cDNA and plasmids
Tau full-length (FL) 2N4R cDNA in pcDNA four was a kind gift from Luc Buée (INSERM U837, Lille, France). The BIN1 isoform used in the present study corresponds to the longest neuronal isoform 1 and will be denoted as BIN1 FL. For GST pull-down experiments, both Tau FL and Tau sub-fragment cDNA sequences were obtained by PCR with the primers described in Additional file 1, and subcloned into the pGEX-4 T2 vector (General Electric Healthcare Bio-Sciences, Piscataway, NJ, USA) to produce GST-Tau constructs. GST BIN1 FL and GST-BIN1/SH3 were obtained as previously described . BIN1/ΔSH3 cDNA sequence was obtained by PCR from BIN1 FL cDNA (NM_139343.1) in PCMV6-XL5 (Origene, Rockville, MD, USA). For NMR experiments, the BIN1/SH3 domain cDNA was synthesized with optimized codons for recombinant expression in E. coli (Genecust, Dudelange, Luxembourg). The cDNA was subcloned between the NdeI and XhoI restriction sites in pET15b (Novagen, EMD Millipore, Darmstadt, Germany), thus allowing its expression with an N-terminal HisTag under the control of a T7 promoter. Recombinant Tau-F5[165-245] and TauFL were prepared for NMR experiments without a N-terminal tag with a pET15B vector. All cDNAs were checked by sequencing.
Cell cultures and transfection
Human embryonic kidney 293 (HEK293) cells (CRL-1573 from LGC Standards/American Type Culture Collection, Molsheim, France) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (1:1) supplemented with 10 % fetal bovine serum, 2 mM glutamine, 20 units/ml penicillin and 20 μg/ml streptomycin (Gibco, LifeTechnologies, Carlsbad, CA, USA) in 5 % CO2 atmosphere and at 37 °C. Transient transfections were performed using Fugene-HD (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Forty-eight hours later, cells were harvested in Tris-buffered saline (100 mM NaCl, 1 mM EDTA, 50 mM Tris–HCl) and centrifuged at 1000 g for 10 min at room temperature. Cell pellets were stored at−80 °C until processing for GST pull-down.
The GST pull-down assay
The GST fusion proteins were expressed in Escherichia coli BL21(DE3) after induction with isopropyl 1-thio-β-D-galactopyranoside. Proteins were extracted from bacterial inclusion bodies by incubation with lysosyme for 1 h, overnight incubation with N-sarkosyl (0.001 %) and Triton X-100 (0.5 %), sonication and then centrifugation at 12,500 g for 30 min. All steps were performed at 4 °C. The GST fusion proteins were immobilized on glutathione-Sepharose beads (Pierce, ThermoFisher Scientific, Rockford, IL USA) according to the manufacturer’s instructions, and then incubated with HEK293 cell lysates for 1 h at room temperature. Beads were washed in Tris buffered saline, centrifuged at 10,500 g for 1 min and processed for SDS-PAGE analysis.
Isotopic labelling and protein purification
Isotopic labelling of Tau and Tau-F5 was performed by growing recombinant BL21 (DE3) in minimal growth medium supplemented with 15N NH4Cl. The first purification step was performed by heating the bacterial protein extract for 15 min at 75 °C. The 15N Tau protein and 15N Tau[165–245] were recovered in the soluble fraction after centrifugation at 15,000 g for 30 min. The 15N Tau protein and 15N Tau-F5 were purified by cation exchange chromatography in 50 mM phosphate buffer pH 6.3, 1 mM EDTA (5 ml Hitrap SP Sepharose FF, General Electric Healthcare, Little Chalfont, United Kingdom). The pooled fractions from the chromatography purification step were transferred to ammonium bicarbonate by desalting on a 15/60 Hiprep desalting column (G25 resin, General Electric Healthcare) and lyophilized. The His-SH3 protein was purified on Ni-NTA resin, according to the manufacturer’s protocol.
Acquisition and analysis of NMR spectra
1 mM d4-TMSP (3-(trimethylsilyl) propionate was used as an internal reference for proton chemical shifts (CSs) (0 ppm). The NMR buffer was 25 mM Tris-d11 pH 6.6, 30 mM NaCl, 2.5 mM EDTA and 1 mM DTT and 5 % D2O. Two-dimensional [1H, 15N] heteronuclear single quantum coherence (HSQC) spectra were recorded at 298 K on a Bruker 900 spectrometer equipped with a triple-resonance cryogenic probe (Bruker, Karlsruhe, Germany). Spectra were processed using Bruker TopSpin software (version 2.1, Bruker, Karlsruhe, Germany), and peaks were picked using Sparky software (version 3, T. D. Goddard and D. G. Kneller, University of California, San Francisco, CA, USA). The delta (δ) CSs of individual amide resonances of Tau-F5 and Tau FL were calculated with the following equation, while taking account of the relative dispersion of the proton and nitrogen CSs: δ(CS) = [((CS1Hbound- CS1Hfree) + 0.2 (CS15Nbound- CS15Nfree)) 2]1/2. The “bound” and “free” subscripts in the equation correspond to the CSs in the SH3-bound protein or the free protein, respectively.
