DOI: 10.5061/dryad.280gb5mxj
title: Data from: BioID2-based tau interactome reveals novel and known protein interactions associated with multiple cellular pathways

description: Pathological inclusions composed of tau protein are hallmarks of
 neurodegenerative diseases collectively known as tauopathies, of which the
 most common is Alzheimer’s disease. Tau is known as a
 microtubule-associated protein involved in regulating microtubule
 dynamics, but accumulating evidence suggests tau is also involved in a
 multitude of biological functions regulated, in part, by direct and/or
 transient protein interactions. Deciphering the tau protein interactome is
 critical for better understanding the physiological and pathological roles
 of tau. This work aimed to identify potential tau-interacting partners
 using the in situ protein labeling biotin identification (BioID2) method.
 Fusion proteins were created between full-length human tau and BioID2 on
 the N-terminus (BioID2-Tau) or C-terminus (Tau-BioID2) of tau. Advantages
 of this approach include in-cell interactor labeling and enhanced
 likelihood of detecting transient and/or weak interactions. We report a
 total of 324 potential tau interactors by combining the list of proteins
 identified with BioID2-Tau and Tau-BioID2. This list included proteins
 found in the cytoskeleton, mitochondria, synapses, nucleus, and
 ribonucleoprotein complex. Gene ontology molecular function analysis
 identified RNA binding, translation regulation, ubiquitin ligase activity,
 kinase binding, and mitochondrial oxidoreductase. We validated the
 interaction between tau and selected candidates using two independent
 approaches; the proximity ligation assay and co-immunoprecipitation. We
 validated novel and known tau interactions with cytoskeletal proteins
 (MAP2 and MAP6), proteins associated with the nucleus (FUS and prune1),
 and proteins associated with the synapses (synapsin-1 and neurabin-2).
 Importantly, the *in situ *labeling approach revealed potential
 interactors that were not clearly identified by traditional approaches
 such as co-immunoprecipitation. Thus, this approach is a powerful tool to
 identify potential members of the tau interactome via in situ labeling.
 This work helps expand our understanding of tau’s potential functional
 roles, which may also advance our understanding of its role in
 neurodegenerative diseases.
description: <em>Animals</em> Human MAPT (tau)
 knock-in (MAPT KI) mice were obtained from Dr. Karen Duff at Columbia
 University with permission from the Saido group at Riken Center for Brain
 Science. Mouse tau knockout (TKO, strain #007251) mice were obtained from
 Jackson Labs. Homozygous breeding pairs were used to generate an in-house
 colony for both mouse lines, and timed-pregnant females were used to
 obtain fetuses for primary cortical neuron cultures. Animals were housed
 at Michigan State University Grand Rapids Research Center Vivarium in a 12
 h light/dark cycle with access to food and water. All animal procedures
 were performed in accordance with the guidelines approved by the Michigan
 State University Institutional Animal Care and Use Committee.
 <em>BioID2 Constructs</em> The
 original BioID2 expression constructs, MCS-13X Linker-BioID2-HA
 (BioID2-HA, Addgene, #80899) and Myc-BioID2-13X Linker-MCS (Myc-BioID2,
 Addgene, #92308), were a kind gift from the Kyle Roux lab. The MCS-13X
 Linker-BioID2-HA plasmid was used to fuse the BioID2 to the C-terminus of
 human Tau (hT40, 2N4R, 441 amino acids) to create a construct consisting
 of Tau-13xLinker-BioID2-HA (referred to as Tau-BioID2) and the BioID2
 control construct consisted of the 13xLinker-BioID2-HA (referred to as
 BioID2-HA). The mycBioID2-13xLinker-MCS plasmid was used to fuse BioID2 to
 the N-terminus of tau to create the Myc-BioID2-13xLinker-Tau (referred to
 as BioID2-Tau) construct and the BioID2 control construct consisted of the
 Myc-BioID2-13xLinker (referred to as Myc-BioID2). BioID2 constructs
 feature a 13xLinker which is a serine-glycine repeat sequence that acts as
 a flexible spacer sequence to increase the biotinylation range of the
 BioID2 protein. The BioID2 constructs were then cloned into the pFIN
 vector (Addgene, #44352). All plasmid constructs were validated by
 restriction digestion, and Sanger sequencing.
