(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, …
Spreadsheet with the normalized fluorescence polarization data (Figure 1c).
Spreadsheet with the normalized fluorescence polarization data (Figure 1d).
Spreadsheet with the raw fluorescence lifetime data (Figure 1e).
Spreadsheet with the raw fluorescence lifetime data (Figure 1f).
The region in the box corresponds to the selected regions presented in Figure 1b.
(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 …
Spreadsheet with the normalized fluorescence polarization data (Figure 1—figure supplement 2a).
Spreadsheet with the normalized fluorescence polarization data (Figure 1—figure supplement 2b).
Spreadsheet with the normalized fluorescence polarization data (Figure 1—figure supplement 2c).
Spreadsheet with the normalized fluorescence polarization data (Figure 1—figure supplement 2d).
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: …
(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 …
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) amplitude data (Figure 2b).
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) frequency data (Figure 2c).
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) decay data (Figure 2d).
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) rise time data (Figure 2e).
Spreadsheet with the diffusion distribution data (Figure 2g).
Spreadsheet with the percentage of mobile AMPARs data (Figure 2h).
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045002 (Figure 2i).
(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: …
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) amplitude data (Figure 2—figure supplement 1a).
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) frequency data (Figure 2—figure supplement 1b).
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) decay time data (Figure 2—figure supplement 1c).
Spreadsheet with the miniature excitatory postsynaptic currents (mEPSCs) rise time data (Figure 2—figure supplement 1d).
Error bars indicate cell-to-cell variability.
Spreadsheet with the cumulative diffusion distribution data.
(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; …
Spreadsheet with the raw object enrichment data (Figure 3b).
Spreadsheet with the raw object colocalization percentage data (Figure 3c).
Spreadsheet with the raw PSD-95-positive objects percentage data (Figure 3d).
Spreadsheet with the fluorescence recovery data (Figure 3f).
Spreadsheet with the mobile fraction extracted from the fluorescence recovery data (Figure 3g).
(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 …
(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 …
Spreadsheet with the normalized object average intensity data (Figure 4b).
Spreadsheet with the normalized dendrite intensity data (Figure 4c).
Spreadsheet with the normalized fluorescence intensity data (Figure 4e).
Spreadsheet with the normalized fluorescence intensity data (Figure 4f).
(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 …
Spreadsheet with the fluorescence intensity ratio data (Figure 5d).
(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 …
(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 …
Spreadsheet with the number of detection data (Figure 6d).
Spreadsheet with the length and width data (Figure 6e).
Spreadsheet with the diffusion distribution data (Figure 6i).
Spreadsheet with the mobile fraction percentage over time data (Figure 6j).
(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 …
Spreadsheet with the diffusion distribution data (Figure 6—figure supplement 1c).
Spreadsheet with the mobile fraction percentage data (Figure 6—figure supplement 1d).
Spreadsheet with the frequency distribution of single detection data (Figure 6—figure supplement 1e).
(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 …
(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)…
(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 …
Spreadsheet with the synaptic enrichment data (Figure 8d).
(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 …
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' |
FRAP data.
List of plasmids used in this work (c = commercial source; *from this study).
Primers used in this study.