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) …
-
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 …
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 …
-
Figure 2—source data 1
Unedited blot of Figure 2A.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data1-v1.zip
-
Figure 2—source data 2
Unedited blot of Figure 2E.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data2-v1.zip
-
Figure 2—source data 3
Unedited blot of Figure 2K.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data3-v1.zip
-
Figure 2—source data 4
Unedited blot of Figure 2L.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig2-data4-v1.zip
-
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 …
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) …
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 …
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 …
-
Figure 4—source data 1
Unedited blot of Figure 4A.
- https://cdn.elifesciences.org/articles/78360/elife-78360-fig4-data1-v1.zip
-
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) …
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, …
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 …
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 |