 <em>Lentiviral Production in HEK293T
 Cells</em> Lentiviruses were created to express
 Tau-BioID2, BioID2-Tau and the BioID2-HA and Myc-BioID2 controls. For each
 lentiviral preparation, 4 x 150 mm cell culture dishes (Corning, #430599)
 were plated at a density of 1 x 10<sup>6</sup> cells/dish in
 25 ml complete DMEM. Culture dishes were incubated overnight in a
 humidified incubator. Two hours prior to transfection, DMEM was removed,
 and fresh DMEM was added. The following transfection mixture was then
 prepared for each 150 mm culture dish: 45 μg plasmid DNA (22.5 μg of the
 pFIN plasmid expressing the BioID2 protein of interest, 15 μg of pNHP
 lentiviral packaging vector (Addgene, #22500), 7.5 μg of pHEF-VSVG
 lentiviral envelope vector (Addgene, #22501), 300 μl polyethylenimine
 transfection reagent and 6 ml of 150 mM NaCl. The transfection mixture was
 incubated for 20 minutes at room temperature, slowly added to each culture
 dish, and then transfected HEK293T cells were maintained overnight in a
 humidified incubator. Complete DMEM was substituted by freshly prepared
 viral medium (98% DMEM, 1% FBS, and 1% penicillin-streptomycin) and cells
 were maintained for 48 hours. Medium containing lentiviruses was
 centrifuged at 675 x g for 5 minutes and the supernatant containing
 lentiviruses was filtered through a 0.45 μm filter. Lentiviruses were
 harvested by layering on 20% sucrose solution, and ultracentrifugation at
 82,700 x g for 2 hours at 4°C using Sorvall™ WX+ ultracentrifuge
 (ThermoScientific, #75000100). The lentiviral pellets were resuspended in
 500 μl sterile PBS, aliquoted, snap-frozen in crushed dry ice, and stored
 at -80°C until use. <em>Primary Cortical Neuron
 Cell Culture</em> Timed-pregnant female MAPT KI
 and TKO mice were euthanized by intraperitoneal injection of 100 mg/kg
 Fatal-Plus solution diluted in saline. Mouse pups were collected on
 embryonic day 18 (E18) and kept in ice-cold 0.9% saline. Fetal cortical
 tissues were isolated under a dissecting microscope, cut into small
 pieces, and collected in a tube containing ice-cold calcium- and
 magnesium-free solution (CMF; contains 1x DPBS, 1x amphotericin B (Gibco,
 #15290018), 1x gentamicin (Gibco, #15750060), and 10% glucose
 (Sigma-Aldrich, #G8270)). Cortical tissue pieces were
 washed four times in CMF and incubated in 0.25% trypsin solution (Gibco,
 #15090046) for 15 minutes at 37 °C. Trypsin was removed and cortices were
 washed two times in CMF. Trypsin inactivation solution (3 ml) was added
 containing 2.1 ml Hank’s Balanced Salt Solution (Gibco#24020117), 0.6 ml
 newborn calf serum (Gibco#16010167), and 0.3 ml DNase solution
 (Worthington# LS002006). A homogenous cell suspension was obtained by
 gentle trituration of the tissue through a series of progressively smaller
 needles (30 x 14-gauge needle, 30 x 15-gauge needle, 20 x 16-gauge needle,
 20 x 18-gauge needle, and 15 x 21-gauge needle). Cell suspensions were
 gently layered onto 5 ml sterile-filtered FBS and centrifuged at 200 x g
 for 5 minutes. Primary neuron cell pellets were gently resuspended in 1ml
 neurobasal media (NBM; Gibco, #21103049) supplemented with L-glutamine (2
 mM, Gibco, #25030081), amphotericin B (2.5 μg/ml, Gibco, #15290026), B-27
 Supplement (Gibco, #12587001), and gentamicin (50 μg/ml, Gibco, #
 15710064). Cell counts were determined using Countess 3 automated cell
 counter (Invitrogen, #AMQAX2000). Primary cortical neurons were plated at
 density of 600,000 cells per well in a poly D-lysine-coated 6-well plate
 (Corning, #354413) and maintained in a humidified incubator at 37 °C and
 5% CO<sub>2</sub>. <em>Primary Neuron
 Lentiviral Transduction</em> TKO primary cortical
 neurons were treated with lentiviruses to induce expression of BioID2-Tau,
 Tau-BioID2 or the respective Myc-BioID2 and BioID2-HA controls on DIV4.
