Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells
- Institut Cochin, Inserm U1016, CNRS UMR 8104, Université de Paris, France
- Les Laboratoires Servier, France
Figures
Figure 1 with 1 supplement
NanoBiT-based biosensors development and application to monitor the tau seeding response in living cells.
(A) Scheme of NanoBiT-based intramolecular (conformational) and intermolecular (interaction) biosensors. (B) Biosensors were developed for three tau forms: full-length wild-type tau (WT-Tau) corresponding to the 2N4R isoform displaying both of the two domains at the N-terminal part (N1, N2), the proline-rich (PR) region, and four-repeat domains (R1–R4); full-length tau carrying the P301L mutation (Tau(P301L)); and the tau fragment comprising only the four repeat domains (R1–R4) and carrying the P301L mutation (K18(P301L)). (C) Workflow of NanoBiT assay (scheme created with Biorender). (D) Saturation curve of the intermolecular interaction sensor K18(P301L) in HEK293T cells expressing a fixed amount of LgBit-K18(P301L) (250 ng of a transfected vector) and increasing amounts of SmBit-K18(P301L) (0–500 ng of a transfected vector); data are expressed as mean ± SEM of four independent experiments. (E) Representative western blot analysis of the expression of K18(P301L) biosensor pairs used in panel D. (F) Cellular distribution of the K18(P301L) biosensor monitored by immunofluorescence microscopy (tubulin: in green; biosensor: anti-HA antibody, in red; nuclei: DAPI staining, in blue). Scale bar: 10 µm. (G) K18(P301S) interaction biosensor signal in the presence of recombinant monomeric K18 (mK18; 100 nM), aggregated K18 (aggK18; 100 nM) or tau (aggTau; 100 nM), or oligomeric amyloid beta peptide (oAb; 1 µM); data are expressed as mean ± SEM of five independent experiments; **p<0.01, ****p<0.001 by one-ANOVA, followed by Dunnett’s multiple comparisons test to basal condition. (H) K18(P301S) interaction biosensor signal in the presence of increasing concentrations of recombinant oligomeric K18 (0.3 nM to 300 nM); data are expressed as mean ± SEM of four independent experiments; * p<0.05 compared to control (0) by Student t-test.
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Figure 1—source data 1
Unedited blot of Figure 1E.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig1-data1-v1.zip
Figure 1—figure supplement 1
Validation of recombinant proteins and the NanoBiT-based K18(P301S) interaction sensor.
(A) Thioflavin T (ThT) signal of recombinant tau, K18, and Aß proteins (10 µM) prepared as monomeric (m), oligomeric (o), or aggregated forms (agg). (B–C) K18(P301S) interaction signal in biosensor cells treated with recombinant aggregated K18 (aggK18) and in cells expressing only LgBiT-K18(P301L) (B) or only SmBiT-K18(P301L) (C); data are expressed as mean ± SEM of three independent experiments; **p<0.01, by Student t-test. (D) Kinetics of the Nluc enzyme:substrate activity following the addition of Nluc substrate in K18(P301L) expressing cells treated with the indicated concentrations of recombinant aggK18. Data are shown as mean ± SD of triplicates.
Figure 2 with 1 supplement
Tau biosensors monitor conformational change and self-interaction of wild-type (WT) full-length tau.
