Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques
- University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, France
- University of Bordeaux, CNRS, INSERM, Bordeaux Imaging Center, BIC, UMS 3420, US 4, France
- University of Bordeaux, Bordeaux Proteome, France
- University of Bordeaux, Inserm U1212, CNRS UMR 5320, IECB, France
Figures
Figure 1 with 3 supplements
Evaluation of the impact of evolved 10FN3 domains binding on the PDZ domains function.
(a) PSD-95 domain organization and binding models of the three clones investigated. (b) Titrations of a monovalent stargazin-derived peptide against PSD-95-12 in the absence or presence of Xph15, …
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Figure 1—source data 1
Spreadsheet with the normalized fluorescence polarization data (Figure 1c).
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Figure 1—source data 2
Spreadsheet with the normalized fluorescence polarization data (Figure 1d).
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Figure 1—source data 3
Spreadsheet with the raw fluorescence lifetime data (Figure 1e).
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Figure 1—source data 4
Spreadsheet with the raw fluorescence lifetime data (Figure 1f).
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Figure 1—figure supplement 1
[15N]-HSQC spectra collected on 200 µM [15N]PSD-95-12 and titrated in with increasing concentrations of a monovalent stargazin-derived peptide (Stg) (a) in the absence of binder or in the presence of 240 µM Xph15 (b), Xph18 (c), or Xph20 (d).
The region in the box corresponds to the selected regions presented in Figure 1b.
Figure 1—figure supplement 2
Fluorescence polarization titrations.
(a) Direct titration of FITC-labeled stargazin-derived divalent ligand (10 nM) against PSD-95 tandem PDZ domains. Each data point represents the average of three measurements ± SD. The dissociation …
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Figure 1—figure supplement 2—source data 1
Spreadsheet with the normalized fluorescence polarization data (Figure 1—figure supplement 2a).
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Figure 1—figure supplement 2—source data 2
Spreadsheet with the normalized fluorescence polarization data (Figure 1—figure supplement 2b).
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Figure 1—figure supplement 2—source data 3
Spreadsheet with the normalized fluorescence polarization data (Figure 1—figure supplement 2c).
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Figure 1—figure supplement 2—source data 4
Spreadsheet with the normalized fluorescence polarization data (Figure 1—figure supplement 2d).
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Figure 1—figure supplement 3
Expression regulation system.
The system relies on a competition for the expressed probe between binding to its cytosolic target (favored) or preventing further transcription (unfavored until the target is saturated). Xph/X: …
Figure 2 with 3 supplements
Evaluation of monobodies binding to endogenous PSD-95.
(a–e) Synaptic currents in wild-type mouse neurons infected with adeno-associated viruses expressing either eGFP, Xph15, Xph18, or Xph20 (n = 19, 15, 17, and 16, respectively, from three independent …
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Figure 2—source data 1
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) amplitude data (Figure 2b).
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Figure 2—source data 2
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) frequency data (Figure 2c).
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Figure 2—source data 3
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) decay data (Figure 2d).
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Figure 2—source data 4
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) rise time data (Figure 2e).
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Figure 2—source data 5
Spreadsheet with the diffusion distribution data (Figure 2g).
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Figure 2—source data 6
Spreadsheet with the percentage of mobile AMPARs data (Figure 2h).
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Figure 2—source data 7
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045002 (Figure 2i).
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Figure 2—figure supplement 1
Spontaneous miniature excitatory postsynaptic currents properties based on the analysis of the 100 first events of control non-transfected or Xph15 and Xph18 (fused to eGFP and the expression regulation system) transfected rat culture neurons.
(a) Amplitude (in pA, control: 18.92 ± 2.37 [n = 20]; Xph15: 17.58 ± 1.71 [n = 15]; Xph18: 18.52 ± 2.03 [n = 11]; mean ± SEM with p>0.61 by ordinary one-way ANOVA). (b) Frequency (in Hz, control: …
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Figure 2—figure supplement 1—source data 1
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) amplitude data (Figure 2—figure supplement 1a).