 Lentiviruses were diluted in complete NBM, and primary neurons were
 transduced at an MOI of 200 (n=3 independent experiments). The neuronal
 culture was maintained for four days to allow lentiviral transgene
 expression before supplementing exogenous biotin (100 μM) into the medium.
 Lentivirus transduced neurons were maintained until collection for
 biochemical or immunofluorescence assays as described below.
 <em>Biotin-Streptavidin Affinity
 Pulldown</em> Ethanol-cleaned low retention
 microcentrifuge tubes (ThermoScientific, #3453) were placed in a magnetic
 separation stand. Streptavidin magnetic Dynabeads T1 (Invitrogen, #65602)
 were washed twice in 1 ml lysis buffer and then 1 mg of protein lysate
 containing biotinylated proteins (collected as described above) was added
 to the beads and rotated overnight at room temperature. The following day
 unbound supernatant proteins were collected as the post-pulldown sample
 and stored at -80°C until further analysis. The beads containing bound
 biotinylated proteins were washed three times in lysis buffer, then
 resuspended in 1 ml 50 mM Tris.HCl, pH 7.4. Then 900 μl of the resuspended
 beads was transferred to a new low retention tube for mass spectrometry
 analysis (described below). The remaining 100 μl was transferred to a new
 tube and was placed on a magnetic separation stand. The supernatant was
 discarded, and the beads were resuspended in an elution buffer containing
 25 mM biotin prepared in lysis buffer. Efficient elution of biotinylated
 proteins was performed by competition with free biotin in the elution
 buffer and heating to 95°C for 15 minutes. The tube was placed on a
 magnetic separation stand, the supernatant containing biotinylated
 proteins was transferred to a new tube, and western blotting validation
 was performed as described above. Blots were incubated in IRDye® 680LT
 Streptavidin (1:5000, LI-COR Biosciences Cat#926-68031) overnight at 4°C.