(A) Saturation curve of the intermolecular WT-Tau interaction sensor in HEK293T cells expressing a fixed amount of LgBit-Tau (250 ng of a transfected vector) and increasing amounts of SmBit-Tau or SmBit-Halo (0–500 ng of a transfected vector); data are expressed as mean ± SEM of five independent experiments. Bottom: Representative western blot (WB) analysis of the expression of the tau biosensor pairs. (B–C) Signals of WT-Tau (B) and K18(P301L) (C) interaction biosensors in cells treated or not with colchicine (10 µM, 1 hr); data are expressed as mean ± SEM of seven and five independent experiments, respectively; **p<0.005 by two-tailed paired Student t-test; n.s.=not-significant. (D) Cellular distribution of the WT-Tau interaction biosensor monitored by immunofluorescence microscopy in the absence (top panels) or presence (bottom panels) of colchicine (biosensor: anti-HA antibody, in red; tubulin: in green; nuclei: DAPI staining, in blue); Scale bar: 10 µm. (E) WT-Tau conformational sensor signal in HEK293T cells expressing increasing amounts of LgBit-Tau-SmBit vector (0–500 ng of a transfected vector); data are expressed as mean ± SEM of seven independent experiments. Bottom: Representative WB analysis of the expression of the Tau conformational biosensor. (F) Basal luminescence signal of the WT-Tau conformational (INTRA) and WT-Tau interaction (INTER) biosensors at the same expression levels; data are expressed as mean ± SEM of five independent experiments. (G–H) WT-Tau (G) and K18(P301L) (H) conformational biosensors signal in cells treated or not with colchicine; data are expressed as mean ± SEM of seven (G) or five (H) independent experiments; ***p<0.001 by two-tailed paired Student t-test; n.s.=not-significant. (I) Cellular distribution of the WT-Tau conformational biosensor monitored by immunofluorescence microscopy in the absence (top panels) or presence (bottom panels) of colchicine (biosensor: anti-HA antibody, in red; tubulin: in green; nuclei: DAPI staining, in blue); Scale bar: 10 µm. (J) Colocalization analysis (Manders' colocalization coefficient) of the fractional overlap between biosensor molecules and MTs. (K–L) Signals of WT-Tau conformational (K) and interaction (L) biosensors in cells treated or not with forskolin (10 µM, 24 hr); data are expressed as mean ± SEM of six independent experiments; **p<0.01, ***p<0.005 by two-tailed paired Student t-test; right panel: WB analysis and quantification of phosphorylated tau (P-Tau) and Total tau (T-tau) using AT8 and anti-HA antibodies, respectively. L: Lgbit-Tau; S: SmBit-Tau. (M) WT-Tau interaction biosensor response in cells treated with colchicine (10 µM, 1 hr), forskolin (10 µM, 24 hr), or both (colchicine followed by forskolin); data are expressed as mean ± SEM of five independent experiments; ** p<0.01, ***p<0.005, ****p<0.001 by one-way ANOVA followed by Sidak’s multiple comparison test.
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Figure 2—source data 1
Unedited blot of Figure 2A.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data1-v1.zip
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Figure 2—source data 2
Unedited blot of Figure 2E.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data2-v1.zip
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Figure 2—source data 3
Unedited blot of Figure 2K.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data3-v1.zip
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Figure 2—source data 4
Unedited blot of Figure 2L.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data4-v1.zip
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Figure 2—source data 5
Unedited blot of Figure 2M.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data5-v1.zip
Figure 2—figure supplement 1
Effect of microtubules (MT) destabilization and phosphorylation on WT-Tau biosensors.
(A) Thioflavin T (ThT) aggregation kinetics assay of recombinant full-length WT tau (10 µM) in the absence or presence of aggregation inducer (heparin 1:1) and in the absence or presence of colchicine (10 µM). (B–C) WT-Tau interaction (B) and conformation (C) biosensors signal in cells treated or not with colchicine (10 µM, 1 hr) or nocodazole (1 µM, 1 hr); data are expressed as mean ± SD of triplicates. (D) K18(P301L) interaction biosensor signal in cells treated or not with colchicine (10 µM, 1 hr) or nocodazole (1 µM, 1 hr); data are expressed as mean ± SD of triplicates. (E) WT-Tau conformation biosensor response in cells treated with colchicine (10 µM, 1 hr), forskolin (10 µM, 24 hr), or both (colchicine followed by forskolin); data are expressed as mean ± SEM of five independent experiments; **p<0.01 by one-way ANOVA followed by Sidak’s multiple comparison test. (F) WT-Tau interaction biosensor response in cells treated with colchicine (10 µM, 1 h), okadaic acid (OA 100 nM, 24 hr), or both (colchicine followed by OA); data are expressed as mean ± SD of triplicates. (G) Immunofluorescence analysis (red: anti-HA antibody; blue: DAPI staining of nuclei; scale bar: 10 µm) and biosensor signal of the WT-Tau conformation sensor expressed in the SH-SY-5Y neuronal cell line; data are expressed as mean ± SD of triplicates.