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Figure 2—figure supplement 1—source data 2
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) frequency data (Figure 2—figure supplement 1b).
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Figure 2—figure supplement 1—source data 3
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) decay time data (Figure 2—figure supplement 1c).
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Figure 2—figure supplement 1—source data 4
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) rise time data (Figure 2—figure supplement 1d).
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Figure 2—figure supplement 2
Cumulative distribution of average distribution of instantaneous diffusion coefficients obtained by uPAINT of synaptic AMPAR (Figure 2g).
Error bars indicate cell-to-cell variability.
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Figure 2—figure supplement 2—source data 1
Spreadsheet with the cumulative diffusion distribution data.
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Figure 2—figure supplement 3
Rat hippocampal culture transduced with adeno-associated viruses expressing either eGFP or Xph20-eGFP at 3 d in vitro (DIV) and imaged and lysed at 16 DIV for proteomics analysis (scale bar 40 µm).
Figure 3 with 1 supplement
Evaluation of monobodies as intrabody fluorescent reporter probes.
(a) Representative epifluorescence images of the eGFP-fused binding modules vs immunostaining of endogenous PSD-95 domain. For the zoomed regions, top: binding module; middle: antibody staining; …
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Figure 3—source data 1
Spreadsheet with the raw object enrichment data (Figure 3b).
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Figure 3—source data 2
Spreadsheet with the raw object colocalization percentage data (Figure 3c).
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Figure 3—source data 3
Spreadsheet with the raw PSD-95-positive objects percentage data (Figure 3d).
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Figure 3—source data 4
Spreadsheet with the fluorescence recovery data (Figure 3f).
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Figure 3—source data 5
Spreadsheet with the mobile fraction extracted from the fluorescence recovery data (Figure 3g).
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Figure 3—figure supplement 1
Regulated vs non-regulated expression of Xph20-FP fusion.
(a) Representative images obtained on neurons transfected with Xph20-eGFP fusion (regulated with a zinc finger and a transcription repressor fusion together with the corresponding zinc finger …
Figure 4
Evaluation of Xph15 and Xph20 intrabodies specificity.
(a) Representative epifluorescence images showing Xph15-eGFP (top) and Xph20-eGFP (bottom) expressed in pyramidal neuron together with a scramble shRNA or the shRNA against PSD-95 both associated to …
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Figure 4—source data 1
Spreadsheet with the normalized object average intensity data (Figure 4b).
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Figure 4—source data 2
Spreadsheet with the normalized dendrite intensity data (Figure 4c).
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Figure 4—source data 3
Spreadsheet with the normalized fluorescence intensity data (Figure 4e).
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Figure 4—source data 4
Spreadsheet with the normalized fluorescence intensity data (Figure 4f).
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Figure 5 with 1 supplement
Evaluation of probes for stimulated emission depletion (STED) imaging.
(a) Schematic representation of fluorescent protein-fused STED probe. (b) Representative confocal images of a neuron transfected with Xph15-mNeonGreen before and after STED. The yellow box …
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Figure 5—source data 1
Spreadsheet with the fluorescence intensity ratio data (Figure 5d).
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Figure 5—figure supplement 1
Stimulated emission depletion (STED) imaging.
(a) Representative image obtained on fixed neurons transfected with Xph20-eGFP fusion and immunolabeled with an antibody against MAP2 to identify dendritic shaft. Zoom region obtained by confocal …
Figure 6 with 1 supplement
Evaluation of mEos3.2-derived probes for photoactivation localization microscopy (PALM) and spt-PALM applications.
(a) Schematic representation of mEos3.2-fused probe. (b) Representative epifluorescence and PALM images of Xph20-mEos3.2 in fixed culture neurons. Left: epifluorescence image obtained from the …
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Figure 6—source data 1
Spreadsheet with the number of detection data (Figure 6d).