 Blots were imaged using LI-COR Odyssey® infrared system and processed for
 publication using ImageStudio software (v5.2.5, LiCor Biosciences) and
 Adobe Illustrator 2023. <em>Nanoscale liquid
 chromatography coupled to tandem mass spectrometry (nano
 LC-MS/MS)</em> The tube containing the 900 μl of
 resuspended beads was placed on magnetic separation stand and the
 supernatant was discarded. The beads were washed six times in 25 mM
 ammonium bicarbonate (pH 8) and then resuspended in 150 μl of 25 mM
 ammonium bicarbonate -50% acetonitrile (ACN). On-bead protein digestion
 was performed by adding 100 ng of rlys-C (Promega, #V1671) and incubating
 for 90 minutes at 37°C then adding 3 μg trypsin (Promega, #V5280) and
 incubating for 16-18 hours at 37°C. The tubes were placed on a magnetic
 separation stand and the supernatant was collected in a new low retention
 tube. Samples were dried to completion in a speed vacuum centrifuge at 30
 °C before being resuspended in 50 μl of 25 mM ammonium bicarbonate -5%
 ACN. NanoLC-MS/MS separations were performed with a
 Thermo Scientific Ultimate 3000 RSLCnano System. Peptides were desalted
 in-line using a 3 μm diameter bead, C18 Acclaim PepMap trap column (75 μm
 × 20 mm) with 2% ACN, 0.1% formic acid (FA) for 5 min with a flow rate of
 5 μl/min at 40°C. The trap column was then brought in line with a 2 μm
 diameter bead, C18 EASY-Spray column (75 μm × 250 mm) for analytical
 separation over 60 min with a flow rate of 350 nl/min at 40°C. The mobile
 phase consisted of 0.1% FA (buffer A) and 0.1% FA in ACN (buffer B). The
 separation gradient was as follows: 5 min desalting, 40 min 4–40% B, 2 min
 40–65% B, 2 min 65–95% B, 7 min 95% B, 1 min 95–4% B, 3 min 4% B. One
 microliter of each sample was injected. Top 20 data-dependent mass
 spectrometric analysis was performed with a Q Exactive HF-X Hybrid
 Quadrupole-Orbitrap Mass Spectrometer. MS1 resolution was 60K at 200 m/z
 with a maximum injection time of 45 ms, AGC target of 3e6, and scan range
 of 300–1500 m/z. MS2 resolution was 30K at 200 m/z, with a maximum
 injection time of 54 ms, AGC target of 1e5, and isolation range of 1.3
 m/z. HCD normalized collision energy was 28. Only ions with charge states
 from +2 to +6 were selected for fragmentation, and dynamic exclusion was
 set to 30 s. The electrospray voltage was 1.9 kV at a 2.0 mm tip to inlet
 distance. The ion capillary temperature was 280°C and the RF level was
 55.0. All other parameters were set as default.
 <em>Mass Spectrometry Protein
 Identification</em> RAW mass spectrometry files
 were identified using Sequest HT against the reviewed <em>Mus
 musculus</em>Uniprot proteome database (UP000000589, 25,285 unique
 sequences) with Thermo Scientific Proteome Discoverer software (version
 2.5). Enzyme specificity was set to trypsin with an MS1 tolerance of 10
 ppm and a fragment tolerance of 0.02 Da. Oxidation (M), biotinylation (K),
 acetylation (protein N-term), methionine loss (protein N-term), and
 biotinylation (protein N-term) were set as dynamic modifications. Peptide
 and protein false discovery rates (FDR) were 1% with threshold determined
 via decoy search using the Percolator algorithm. At least two peptide
 identifications were required per protein identification. All other
 parameters were set as default. Mass spectrometry RAW files from each
 experimental sample (n=3) or control sample (n=3) were analyzed by
 qualitative protein identification or LFQ (see details below). For the
 qualitative method, proteins identified in only one independent
 experimental replicate were excluded from further analysis, as were all
 keratins. To identify potential tau protein interacting partners, protein
 lists from Tau-BioID2 and BioID2-Tau experiments were compared to the
 respective controls BioID2-HA and Myc-BioID2. Proteins identified in the
 controls were removed and the curated Tau-BioID2 and BioID2-Tau lists were
 combined to generate the full list of potential tau interactors.
 <em>LFQ of Identified Proteins</em>
 LFQ was performed using the Precursor Ions Quantifier node.
 Precursor abundance was based on peak intensity. Protein abundance was
 normalized by total peptide amount. The protein abundance ratio was
 calculated using pairwise ratios, whereby the median peptide ratio is
 selected as the protein abundance ratio. The following criteria were used
 to define proteins as Tau-BioID2 or BioID2-Tau interactors: 1) being
 identified in at least two of the independent experimental replicates, and
 2) being detected at ≥ 3-fold increase compared to the respective BioID2
 control. Proteins identified in only one independent replicate as well as
 contaminant keratins were removed from further analysis. Data analysis
 output in .mzTab files contains values of "null" for numerical
 categories when there is an undetected measurement or for
 descriptive categories when those descriptors were not assigned.
