT) adjacent to the nucleus, and small, dimmer accumulations right at and along the nuclear envelope (NE). Cells treated with Tau aggregates (hepAGG) had mainly large cytoplasmic (CYT) Tau inclusions. The different Tau accumulations coexisted in most cells, and also more than one accumulation of the same type could occur. Nuclei were stained with propidium iodide (PI, pink). Scale bars=10 μm. Fluorescence intensity of NE and CYT Tau compared to soluble Tau in the cytosol of cells without accumulations. N=3 experiments, and 5-6 images per experiment. Data shown as mean±SD. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Immunolabeling of lamin-B1 and nuclear pore complexes (NPC) shows the location of CYT and NE Tau in relation to the nuclear envelope in seeded HEK sensor cells. White arrows and boxes show position and width of cross-sectional profiles shown in (D). Scale bar=10 μm. Cross sectional profiles show the localization of Tau (green) in CYT accumulations adjacent to the nucleus in the cytosol, and the colocalization of NE Tau with lamin (pink) and NPCs (blue). Notably, CYT-1 also contains NPC signal. Dashed vertical lines indicate x-positions of highest intensity values for respective intensity profiles. Seeded HEK sensor cells stained with the amyloid dye (Amytracker-680). Scale bar=10 μm. HEK sensor cells treated with AD brain TBS extract show similar CYT, NUC, and NE accumulations as Tau condensate treated cells. Arrow heads and arrows indicate positions of respective Tau accumulation types. Scale bars=10 μm. Immunolabeling of phosphorylated Tau (PHF1=pS396/404, AT8=pS199/pS202/pT205, AT180=pT231) in AD brain section. Both 3,3′-Diaminobenzidine (DAB) and fluorescence labeling show Tau accumulating in a layer at the nuclear envelope (NE) in non-tangle neurons with or without cytosolic (CYT) Tau accumulations. Control brain does not show Tau labeling in neuronal cell bodies. Scale bars= 20 and 10 μm, as indicated. High resolution confocal images show colocalization (arrow heads) of small NE Tau clusters with nuclear pore proteins (NPC marker, POM121). Scale bars=10 μm. Reduced nucleo-cytoplasmic Ran gradient (nuclear/cytosolic Ran intensity) in HEK sensor cells having CYT Tau (CYT), or NE Tau (NE), compared to non-treated (NT) or control treated (lipo) sensor cells. Wildtype (WT) HEK cell Ran gradient was analyzed for the effect of TauRDP301S expression in general. N=3 experiments, 7-10 cells analyzed per experiment and condition. Data shown as mean±SD. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Frequency of Tau accumulation types observed in sensor cells treated with Tau aggregates (hepAGG), AD lysate, or Tau condensates. In cells having Tau accumulations we determined which accumulation types were present, regardless of the co-presence of other accumulation types and if multiple accumulations of the same type were present. n=number of cells with accumulations analyzed, resulted from the actual number of cells in the three replicates.