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Figure 6—source data 2
Spreadsheet with the length and width data (Figure 6e).
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Figure 6—source data 3
Spreadsheet with the diffusion distribution data (Figure 6i).
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Figure 6—source data 4
Spreadsheet with the mobile fraction percentage over time data (Figure 6j).
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Figure 6—figure supplement 1
Evaluation of mEos3.2-derived probes for photoactivation localization microscopy (PALM) applications.
(a, b) Representative spt-PALM images of fixed culture neurons expressing Xph20-mEos3.2. Super-resolution intensity map obtained by sptPALM from a sequence of 4000 images of sparse single molecules …
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Figure 6—figure supplement 1—source data 1
Spreadsheet with the diffusion distribution data (Figure 6—figure supplement 1c).
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Figure 6—figure supplement 1—source data 2
Spreadsheet with the mobile fraction percentage data (Figure 6—figure supplement 1d).
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Figure 6—figure supplement 1—source data 3
Spreadsheet with the frequency distribution of single detection data (Figure 6—figure supplement 1e).
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Figure 7 with 1 supplement
Evaluation of SNAP-tag-derived probes for DNA-PAINT super-resolution microscopy.
(a) Probe design and labeling scheme (BG: benzylguanine). (b) Reconstructed DNA-PAINT image (10 Hz, 32,000 frames) of Xph20-SNAP-tag in the dendrites of a 14 day in vitro (DIV) hippocampal primary …
Figure 7—figure supplement 1
DNA-PAINT imaging.
(a) Representative DNA-PAINT image obtained on neurons co-transfected with a soluble GFP marker and Xph20-SNAP-tag, not incubated with BG-docking strand and in the presence of Cy3-imaging strand. (b)…
Figure 8 with 3 supplements
Application of the ReMoRa method for the synaptic targeting of calcium reporters.
(a) Schematic representation of calcium signaling probe. (b) Comparison of the expression profile of targeted and regulated (Xph20-GCaMP7f, bottom panel) vs parental calcium sensor (GCaMP7f, top …
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Figure 8—source data 1
Spreadsheet with the synaptic enrichment data (Figure 8d).
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Figure 8—figure supplement 1
Engineered calcium reporters expression.
(a, b) Maximum projection of the fluorescence movies collected for hippocampal neurons expressing GCaMP6f (a) or Xph15-GCaMP6f* (non-regulated) (b). In the insets, the blue arrows indicate …
Figure 8—video 1
Spontaneous responses of GCaMP6f.
Figure 8—video 2
Xph15-GCaMP6f expressed in hippocampal Banker cultures and recorded at 50 Hz.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21 CodonPlus (DE3)-RIPL | Agilent | Cat# 230280 | |
| Strain, strain background (E. coli) | T7 Express lysY | New England Biolabs | Cat# C3010I | |
| Cell line (simian) | COS-7 | ECACC-87021302 | ||
| Antibody | Anti-GluA2 ATTO-647N (mouse monoclonal) | Gift from Eric Gouaux, coupled in lab. | PMID:23926273 | 30 ng/ml |
| Antibody | Anti-PSD-95 (mouse monoclonal) | Thermo Fisher | Cat# MA1-046 | 1:1000 |
| Antibody | Goat anti-mouse Alexa Fluor 568 (goat polyclonal) | Thermo Fisher | Cat# A-11031 | 1:1000 |
| Antibody | Anti-MAP2 (chicken polyclonal) | Synaptic Systems | Cat# 188 006 | 1:2000 |
| Antibody | Goat anti-chicken Alexa Fluor 594 (goat polyclonal) | Thermo Fisher | Cat# A-11042 | 1:800 |
