Materials – All chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany), Fluka (Seelze, Germany), and Roth (Karlsruhe, Germany) in highest purity if not stated otherwise. Heparin 5000 or 16000 was purchased from Fisher Scientific (#BP2524-10). MSNL-10 probe for AFM measurements were from Bruker, USA.
Cells and viruses
Sf9 cells were obtained from Invitrogen (Life Technologies, DE) and grown at 27°C in monolayer culture with Grace's medium (Sigma /Life Technologies TM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (PS).
Sapphire Baculovirus DNA was obtained from Orbigen/Biozol (Eching, DE) and pVL1392 from Invitrogen (Life Technologies, EM). MultiBacTurbo system contains the acceptor plasmid pACEBac1 and the E.coli strain DH10MultiBac /Turbo contains the acceptor bacmid. MultiBacTurbo baculoviral DNA were obtained from EMBL Grenoble Outstation(Bieniossek et al., 2012).
Plasmids and baculovirus construction
The hTau40 cDNA, the longest Tau isoform in human CNS (2N4R), was tagged with 6xHis at the C-terminus using PCR amplification and the modified Tau-His cassette was introduced into pET3a plasmid (Novagen, DE) linearized with NdeI enzyme succeeding in pET3a/htau40/6xHis plasmid for the protein expression in E.coli. The cDNA cassette of GFP-hTau40 (consisting of M1 – L239 of GFP sequence bridged by one aa: His to htau40 (M1 – L441) sequence elongated with 6x His at the C-terminus was inserted into pET3a plasmid succeeding in E.coli expression vector pET3a GFP/htau40/6xHis.
The cDNA cassette of htau40-GFP (consisting of htau40 (M1-L441) bridged by 13 aa AH linker sequence: GAPGSAGSAAGSG to M1- L239 GFP terminated with 6xHis Tag was also inserted into pET3a plasmid leading to the pET3a htau40/GFP/6xHis expression vector for E.coli. Similarly, the cDNA cassette of GFP-TauRDΔK-6xHis consisting of GFP, pro-aggregant Tau repeat domain (4-repeat construct K18, M-Q244-E372, with the deletion mutation ΔK280 terminated with 6xHis Tag (GFP-TauRDΔK) was also inserted into pET3a plasmid, yielding the pET3a GFP-TauRDΔK-6xHis expression vector. This mutation strongly accelerates aggregation due to pronounced β propensity (von Bergen et al., 2001).
A second group of proteins: TauRDΔK –GFP and GFP/6xHis proteins was expressed in the baculovirus expression system. A cDNA cassette containing TauRDΔK tagged with GFP at its C-terminus (TauRDΔK–GFP) followed by a 6xHis tail was inserted into pVL1392 vector resulting in the plasmid pVL1392 TauRDΔK/GFP/6xHis. After mixing with Saphire TM baculovirus DNA, this plasmid was used for the generation of baculovirus and protein expression in Sf9 insect cells as described before (Biernat et al., 1993).
The GFP cDNA sequence fused to a 6xHis tail at its C-terminus was inserted into the pACEBac-1 acceptor vector to generate the pACEBac/GFP/6xHis plasmid. This plasmid was subsequently subjected for the Tn7- dependent integration into the baculoviral genome of DH10 MultiBacTurbo E.coli cells for the generation of baculovirus encoding GFP-His-tagged protein.
The purified MultiBacTurbo bacmid encoding GFP protein was transfected into Sf9 cells for the generation of baculoviruses and subsequent protein expression (Bieniossek et al., 2012).
An overview of the Tau constructs is shown in Fig. 2 (for sequence see Supplement Table S1).
Protein preparation and purification
Proteins were expressed either in E.coli or in insect Sf9-cells using the baculovirus expression system. The Tau proteins hTau40wt (2N4R) and TauRDΔK (K18 construct, (M) Q244-E372 with deletion of K280) were expressed in E.coli and purified as described earlier (Barghorn et al., 2005).