 <em>Functional Protein Association
 Networks</em> Functional protein-protein
 interaction networks were visualized by the Search Tool for the Retrieval
 of Interacting Genes/Proteins (STRING) analysis. STRING V12.0 was used to
 visualize the protein interactome network including both functional
 associations retrieved from published databases, text mining,
 computational prediction methods and physical interactions retrieved from
 experimental data (genetic, biochemical, and biophysical techniques) with
 a minimum required confidence interaction score set at 0.7 and above (high
 confidence interaction). STRING V12.0 was used to perform Markov
 Clustering (MCL) with inflation value of 3.0 to reduce the cluster size.
 STRING V12.0 was used to perform the Gene Ontology (GO) enrichment
 analysis and KEGG pathways analysis. GO cellular component, molecular
 function, biological process, and KEGG pathways were performed showing
 annotations with FDR ≤0.05. <em>Statistical
 Methods</em> GO cellular component and molecular
 function showed enriched annotations with FDR ≤ 0.05 and graphed as
 -Log<sub>10</sub>(FDR). Statistical tests and graphs were
 created using GraphPad Prism (V.10.0.2; GraphPad Software, La Jolla
 California USA). <em>Experimental Design and
 Statistical Rationale</em> Mass spectrometry
 identification of biotinylated proteins in BioID2 experiments was
 performed in three independent biological replicates per experimental or
 control condition. Experimental conditions included Tau-BioID2 and
 BioID2-Tau, while the controls were BioID2-HA and Myc-BioID2. Two control
 proteins were utilized because the position of the 13X linker as well as
 the tags used (Myc and HA) were different. For each experimental and
 control condition, peptides identified by mass spectrometry were filtered
 by 1% FDR and at least two peptides were required to identify a protein.
 For both the qualitative and LFQ analyses, proteins identified in less
 than two experimental replicates and two control replicates were omitted
 from further analysis. LFQ protein ratios were measured relative to the
 respective control (Tau-BioID2 versus BioID2-HA or BioID2-Tau versus
 Myc-BioID2). For the LFQ analysis, we used a cutoff abundance ratio of ≥
 3-fold. It is important to note that we are using the ratios as a cutoff
 for GO grouping and designating proteins for follow-up validation
 experiments to confirm interacting partners, and thus, did not consider
 the LFQ p-values. Validation of tau interacting partners using either
 co-IP or PLA were performed in three independent biological replicates per
 condition. Antibody isotype controls were used for co-IP experiments. PLA
 experimental controls included omission of each primary antibody and
 omission of all primary antibodies in MAPT KI neurons, as well as the full
 PLA reaction in TKO neurons. Lists of identified proteins were curated and
 filtered according to the defined criteria using RStudio (V.1.4.1717).
 Schematic figures were created with BioRender.com.
