Secondary antibody was removed, cells rinsed with PBS 3 times and imaged via fluorescence microscopy

Secondary antibody was removed, cells rinsed with PBS 3 times and imaged via fluorescence microscopy. seeded aggregation. Our findings establish entry as a rate-limiting step to seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation by promoting entry of tau assemblies into the cell interior. disease models (Clavaguera et?al., 2009, 2013; Guo et?al., 2016; Iba et?al., 2013). The relative contributions of these two mechanisms to human disease progression remain unknown (Mudher et?al., 2017). Tau assemblies are taken up into membrane-bound vesicles after interactions between tau and cell-surface heparan sulfate proteoglycans (HSPGs) as well as the recently identified low-density lipoprotein receptor LRP1 (Holmes et?al., 2013; Rauch et?al., 2020). Tau is then incorporated into membrane-bound compartments via endocytosis and macropinocytosis (Evans et?al., 2018; Falcon et?al., 2018; Holmes et?al., 2013; Wu et?al., 2012). For a prion-like mechanism to occur, LEQ506 tau assemblies must gain access to the cytosol, somehow breaching these cell-limiting membranes. Although the process of tau filament uptake has been comparatively well studied, the transfer of tau from intracellular membrane-bound vesicles to the cytosol is largely unexplored and remains a critical missing step for assessing the relevance of seeded aggregation to disease progression (De La-Rocque et?al., 2021; Mudher et?al., 2017). Cholesterol is a critical determinant of membrane bilayer structural integrity LEQ506 and a known risk factor in neurodegenerative disease (Arenas et?al., 2017; Dai et?al., 2021; Notkola et?al., 1998; Valenza and Cattaneo, 2006). Cholesterol is depleted from the brain in an age-dependent manner, resulting in impaired intracellular signaling and synaptic plasticity (Egawa et?al., 2016; Martn-Segura et?al., 2019; Palomer et?al., 2016). Variation in and in cells Study of tau entry to cells has been complicated by the difficulty of CUL1 reliably distinguishing cytosolic populations from vesicular populations (De La-Rocque et?al., 2021). To specifically detect the cytosolic fraction of exogenously supplied tau assemblies, we established a live-cell assay relying on the split luciferase NanoLuc binary technology system (NanoBiT) (Figure?1A). The NanoBiT system relies on the NanoLuc (Nluc) enzyme, which is a 19-kDa luminescent protein engineered from the luciferase of the deep-sea shrimp This enzyme has been split into a larger 18-kDa subunit (LgBiT) and an 11-amino-acid high-affinity peptide (HiBiT) that interact with subnanomolar affinity. Reconstitution results in complementation of activity and luminescence in the presence of substrate (Dixon et?al., 2016). Previous studies have demonstrated the applicability of split luciferase as a tool to monitor tau aggregation and propagation mechanisms (Mirbaha et?al., 2015; Wegmann et?al., 2016). We expressed recombinant P301S tau (0N4R isoform) in fusion with a HiBiT tag at the C terminus in (Figures?1B and 1C)Assemblies of tau-HiBiT were produced by incubation with heparin and aggregation kinetics were quantified by thioflavin T fluorescence. Tau-HiBiT assembled into filaments, as seen by electron microscopy, and had similar aggregation profiles as tagless tau (Figures?1D and 1E). Open in a separate window Figure?1 Characterization of LEQ506 HiBiT-tagged tau assemblies and their entry into the cytosol of HEK293 cells (A) Cartoon depicting the intracellular reconstitution of Nluc and the enzymatic production of light through interaction of exogenously supplied assemblies of tau-HiBiT with intracellular LgBiT. (B) Depiction of the His6-0N4R-P301S-Tau-HiBiT construct and the amino acid sequence of the HiBiT peptide. (C) Western blot of 50?ng recombinant tau or tau-HiBiT monomers with anti-tau (Dako) or anti-HiBiT antibody. (D) Time course of 5?M tau-HiBiT and tagless tau aggregation kinetics monitored by thioflavin T (15?M) fluorescence; n?= 4. (E) Representative transmission electron micrographs of heparin-induced tau-HiBiT assemblies before and after sonication. Scale bar, 200?nm. (F) Titration of tau-HiBiT assemblies complexed with recombinant LgBiT (0.2?L/well) for 30?min; n?= 4. (G) Confocal microscopy images of HEK293T cells expressing NLS-EGFP-LgBiT (HEK-NGL), immunostained with anti-GFP and anti-LgBiT antibodies. Scale bars, 50?m. (H) Western blot of cytosolic and nuclear fractions of NGL lysates probing for LgBiT, GFP, the nuclear marker histone H3, and the cytosolic marker tubulin. (I) Effect of trypsin protease (Try) treatment, which degrades extracellular luciferase, on the luminescent signal in NGL cells; n 3. (J) Titration of tau-HiBiT assemblies on NGL cells for 1 h; n?= 3. (K) Time course of entry of 50?nM tau-HiBiT assemblies added to NGL cells; n?= 3. All error bars indicate mean SEM. To assess the ability of tau-HiBiT assemblies to reconstitute Nluc,.