The proteins Tau-His (hTau40/6xHis; 2N4R), GFP-TauRDΔK (GFP- (M)Q244-E372 with deleted K280/6xHis) and GFP-Tau (GFP- hTau40/6xHis; 2N4R) and Tau-GFP (hTau40/-GFP/6xHis; 2N4R) were expressed as fusion proteins with 6x polyHis tail at the C-terminus in E.coli strain BL21(DE3) (Merck-Novagen, Darmstadt). After harvesting, the E.coli bacteria cell pellet was directly re-suspended in lysis buffer [50 mM Tris HCl pH 7.3, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP, 10 mM Imidazol, 1 mM Benzamidin, 1 mM PMSF and 10µg/ml each of protease inhibitors leupeptin, aprotinin, and pepstatin], in the ratio 1g E.coli pellet to 10 ml lysis buffer and disrupted by French press.
Protein TauRDΔK-GFP (M) Q244-E372 with deletion of K280-GFP/6xHis), GFP/6xHis and also GFP-Tau (GFP- hTau40/6xHis; 2N4R) and (hTau40/-GFP/6xHis; 2N4R) were expressed in Sf9 insect cells from Invitrogen. Sf9 cells were infected with recombinant virus at a MOI of > 1 typically in six T150 cell culture flasks containing 75% confluent Sf9 cells. Cells were incubated for three days at 27°C, collected and re-suspended for preparation in the lysis buffer [50 mM Tris HCl pH 7.3, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP, 10 mM Imidazol, 1 mM Benzamidin, 1 mM PMSF and 10 µg/ml each of protease inhibitors leupeptin, aprotinin, and pepstatin], in the ratio 1g Sf9 pellet to 10 ml lysis buffer and disrupted by French press.
The lysates of the E.coli bacteria and Sf9 cells were cleared by centrifugation in a Beckman Optima L- 80 XP Ultracentrifuge with a Ti45 rotor (40,000 rpm. for 60 min at 4 °C), applied to Ni2+ ion affinity chromatography His Trap FF column (GE Healthcare), and purified using an Äkta pure chromatography system (GE Healthcare). Following extensive wash with 12 column volumes (CV) of wash buffer (50mM Na phosphate buffer pH 7.2, 300 mM NaCl, and 25 mM imidazole) the protein was eluted with elution buffer (50mM Na Phosphate buffer pH 7.2, 300 mM and 1 M imidazol).
If necessary the protein breakdown products were separated in the second chromatography step using gel filtration on a Superdex G200 column (GE Healthcare, Freiburg). PBS buffer was used for gel filtration column (137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7,4) with 1 mM DTT (freshly added).
Light scattering: Tau aggregation was monitored by 90° angle light scattering at 350nm in a FluoroMax spectrophotometer (HORIBA). 50 µM Tau protein in suspended in BES buffer, pH 7.0
(20 mM BES, 25 mM NaCl) and supplemented with 12.5µM heparin 16000 or heparin 5000. Heparin 5000 and 16000 induce the aggregates at the same level and there is no difference between the aggregates formed under these conditions. This mixture was incubated at 37°C. At different time points 20µl of the samples were analyzed by light scattering at 350 nm and then the samples were brought back to original tube for further aggregation.
Sedimentation assay and western blotting: The aggregated Tau samples (t=48 h) were sedimented at 61,000 rpm (TLA 100.3 rotor) for 1h at 4°C. The supernatant was collected and the pellet was resuspended to the same volume as supernatant. 5µl of the samples were resolved on 10% SDS gel and immunoblotted as described in (Kaniyappan et al., 2017). Later the blot was probed with K9JA (1:5000 dilution) and GFP (1:1000 dilution) antibodies and the signal was detected by chemiluminescence method.