description: # Mass Spectrometry RAW Data Files
 [https://doi.org/10.5061/dryad.280gb5mxj](https://doi.org/10.5061/dryad.280gb5mxj) The files contained here are the mass spectrometry (MS)-derived file sets used to generate the data reported in the manuscript. This study used the BioID2 (a biotin ligase proximity labeling) approach to identify proteins interacting with tau protein. Tau knockout primary neurons were transduced with lentiviruses to express two sets of proteins (one control construct and one related tau construct). The first set of constructs was a Myc-BioID2 alone control construct and the related BioID2-Tau fusion construct (BioID2 fused to the N-terminus of tau protein. The second set of constructs was a BioID2-HA alone tau construct and the related Tau-BioID2 fusion construct (BioID2 fused to the C-terminus of tau protein). Each construct condition included three independent experimental replicate samples. Biotin-labeled proteins were extracted from cell lysates and analyzed using mass spectrometry. RAW files were obtained using MS as described in the methods. The files include the MS/MS peak lists (.mzML), the peptide/protein ID files (.mzid), the label-free quantitation (LFQ) data (.mzTab), and the RAW MS files for each of the experimental replicates (.RAW). Protein ID and LFQ results were obtained by comparing each of the BioID2 tau fusion proteins to their respective controls (Tau-BioID2 versus BioID2-HA or BioID2-Tau versus Myc-BioID2). The .RAW files can be used in open source software, such as MetaMorpheus ([https://github.com/smith-chem-wisc/MetaMorpheus](https://github.com/smith-chem-wisc/MetaMorpheus)). NanoLC-MS/MS separations were performed with a Thermo Scientific Ultimate 3000 RSLCnano System. Top 20 data-dependent mass spectrometric analysis was performed with a Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer. ## Description of the data and file structure Data File Descriptions: * MYC-BioID2.mzML: MS/MS peak lists for Myc-BioID2 control construct. Counting the peptide/protein matching spectra data. * MYC-BioID2.mzid: Myc-BioID2 control construct results file. Reports the peptide/protein IDs * BioID2-Tau.mzML: MS/MS peak lists for BioID2-Tau construct. Counting the peptide/protein matching spectra data. * BioID2-Tau.mzid: BioID2-Tau construct results file. Reports the peptide/protein IDs * BioID2-HA.mzML: MS/MS peak lists for BioID2-HA control construct. Counting the peptide/protein matching spectra data. * BioID2-HA.mzid: BioID2-HA control construct results file. Reports the peptide/protein IDs * Tau-BioID2.mzid: Tau-BioID2 construct results file. Reports the peptide/protein IDs * Tau-BioID2.mzML: MS/MS peak lists for Tau-BioID2 construct. Counting the peptide/protein matching spectra data. * LFQ_Analysis.mzML: MS/MS peak lists for the LFQ analysis. Counting the peptide/protein matching spectra data. * LFQ_Analysis.mzid: LFQ analysis results files. Reports the peptide/protein IDs * LFQ_Analysis.mzTab: Reports quantification data from LFQ analysis (available for LFQ only) * MYC-BioID2-Replicate_1.raw: Replicate sample #1 of Myc-BioD2 control construct; mass spec file generated by the instrument. Thermo's proprietary format. * MYC-BioID2-Replicate_2.raw: Replicate sample #2 of Myc-BioD2 control construct; mass spec file generated by the instrument. Thermo's proprietary format. * MYC-BioID2-Replicate_3.raw: Replicate sample #3 of Myc-BioD2 control construct; mass spec file generated by the instrument. Thermo's proprietary format. * BioID2-Tau-Replicate_1.raw: Replicate sample #1 of BioD2-Tau construct; mass spec file generated by the instrument. Thermo's proprietary format. * BioID2-Tau-Replicate_2.raw: Replicate sample #2 of BioD2-Tau construct; mass spec file generated by the instrument. Thermo's proprietary format. * BioID2-Tau-Replicate_3.raw: Replicate sample #3 of BioD2-Tau construct; mass spec file generated by the instrument. Thermo's proprietary format. * BioID2-HA-Replicate_1.raw: Replicate sample #1 of BioD2-HA control construct; mass spec file generated by the instrument. Thermo's proprietary format. * BioID2-HA-Replicate_2.raw: Replicate sample #2 of BioD2-HA control construct; mass spec file generated by the instrument. Thermo's proprietary format. * BioID2-HA-Replicate_3.raw: Replicate sample #3 of BioD2-HA control construct; mass spec file generated by the instrument. Thermo's proprietary format. * Tau-BioID2-Replicate_1.raw: Replicate sample #1 of Tau-BioD2 construct; mass spec file generated by the instrument. Thermo's proprietary format. * Tau-BioID2-Replicate_2.raw: Replicate sample #2 of Tau-BioD2 construct; mass spec file generated by the instrument. Thermo's proprietary format. * Tau-BioID2-Replicate_3.raw: Replicate sample #3 of Tau-BioD2 construct; mass spec file generated by the instrument. Thermo's proprietary format.