Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells

  1. Erika Cecon
  2. Atsuro Oishi
  3. Marine Luka
  4. Delphine Ndiaye-Lobry
  5. Arnaud François
  6. Mathias Lescuyer
  7. Fany Panayi
  8. Julie Dam
  9. Patricia Machado
  10. Ralf Jockers  Is a corresponding author
  1. Institut Cochin, Inserm U1016, CNRS UMR 8104, Université de Paris, France
  2. Les Laboratoires Servier, France
5 figures, 1 table and 1 additional file

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—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—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—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 resourceDesignationSource or referenceIdentifiersAdditional information
gene (Homo sapiens)MAPTGenBankNCBI Entrez Gene: 4137
strain, strain background (Escherichia coli)BL21(DE3)Sigma-AldrichCMC0016Electrocompetent cells
cell line (Homo sapiens)Human Embryonic Kidney (HEK) 293Sigma-AldrichRRID: CVCL_0063
cell line (Homo sapiens)Human neuroblastoma SH-SY5Y cellsSigma-AldrichRRID:CVCL_0019
transfected construct (Tag constrcut)NanoBiT system (SmBit and LgBit)Promega
transfected constructo (Homo sapiens)LgBit-HA-K18(P301L)This papersee Methods
transfected construct (Homo sapiens)SmBit-HA-K18(P301L)This papersee Methods
transfected construct (Homo sapiens)LgBit-HA-Tau(P301L)This papersee Methods
transfected constructo (Homo sapiens)SmBit-HA-Tau(P301L)This papersee Methods
transfected constructo (Homo sapiens)LgBit-HA-TauThis papersee Methods
transfected constructo (Homo sapiens)SmBit-HA-TauThis papersee Methods
antibodyanti-HA antibody (rabbit monoclonal)Cell SignalingCat# 3724 SIF(1:500), WB (1:1000)
antibodyanti-HA antibody (mouse monoclonal)BiolegendCat#. 901514IF(1:500)
antibodyanti-tubulin (rat polyclonal)MilliporeCat#: MAB1864IF(1:200)
antibodyanti-phospho tau antibody AT8 (Mouse monoclonal)ThermofisherMN1020;IF(1:200), WB (1:500)
antibodyanti-aggregated tau MC-1 antibody (Mouse monoclonal), conformation-specific anti-tau antibodyPMID:9349554IF(1:500)
antibodyIRDye 800CW anti-Rabbit IgG (Goat polyclonal) Secondary AntibodyLI-COR Biosciences - GmbHCat.# 926–32211WB (1:10,000)
antibodyIRDye 680RD anti-Mouse IgG
(Goat polyclonal) Secondary Antibody
LI-COR Biosciences - GmbHCat.# 926–68070WB (1:10,000)
commercial assay or kitnanoluciferase substrate Nano-Glo Live CellPromegaCat.# N2012
commercial assay or kitDuolink PLASigma-AldrichCat.# DUO92101
chemical compound, drugthioflavin SSigma AldrichCat.# T1892
chemical compound, drugthioflavin TSigma AldrichCat.# T3516
software, algorithmImage Jhttps://doi.org/10.1038/nmeth.2089RRID: SCR_003070
https://imagej.nih.gov/ij/download.html
software, algorithmGraphPad Prism 6GraphPad Software IncRRID:SCR_002798; https://www.graphpad.com/scientificsoftware/-prism/
otherEnVision Plate ReaderPerkinElmersee Methods

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