Article Figures & Data
Figures
- Figure 1.
Tau interactome in human fetal, adult, and Alzheimer’s disease brain. A, Total tau levels are not significantly different in immunoprecipitated samples from different groups, y-axis shows log2 transformed LFQ intensity. Volcano plot showing differentially expressed genes (red) between fetal and AD (B), fetal and adult control (C), and number of shared genes between comparisons (D). Significant interactors are determined using a permutation-based FDR (0.05), 14-3-3-β is indicated as a blue diamond. The horizontal axis in all volcano plots shows the difference of log2 transformed LFQ intensity as previously described (Zhang et al., 2019). All differentially expressed genes are shown in Extended Data Table 1-1.
- Figure 2.
Tau-14-3-3-β interaction depends on tau splicing and phosphorylation. A, HEK 293T cells were transfected with the indicated constructs, immunoprecipitated with anti-FLAG antibodies, and then probed with the indicated antibodies. Uncropped blots are shown in Extended Data Figure 2-1, and an additional replicate (uncropped) in Extended Data Figure 2-2. B, Representative mass photometry density plots of phosphorylated (magenta) and unphosphorylated (cyan) tau 14-3-3-β one representative plot is shown out of three replicates, gray bars indicate windows of predicted MW ±10 kDa and quantification across three replicates is shown in upper right using Student’s t test, validation of phosphorylation is shown in Extended Data Figure 2-3 and a phosphorylated tau only plot on the same axes is shown in Extended Data Figure 2-4. C, Recombinant 15N-labeled tau441 NMR with and without ERK1 phosphorylation (validated in Extended Data Fig. 2-5). D, Phosphorylated 15N-labeled tau 441 showing residue shifts after incubation with 2:1 ratio of 14-3-3-β to tau, green lines indicate predicted 14-3-3-pred binding sites, black indicates variably spliced second microtubule binding repeat (R2).
- Figure 3.
Fraction of shared interactors between human 14-3-3 isoforms (BioGRIDv4.4).
Tables
Case ID Age Sex Brain diagnosis Source Cause of death BBID Fetal 1 0 F None NIH Prematurity 4510 Fetal 2 0 F None NIH Prematurity 1756 Fetal 3 0 M None NIH Prematurity 1085 Fetal 4 0 M None NIH Prematurity 4711 Adult 1 66 F None University of Iowa Interstitial lung disease P0002 Adult 2 58 M None University of Iowa Cardiovascular disease P0005 Adult 3 60 F None University of Iowa Overdose P0006 AD 1 59 M AD University of Iowa c/o AD P0036 AD 2 65 M AD University of Iowa c/o AD P0053 AD 3 67 F AD University of Iowa c/o AD P0062 Name Catalog # Manufacturer RRID Concentration HT7 MN1000 Invitrogen RRID:AB_2314654 1:500 (WB), 5 μg/500 μg (co-IP) ANTI-FLAG M2 F1804 Sigma-Aldrich RRID:AB_262044 10 μg/500 μg beads (co-IP) 14-3-3-β Ab15260 Abcam RRID:AB_301799 1:1000 (WB) p-Ser/Thr 