Figure 3 with 1 supplement
Tau seeding response monitored by Nanobinary technology (NanoBiT) tau biosensors.
(A) WT-Tau interaction biosensor signal in cells treated with monomeric (m) or aggregated (agg) forms of recombinant K18 or tau; data are expressed as mean ± SEM of five independent experiments. (B) K18(P301L) and WT-Tau interaction biosensors signal in cells treated with brain lysates (3 µg of total protein, 24 hr) obtained from wild-type (WT) or transgenic (Tg) mice; data are expressed as mean ± SEM of seven independent experiments; ****p<0.001 by one-ANOVA, followed by Sidak’s multiple comparison tests. (C) K18(P301L) interaction biosensor signal in the presence of increasing concentrations of brain lysates (in µg of total protein) obtained from WT or Tg mice; the representative curve of four independent experiments expressed as mean ± SD of triplicates. Insert: zoom of data points indicated by the square. (D) LgBit-Tau +SmBit-K18 interaction biosensor signal in the presence of brain lysates obtained from WT or Tg mice (3 µg); data are expressed as mean ± SEM of five independent experiments; ***p<0.005 by one-ANOVA, followed by Sidak’s multiple comparison tests.
Figure 3—figure supplement 1
Specificity and the seeding response of the K18(P301L) conformation and interaction sensors.
(A) Biosensor signal in cells expressing K18(P301L) interaction biosensor or the non-related LgBiT-K18(P301L)+SmBiT Halo biosensor interaction pair following treatment with brain lysates obtained from wild-type (WT) or transgenic (Tg) mice; data are expressed as mean ± SEM of five (SmBiT-K18(P301L)) or three (SmBiT-Halo) independent experiments; *p<0.05, by Student t-test compared to WT group. (B) Reproducibility of the K18(P301L) interaction biosensor response towards seeds from brain lysates obtained from four independent WT (WT 1–4) or Tg (Tg 1–4) mice; data are expressed as mean ± SEM of hree independent experiments; ****p<0.001, by one-way ANOVA followed by Sidak’s multiple comparison tests. (C) Assay miniaturization of K18(P301L) interaction biosensor: sensor response towards seeds from brain lysates obtained from WT or Tg mice in 384-well plate format; data are expressed as mean ± SD of quadruplicates. (D-E) K18(P301L) conformation biosensors signal in cells treated with monomeric (m) or aggregated (agg) forms of recombinant K18 or tau (D), or with brain lysates obtained from WT or Tg mice (E). Data are expressed as mean ± SEM of three to five independent experiments. **p<0.01 by one-ANOVA, followed by Sidak’s multiple comparison tests.
Figure 4 with 1 supplement
Tau(P301L) self-interaction and seeding responses monitored by Nanoluciferase (Nluc) biosensors.