| Recombinant DNA reagent | Numerous | See Supplementary file 2 | https://www.addgene.org/Matthieu_Sainlos/ | |
| Sequence-based reagent | Numerous | See Supplementary file 3 | ||
| Peptide, recombinant protein | PSD-95-12 [61-249] | Rimbault et al., 2019 | PMID:31586061 | |
| Peptide, recombinant protein | Xph20 | Rimbault et al., 2019 | PMID:31586061 | https://www.addgene.org/Matthieu_Sainlos/ |
| Peptide, recombinant protein | Xph18 | Rimbault et al., 2019 | PMID:31586061 | https://www.addgene.org/Matthieu_Sainlos/ |
| Peptide, recombinant protein | Xph15 | Rimbault et al., 2019 | PMID:31586061 | https://www.addgene.org/Matthieu_Sainlos/ |
| Peptide, recombinant protein | Xph0 | Rimbault et al., 2019 | PMID:31586061 | https://www.addgene.org/Matthieu_Sainlos/ |
| Peptide, recombinant protein | Stg15 (Ac-YSLHANTANRRTTPV) | Rimbault et al., 2019 | PMID:31586061 | |
| Peptide, recombinant protein | FITC-Stg15 (FITC-PEG-YSLHANTANRRTTPV) | Rimbault et al., 2019 | PMID:31586061 | |
| Peptide, recombinant protein | [Stg15]2 | Rimbault et al., 2019 | PMID:31586061 | |
| Peptide, recombinant protein | FITC-[Stg15]2 | Rimbault et al., 2019 | PMID:31586061 | |
| Commercial assay or kit | X-treme GENE HP DNA transfection reagent | Roche | ||
| Commercial assay or kit | Effectene Kit | QIAGEN | ||
| Chemical compound, drug | SNAP-Cell 647-SiR (BG-SiR) | New England Biolabs | Cat# S9102S | |
| Software, algorithm | PyMOL | Warren DeLano | RRID:SCR_000305 | |
| Software, algorithm | Fiji | PMID:22743772 | RRID:SCR_002285 | |
| Software, algorithm | Adobe Illustrator | Adobe Systems | RRID:SCR_010279 | |
| Software, algorithm | Prism 7.04, 8 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | TopSpin v4.0 | Bruker | RRID:SCR_014227 | |
| Software, algorithm | MetaMorph v7.8.10.0 | Molecular Devices | RRID:SCR_002368 | |
| Software, algorithm | LI-FLIM v1.2.12 | Lambert Instruments | ||
| Software, algorithm | POLARstar Omega v5.11 | BMG Labtech | ||
| Software, algorithm | NMRPipe v8.6 | Delaglio et al., 1995 | PMID:8520220 | |
| Software, algorithm | Sparky v3.113 | D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco | RRID:SCR_014228 | |
| Software, algorithm | POLARstar MARS data analysis software v3.20 | BMG Labtech | ||
| Software, algorithm | Patchmaster | Heka Elektronik | ||
| Software, algorithm | SR-Tesseler | Levet et al., 2015 | PMID:26344046 | |
| Software, algorithm | IJ-Macro_FRAP-MM | https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM | ||
| Software, algorithm | IJ-Plugin_Metamorph-Companion | https://github.com/fabricecordelieres/IJ-Plugin_Metamorph-Companion | ||
| Software, algorithm | PICASSO | Schnitzbauer et al., 2017 | PMID:28518172 | |
| Other | SNAPligand-modified DNA oligomer | Schnitzbauer et al., 2017 | PMID:28518172 | 5' BG-TTATACATCTA 3' |
| Other | Cy3b-labeled DNA imager strands | Schnitzbauer et al., 2017 | PMID:28518172 | 5' CTAGATGTAT-Cy3b 3' |
Additional files
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Supplementary file 1
FRAP data.
- https://cdn.elifesciences.org/articles/69620/elife-69620-supp1-v2.xlsx
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Supplementary file 2
List of plasmids used in this work (c = commercial source; *from this study).
- https://cdn.elifesciences.org/articles/69620/elife-69620-supp2-v2.xlsx
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Supplementary file 3
Primers used in this study.
- https://cdn.elifesciences.org/articles/69620/elife-69620-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69620/elife-69620-transrepform1-v2.docx