9631 Cell Signaling RRID:AB_330308 1:1000 (dot blot) pSer214-tau Ab170892 Abcam RRID:AB_2905610 1:1000 (dot blot) Mouse TrueBlot 88-8887-31 Rockland Antibodies RRID:AB_2614895 1:1000 (WB) Rabbit TrueBlot 88-8886-31 Rockland Antibodies RRID:AB_2614893 1:1000 (WB) Goat anti-rabbit Ab205718 Abcam RRID:AB_2819160 1:1000 (dot blot) Data structure Test used Location Power a Normal distribution Permutation-based FDR Extended Data Table 1-1 80% for 2-fold difference, α = 0.043 b List FDR enrichment Extended Data Table 4-1 NA c List FDR enrichment Extended Data Table 4-2 NA d Normal distribution t test Figure 2B 90% power for 2-fold difference, α = 0.001 - Table 4
List of top five enriched gene ontology terms in genes showing increased tau interaction in fetal brain (top) or AD brain (bottom)
GO Term Total Expected FE FDR Fetal BP Positive regulation of axon extension (GO:0045773) 3 0.05 58.66 4.98E-02 BP Positive regulation of response to biotic stimulus (GO:0002833) 5 0.25 20.17 3.75E-02 BP Negative regulation of biological process (GO:0048519) 19 6.97 2.73 2.51E-02 MF Structural molecule activity (GO:0005198) 9 1.04 8.62 1.25E-03 MF Protein-containing complex binding (GO:0044877) 10 1.72 5.81 5.80E-03 MF RNA binding (GO:0003723) 12 2.18 5.49 2.21E-03 CC Paraspeckles (GO:0042382) 3 0.01 >100 2.24E-04 CC Nuclear matrix (GO:0016363) 6 0.17 36.31 3.33E-05 CC Growth cone (GO:0030426) 5 0.22 22.56 1.43E-03 CC Cortical cytoskeleton (GO:0030863) 3 0.15 20.61 4.47E-02 CC Nuclear speck (GO:0016607) 6 0.55 10.92 3.88E-03 Alzheimer’s
diseaseBP Neurofilament bundle assembly (GO:0033693) 3 0.01 >100 3.28E-04 BP Axon target recognition (GO:0007412) 2 0.01 >100 1.38E-02 BP Postsynaptic intermediate filament cytoskeleton organization (GO:0099185) 2 0.01 >100 1.37E-02 BP Isocitrate metabolic process (GO:0006102) 2 0.02 >100 3.10E-02 BP Fructose 1,6-bisphosphate metabolic process (GO:0030388) 3 0.03 >100 1.78E-03 CC Postsynaptic intermediate filament cytoskeleton (GO:0099160) 3 0.01 >100 6.80E-05 CC Laminin-11 complex (GO:0043260) 2 0.01 >100 3.51E-03 CC Neurofibrillary tangle (GO:0097418) 3 0.02 >100 9.71E-05 CC Internode region of axon (GO:0033269) 2 0.01 >100 4.70E-03 CC Calcium- and calmodulin-dependent protein kinase complex (GO:0005954) 2 0.02 >100 6.11E-03 MF Fructose-bisphosphate aldolase activity (GO:0004332) 2 0.01 >100 1.35E-02 MF Protein kinase C inhibitor activity (GO:0008426) 2 0.01 >100 1.31E-02 MF Structural constituent of postsynaptic intermediate filament cytoskeleton
(GO:0099184)2 0.01 >100 1.28E-02 MF Cytoskeletal protein-membrane anchor activity (GO:0106006) 2 0.01 >100 1.74E-02 MF Low-density lipoprotein particle receptor binding (GO:0050750) 3 0.08 36.88 1.36E-02 See row b in statistical table for analysis and Extended Data Tables 4-1 and 4-2 for complete lists of all enriched terms.
