Figure 4(A) Strategy used to purify and identify neuron intrinsic membrane proteins with extracellular domain that interact specifically with Fib-Tau-1N3R. Fib-Tau were labeled 1h with 10 molar equivalents of NHS-S-S-Biotin. Mice cortical neuron cultures were exposed for 10 min to biotinylated Fib-Tau (14.4nM). Fresh protein extracts from those neurons were incubated with streptavidin magnetic beads to pull down Fib-Tau together with their specific protein partners. Unexposed neuron extracts were used as a control. Proteins bound to the streptavidin magnetic beads were eluted with Laemmli buffer and subjected to short migration on a SDS-PAGE gel. After coomassie blue staining, proteins were subjected to in-gel digestion using trypsin and subsequently identified by nanoLC-MS/MS analysis, using a nanoLC-TripleTOF mass spectrometer. Relative quantification between control and exposed neuron samples was performed using a label-free MS based approach. Six independent replicates were analyzed. (B) Venn Diagram of 968 proteins identified in Fib-Tau pull-downs only (red), in control pull-downs only (grey) or in both samples (overlap). Of the 92 proteins identified in both samples, 45 proteins were significantly enriched in Fib-Tau pull-downs (t-test with p-values <0.05, Benjamini-Hochberg, Fold Change >2). (C) Distribution of the 372 synaptic and membrane protein interactors of Fib-Tau identified in the pull-down experiments. Locations of proteins at the levels of subcellular structures were annotated using the Gene Ontology Cell Component annotation tool of AMIGO 2 Distribution of Fib-Tau interactors in the plasma membrane, pre-synaptic membrane, post-synaptic membrane, pre-synapse and post-synapse is shown. (D) List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull-downs from neurons exposed to Fib-Tau. For each identified protein, the name of the protein, the gene name, the p-value (t-test with Benjamini-Hochberg correction), and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given. In an independent analysis, after 10 min exposure of neurons to biotinylated Fib-Tau, a cross-linking step was performed during 20 min using 1mM of DTSSP added in the culture medium, in order to cross-link the protein complexes formed at the cell surface using a membrane impermeable cross-linker. After cross-linking, proteins were analyzed and identified exactly as non-cross-linked samples. Proteins identified with at least 2 peptides are labeled "+", the other are labeled "-". (E). Co-immunoprecipitation of exogenous biotin-labelled Fib-Tau with α3-NKA, GluA2 and GluN1. α3-NKA, GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section. The presence of Fib-Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin-HRP. A 2.4-, 2.3-, and 1.8- fold enrichment in Tau band-intensity is observed in α3-NKA, GluA2 and GluN1 immunoprecipitates, respectively, compared to controls performed with pre-immune goat or rabbit IgGs. Co-immunoprecipitates of exogenous biotin-labelled Fib-Tau with anti α3-NKA, GluA2 and GluN1 specific antibodies were also subjected to SDS-PAGE and Western blot analysis. The presence of Fib-Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin-HRP. Fib-Tau co-immunoprecipitates with α3-NKA and GluA2-AMPA receptor but not with GluN1-NMDA receptor. (F) Network describing the interconnectivity of intrinsic membrane proteins extracellularly exposed (presented in panel D, labeled in yellow) and post-synaptic proteins (proteins with a post-synapse or a post-synaptic membrane annotation, presented in panel C and labeled in blue) that interact with Fib-Tau-1N3R. This Fib-Tau-1N3R interactome was input in the String database (String v10, https://string-db.org/) and exported to Cytoscape (version 3.5.1 at http://www.cytoscape.org/) to visualize interactions between the identified proteins. 121 proteins were evaluated. We set parameters to only detect interactions that were validated experimentally or described in databases. The thickness of the line corresponds to the confidence of interaction (thin lines, >0.4; medium lines, >0.7; thick lines, >0.9).