(A–B) Tau(P301L) interaction (A) and conformation (B) biosensor signal in cells treated or not with forskolin (10 µM, 24 hr); data are expressed as mean ± SEM of four (A) or five (B) independent experiments; *p<0.05, ** 0.01 by Student t-test. Right panel: western blot analysis of phosphorylated tau (P-Tau) and total tau (T-tau) using AT8 and anti-HA antibodies, respectively; L: Lgbit-Tau; S: SmBit-Tau. (C) Cellular distribution of the Tau(P301L) interaction biosensor monitored by immunofluorescence microscopy in the absence (top panels) or presence (bottom panels) of colchicine (biosensor: anti-HA antibody, in red; tubulin: in green; nuclei: DAPI staining, in blue); Scale bar: 10 µm. Insets: colocalization analysis (Manders' colocalization coefficient) of the fractional overlap between biosensor molecules and microtubules (MTs). (D–E) Tau(P301L) and WT-Tau conformation (D) and interaction (E) biosensors signal in cells treated or not with colchicine (10 µM, 1 hr); data are expressed as mean ± SEM of three independent experiments; *p<0.05, **p<0.01, ***p<0.005 by Student t-test. (F) Proximity of Tau(P301L) biosensor molecules to MTs by PLA assay (PLA signal in red; tubulin in green; nuclei in blue by DAPI staining); i. negative control (absence of primary antibodies); ii. positive PLA signal; iii. zoomed image of the white square area shown in ii; white arrows show an overlap of PLA signal and MTs; scale bar: 10 µm. Inset graph: colocalization analysis (Manders' colocalization coefficient) of the fractional overlap between PLA signal and MTs; data are expressed as mean ± SEM of five images. (G–H) Tau(P301L) interaction biosensor signal in cells treated for 24 hr with recombinant proteins (G) or brain lysates obtained from wild-type (WT) or transgenic (Tg) mice (H); data are expressed as mean ± SEM of four (G) or five (H) independent experiments; **p<0.01 by one-ANOVA, followed by Sidak’s multiple comparison tests to the indicated group. (I) Immunofluorescence microscopy detection of tau pathological conformation (MC-1 antibody, green, upper panel) or phospho-tau (AT8 antibody, green, bottom panel) in cells expressing Tau(P301L) or WT-Tau interaction biosensors (HA antibody, red) and treated with brain lysates obtained from WT or Tg mice for 24 hr; nuclei are stained with DAPI (blue); scale bar: 10 µm.
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Figure 4—source data 1
Unedited blot of Figure 4A.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig4-data1-v1.zip
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Figure 4—source data 2
Unedited blot of Figure 4B.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig4-data2-v1.zip
Figure 4—figure supplement 1
Characterization of Tau(P301L) biosensors.
(A) Thioflavin T (ThT) aggregation kinetics assay of recombinant full-length tau(P301L) and LgBit-Tau(P301L) proteins (10 µM), in the presence of aggregation inducer (heparin 1:1). (B–C) Tau(P301L) conformation biosensors signal in cells treated with monomeric (m) or aggregated (agg) forms of recombinant K18 or tau (B), or with brain lysates obtained from wild-type (WT) or transgenic (Tg) mice (C). Data are expressed as mean ± SEM of three to six independent experiments. **p<0.01 by one-ANOVA, followed by Sidak’s multiple comparison tests. (D) Thioflavin S (ThS) detection (blue) of tau fibrils in cells expressing the Tau(P301L) interaction biosensor (anti-HA staining, in red) in the absence (top panel) or presence (middle panel) of Tg brain lysates (12 ug of total protein per well in a 24-well plate; 24 hr), or in cells not expressing the biosensor and treated with Tg brain lysates (bottom panel). Merged image on the right of the ThS and HA fluorescence together with bright field image; scale bar: 10 µm. Inset graphs on the right show the respective biosensor luminescence signal of each condition.
Figure 5 with 1 supplement
Tau Nano binary technology (NanoBiT) biosensors application in the identification of inhibitors of tau seeding and self-interaction.
(A–B) Determination of the assay Z-factor by collecting biosensor signals for K18(P301L) (A) or Tau(P301L) (B) interaction biosensor cells in the presence of brain lysates from wild-type (WT) (red, low reference signal) or transgenic (Tg) (blue, high reference signal) mice. (C–D) K18(P301L) (C) or Tau(P301L) (D) interaction biosensor signal in the presence of candidate inhibitors (10 µM, simultaneously to Tg treatment, 24 hr) of tau aggregation; data are expressed as mean ± SEM of four (C) or three (D) independent experiments; *p<0.05, ***p<0.005, ****p<0.001 by one-ANOVA, followed by Sidak’s multiple comparison tests to the vehicle group. (E–G) Concentration-response curves of the effect of compounds ANLE138b (E), LMTMeSO4 (F), and bb14 (G) on the K18(P301L) biosensor signal; data are expressed as mean ± SEM of four independent experiments.