Turbidity assays for MT assembly: Tau-induced microtubule assembly was monitored by 90° angle light scattering at 350nm in a FluoroMax spectrophotometer (HORIBA). 10 µM PC-purified Tubulin were mixed with 5 µM Tau protein in RB-Buffer (100 mM PIPES pH 6.9, 1 mM DTT, 1mM MgSO4, 1mM EGTA, 1 mM GTP). The polymerization was started by transferring the ice-cold Tubulin-Tau-solution to the 37°C warm cuvette-holder and the reaction started once the temperature was reached. Tubulin assembly was monitored for 15 min.
Electron microscopy and mass per length analysis
Sample preparation: Holey carbon grids (Quantifoil R2/1) covered with either 2 nm amorphous carbon (Quantifoil, R2/1+2 nm C) or graphene were used for sample preparation. The preparation of graphene grids was done according to a method described earlier (Pantelic et al., 2011).The sample concentration was adjusted for MPL measurements to a final concentration of 5–10 molecules / grid hole. The protein solution was mixed with Tobacco mosaic virus (TMV) prior to application to the grids. The TMV was used to calibrate the mass measurements. 10 µl sample was applied to the grids for 2 min. Excess liquid was blotted away using filter paper. Samples were washed 3 times with doubled distilled water to remove buffer salts and afterwards air dried or frozen and vacuum dried at – 80 °C for 12 h.
Scanning-transmission electron microscopy (STEM): For the MPL experiments, a Zeiss Libra200 MC Cs-STEM CRISP (Corrected Illumination Scanning Probe) was used. The instrument was operated at 200 kV. The CRISP is equipped with a monochromated Schottky-type field emission cathode and a double hexapole-design corrector for spherical aberrations of the illumination system (Cs-corrector). A high-angle annular dark field (HAADF) detector (Fischione Instruments, USA) was used for imaging. The images were recorded at a convergence angle of 16 mrad and an acceptance angle of 20 mrad. Images were recorded at a pixel size of 0.6 nm with a dwell time of 70 µs/px. The total dose per image was 400 e/nm2.
Mass determination was done using the software PCMASS32 (Schutz et al., 2015). In a first step images were calibrated using tobacco mosaic virus as calibration sample. Filament regions were manually selected and masked. The full datasets were plotted as histograms and fitted to Gaussian curves.
Transmission electron microscopy (TEM): Samples for negative stain transmission electron microscopy were placed on 200 mesh formvar-carbonated copper grids with (Plano, Quantifoil #S162). Grids were glow-discharged for 30 s (Baltec, MED010) prior to sample addition. The protein solution was removed after 3 min incubation time by filter-paper and the grids washed 3 times with ddH2O (grid on top of each drop) to remove buffer salts, then followed by staining with 2% uranyl acetate for 60 seconds and a finally removing the stain slowly with a wet-filter paper and rapid air drying. To prepare grids with microtubules for TEM, all steps were carried at out at 37°C and with pre-warmed solutions. All specimens were analyzed at 200kV using a JEOL JEM-2200FS TEM.
Atomic force microscopy (AFM)
AFM measurements were performed as described earlier (Kaniyappan et al., 2018). Briefly, Tau fibril samples were diluted in PBS buffer for a final concentration of 0.5 – 1µM. 30µl of the sample was placed onto freshly cleaved mica and allowed to adsorb for 10 minutes at room temperature. Unattached and excess proteins were removed by rinsing the sample with PBS for 4-5 times. Finally, the sample on the mica was filled with imaging buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl). AFM imaging was performed in oscillation mode using JPK NanoWizard® ULTRA Speed AFM system and MSNL-10 probe with “F” cantilever. The amplitude set point and the gains were adjusted manually to control the thermal drift and to achieve the minimal force between the cantilever and the sample. AFM images were processed by JPK data processing software. Fibril heights/widths were measured by the inbuilt cross-sections method of JPK data processing software. For the analysis of fibril width 50 fibrils per condition were measured .