Phosphorylation of Tau protein
The CDK2/CycA3 protein was prepared and Tau was phosphorylated in vitro as previously described . Enzymatic reactions were terminated by heating for 15 min at 75 °C and then centrifuged. The phosphorylation mixture was buffer-exchanged for NMR buffer using centrifugal desalting columns (Zeba Desalting Columns, with a 0.5 ml bed of G25 resin and a 7 kDa cut-off (Thermofisher Scientific, Waltham, MA USA)).
Electrophoresis and Western blots
Samples were resuspended in Lithium Dodecyl Sulfate buffer supplemented with Nupage antioxidant, heated for 10 min at 95 °C, loaded and separated on a 4–12 % acrylamide gel (Nupage, Novex, Life Technologies, Carlsbad, CA, USA) and blotted on nitrocellulose membranes using a BioRad Trans-Blot transfer system kit (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. Membranes were blocked and probed with antibodies diluted at the concentration indicated in Additional file 2. All antibodies were purchased directly from the provider (described in Additional file 2), except for CP13 , RZ3  and PHF1 . Membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA), and revealed by chemiluminescence (Luminata ClassicoTM, EMD Millipore) in a BioRad Chemidoc XRS system (BioRad). Immunoblot data were quantified with ImageLab software (BioRad). Coomassie staining was performed with 0.05 % Brilliant Blue G (mass/volume) in 50 % methanol (vol/vol) and 10 % acetic acid (v/v). Destaining was performed with a 25 % methanol (v/v) and 7 % acetic acid (v/v) solution.
Immunofluorescence assays and PLAs
Cultured cells were fixed on glass coverslips with either 4 % paraformaldehyde (EMS, Hatfield, PA, USA) in Phosphate Buffered Saline (PBS) (Life Technologies). Fixed cells were washed and permeabilized for 10 min in PBS supplemented with 0.25 % Triton X-100, and then blocked in PBS supplemented with 2 % bovine serum albumin for 2 h at room temperature. Coverslips were incubated overnight at 4 °C with primary antibodies (as specified in Additional file 2), washed in PBS and incubated for 45 min with Alexa Fluor antibodies diluted at 1/400 from stock (Molecular Probes, Life Technologies). Alexa Fluor 647 phalloidin (Molecular Probes, Life Technologies)was used as per the manufacturer’s instructions to stain the actin cytoskeleton. For PLAs, the initial steps (from fixation to primary antibody incubation) were the same as those described above for immunofluorescence assays. The following steps (i.e., secondary antibody incubation, ligation, amplification and probing) were performed according to the manufacturer’s instructions (Olink Bioscience, Uppsala, Sweden). Images were acquired with a confocal microscope (LSM 710, Zeiss, Oberkochen, Germany) and processed using ZEN 2012 software (Zeiss). Three independent experiments were performed for each condition. The mean intensity per pixel in three different fields was measured using ImageJ software. Imaris software (Bitplane, Zurich, Switzerland) was used for three-dimensional (3D) image processing.
Primary neuron cultures
Mixed cortical and hippocampal primary cultures were obtained from P0 rats, according to previously described procedures . Briefly, hippocampi and cortices were isolated from newborn rats, and neurons were dissociated by trypsin digestion. Neurons were plated on poly-L-lysine-coated coverslips or six-well plates, and were incubated with Minimal Essential Medium (MEM) supplemented with 10 % fetal bovine serum, Glutamax, MEM vitamins and penicillin/streptomycin (Life Technologies), according to the manufacturer’s instructions. After 24 h, neurons were transferred into serum-free Neurobasal-A medium supplemented with B27 (Gibco, Life Technologies), Glutamax and uridine-deoxyfluorouridine for 14 days of in vitro culture. For Western blot analysis, primary neurons were directly harvested in LDS buffer supplemented with Nupage antioxidant (Life Technologies) and processed as described above in the “Electrophoresis and Western blots” section. For immunofluorescence assays, cells were fixed in 4 % paraformaldehyde (EMS) and processed as described above in the “Immunofluorescence assays and PLAs” section.