Figure 5—figure supplement 1
Effect of tested compounds on Nanoluciferase (Nluc) activity and cell viability.
(A) Cell viability assessed by MTT test in HEK293T cells incubated with vehicle (DMSO 0.1%) or the corresponding compounds (10 µM, 24 h); Triton 20% was used as a positive control of the assay; data are expressed as mean ± SD of triplicates. (B) Nluc activity in HEK293T cells expressing Nluc construct only (no tau) and incubated with vehicle (DMSO 0.1%) or the corresponding compounds (10 µM, overnight); data are expressed as mean ± SD of triplicates.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Homo sapiens) | MAPT | GenBank | NCBI Entrez Gene: 4137 | |
| strain, strain background (Escherichia coli) | BL21(DE3) | Sigma-Aldrich | CMC0016 | Electrocompetent cells |
| cell line (Homo sapiens) | Human Embryonic Kidney (HEK) 293 | Sigma-Aldrich | RRID: CVCL_0063 | |
| cell line (Homo sapiens) | Human neuroblastoma SH-SY5Y cells | Sigma-Aldrich | RRID:CVCL_0019 | |
| transfected construct (Tag constrcut) | NanoBiT system (SmBit and LgBit) | Promega | ||
| transfected constructo (Homo sapiens) | LgBit-HA-K18(P301L) | This paper | see Methods | |
| transfected construct (Homo sapiens) | SmBit-HA-K18(P301L) | This paper | see Methods | |
| transfected construct (Homo sapiens) | LgBit-HA-Tau(P301L) | This paper | see Methods | |
| transfected constructo (Homo sapiens) | SmBit-HA-Tau(P301L) | This paper | see Methods | |
| transfected constructo (Homo sapiens) | LgBit-HA-Tau | This paper | see Methods | |
| transfected constructo (Homo sapiens) | SmBit-HA-Tau | This paper | see Methods | |
| antibody | anti-HA antibody (rabbit monoclonal) | Cell Signaling | Cat# 3724 S | IF(1:500), WB (1:1000) |
| antibody | anti-HA antibody (mouse monoclonal) | Biolegend | Cat#. 901514 | IF(1:500) |
| antibody | anti-tubulin (rat polyclonal) | Millipore | Cat#: MAB1864 | IF(1:200) |
| antibody | anti-phospho tau antibody AT8 (Mouse monoclonal) | Thermofisher | MN1020; | IF(1:200), WB (1:500) |
| antibody | anti-aggregated tau MC-1 antibody (Mouse monoclonal), conformation-specific anti-tau antibody | PMID:9349554 | IF(1:500) | |
| antibody | IRDye 800CW anti-Rabbit IgG (Goat polyclonal) Secondary Antibody | LI-COR Biosciences - GmbH | Cat.# 926–32211 | WB (1:10,000) |
| antibody | IRDye 680RD anti-Mouse IgG (Goat polyclonal) Secondary Antibody | LI-COR Biosciences - GmbH | Cat.# 926–68070 | WB (1:10,000) |
| commercial assay or kit | nanoluciferase substrate Nano-Glo Live Cell | Promega | Cat.# N2012 | |
| commercial assay or kit | Duolink PLA | Sigma-Aldrich | Cat.# DUO92101 | |
| chemical compound, drug | thioflavin S | Sigma Aldrich | Cat.# T1892 | |
| chemical compound, drug | thioflavin T | Sigma Aldrich | Cat.# T3516 | |
| software, algorithm | Image J | https://doi.org/10.1038/nmeth.2089 | RRID: SCR_003070 https://imagej.nih.gov/ij/download.html | |
| software, algorithm | GraphPad Prism 6 | GraphPad Software Inc | RRID:SCR_002798; https://www.graphpad.com/scientificsoftware/-prism/ | |
| other | EnVision Plate Reader | PerkinElmer | see Methods |
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Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells
eLife 12:e78360.
https://doi.org/10.7554/eLife.78360