ID Description Number Strength FDR PF00076 RNA recognition motif (also known as RRM, RBD, or RNP domain) 17 0.87 1.73E-06 PF00244 14-3-3 protein 6 1.87 5.59E-06 PF04732 Intermediate filament head (DNA binding) region 5 1.79 0.00015 PF00038 Intermediate filament protein 9 1.04 0.0003 PF00538 Linker histone h1 and h5 family 5 1.6 0.00049 PF01391 Collagen triple helix repeat (20 copies) 8 0.96 0.0029 PF00052 Laminin B (Domain IV) 4 1.64 0.0037 PF01979 Amidohydrolase family 4 1.54 0.0064 PF00418 Tau and MAP protein, tubulin-binding repeat 3 1.94 0.0108 PF08075 NOPS (NUC059) domain 3 1.94 0.0108 PF08702 Fibrinogen α/β chain family 3 1.94 0.0108 PF00053 Laminin EGF domain 5 1.15 0.0141 PF08332 Calcium/calmodulin dependent protein kinase II association domain 3 1.81 0.0141 PF13474 SnoaL-like domain 3 1.81 0.0141 PF14534 Domain of unknown function (DUF4440) 3 1.81 0.0141 PF00055 Laminin N terminal (Domain VI) 4 1.34 0.0147 PF13671 AAA domain 4 1.34 0.0147 PF00932 Lamin tail domain 3 1.72 0.0149 PF03953 Tubulin C-terminal domain 4 1.18 0.0422 IPR000504 RNA recognition motif domain 18 0.84 2.95E-06 IPR012677 Nucleotide-binding α-β plait domain superfamily 18 0.81 3.61E-06 IPR035979 RNA-binding domain superfamily 18 0.81 3.61E-06 IPR000308 14-3-3 protein 6 1.87 5.49E-06 IPR023409 14-3-3 protein, conserved site 6 1.87 5.49E-06 IPR023410 14-3-3 domain 6 1.87 5.49E-06 IPR036815 14-3-3 domain superfamily 6 1.87 5.49E-06 IPR018039 Intermediate filament protein, conserved site 9 1.1 9.24E-05 IPR006821 Intermediate filament head, DNA-binding domain 5 1.79 0.0001 IPR039008 Intermediate filament, rod domain 9 1.03 0.00026 IPR005818 Linker histone H1/H5, domain H15 5 1.52 0.00085 IPR011778 Hydantoinase/dihydropyrimidinase 4 1.76 0.0018 IPR008160 Collagen triple helix repeat 8 0.96 0.0029 IPR000034 Laminin IV 4 1.64 0.0036 IPR005819 Linker histone H1/H5 4 1.64 0.0036 IPR011059 Metal-dependent hydrolase, composite domain superfamily 4 1.54 0.0063 IPR043129 ATPase, nucleotide binding domain 7 0.97 0.007 IPR006680 Amidohydrolase-related 4 1.5 0.0076 IPR001084 Microtubule associated protein, tubulin-binding repeat 3 1.94 0.0101 IPR012290 Fibrinogen, α/β/γ chain, coiled coil domain 3 1.94 0.0101 IPR012975 NOPS 3 1.94 0.0101 IPR013543 Calcium/calmodulin-dependent protein kinase II, association-domain 3 1.81 0.0151 IPR008211 Laminin, N-terminal 4 1.34 0.0201 IPR001322 Lamin tail domain 3 1.72 0.022 IPR036415 Lamin tail domain superfamily 3 1.72 0.022 IPR002049 Laminin EGF domain 5 1.07 0.0286 IPR037103 Tubulin/FtsZ, C-terminal domain superfamily 4 1.22 0.0428
Extended Data Table 1-1
List of all proteins and statistical analyses (shown in Fig. 1A–C). Download Table 1-1, DOC file.
Extended Data Table 4-1
Table of all gene ontology term enrichments (AD). Download Table 4-1, DOC file.
Extended Data Table 4-2
Table of all gene ontology term enrichments (Fetal). Download Table 4-2, DOC file.
Extended Data Figure 2-1
Uncropped western blot images for Figure 2A. Red boxes indicate irrelevant bands from unrelated experiment. Boxes indicate bands shown in Figure 2A. Additional bands in top left, and bottom left, right side represent tau degradation products. Download Figure 2-1, TIF file.
Extended Data Figure 2-2
Replication of co-IP in Figure 2A, with uncropped blots. Download Figure 2-2, TIF file.
Extended Data Figure 2-3
Validation of tau phosphorylation by PKA for mass photometry. We added 1 μg of the phosphorylated, recombinant tau to a nitrocellulose membrane and probed with anti-tau Ser214 and total pSer/pThr antibodies as indicated. Control, unphosphorylated tau. Download Figure 2-3, TIF file.
Extended Data Figure 2-4
Mass photometry histogram of ptau only, as in Figure 2B. Download Figure 2-4, TIF file.
Extended Data Figure 2-5
Validation of tau phosphorylation by ERK2 for NMR. We loaded 10 μg of phosphorylated, recombinant tau and stained the resulting gel with Coomassie blue as an initial validation of phosphorylation efficiency (see Fig. 2C and text for site-specific mapping of phosphoepitopes). Control, unphosphorylated tau. Download Figure 2-5, TIF file.
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