guinea pig anti homer 1  (Alomone Labs)

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Nlg1 mutants differentially associate with gephyrin and PSD-95 scaffolds. a Schematics of Nlg1 structure. The sequence of the gephyrin-binding motif (blue) is shown with Y782 in red; AchE: acetylcholine esterase-like domain; TM: transmembrane domain. b Coomassie gel loaded with GST or GST-Nlg1 fusion proteins (Nlg1-WT, Y782A, or Y782F). These proteins migrate as three major bands between 25 kD and 50 kD, suggesting that they experience some degradation. The upper bands at 50 kD corresponding to the full length molecule are expected to bind PSD-95. This upper band plus the major band at ~40 kD are expected to bind gephyrin. c , d Immunoblots of gephyrin-Venus and PSD-95-mCherry pulled down by GST-Nlg1 proteins, respectively, and corresponding graphs of bound gephyrin and PSD-95, normalized to GST-Nlg1-WT (mean ± SEM of 2 independent experiments). e , g Dendrites from DIV 14–15 cultured neurons electroporated at DIV 0 with AP-tagged Nlg1-WT, -Y782A, or -Y782F (red) along with BirA ER and GFP (blue). Surface Nlg1 was labeled with Atto647-conjugated mSA (red) and endogenous gephyrin or PSD-95 were immunostained, respectively (green). Scale bars, 10 µm. f , h Percentage of surface Nlg1 clusters positive for gephyrin or PSD-95 for the three conditions (number of cells indicated within the bars, from 3 independent experiments). i Representative trajectories of individual Nlg1 molecules tracked by uPAINT (see Methods) at the surface of neurons at DIV 14–15 expressing Nlg1-WT, -Y782A, or -Y782F along with BirA ER and Homer1c-GFP (white), sparsely labeled with Atto594-conjugated mSA. Magenta and green represent fast ( D > 0.01 µm²/s) and slow ( D ≤ 0.01 µm²/s) diffusing molecules, respectively. Scale bar, 2 µm. j Distributions of individual diffusion coefficients in log scale (26013, 28756, and 17894 trajectories from 7, 10, and 9 cells for Nlg1-WT, Nlg1-Y782A, and Nlg1-Y782F, respectively). k Median diffusion coefficient for the 3 conditions (from one experiment). Data in graphs f , h , and k were compared by a Kruskal–Wallis test followed by Dunn’s multiple comparison test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data represent mean ± SEM

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: Sequencing, Binding Assay, Western Blot, Cell Culture, Labeling, Expressing, Diffusion-based Assay

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Effects of Nlg1 mutants on excitatory synapse differentiation. a Dendrites from DIV 14–15 cultured neurons co-transfected with GFP (green) and either EV, Nlg1-WT, -Y782A, or -Y782F. Neurons were live stained with an N-terminal GluA1 antibody (red) and counterstained with a VGlut1 antibody (blue). Scale bar, 5 µm. b , c Density of VGlut1 or GluA1 clusters, respectively, normalized to the EV condition (number of cells indicated within the bars, from 3 independent experiments). d Representative traces of mEPSCs recordings from DIV 14–15 neurons expressing EV, Nlg1-WT, -Y782A, or -Y782F clamped at −70 mV in the presence of TTX and bicuculline. e Cumulative distributions of the mEPSCs inter-event intervals for the corresponding conditions. f , g Mean mEPSC frequencies and amplitudes, respectively, for each condition (number of cells indicated within the bars, from 3 independent experiments). Data in graphs b , c , f , and g represent mean ± SEM and were compared by a Kruskal–Wallis test followed by Dunn’s multiple comparison test (ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: Cell Culture, Transfection, Staining, Expressing

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Nlg1-Y782A but not –Y782F trap surface-diffusing AMPARs. a Neurons were live labeled for endogenous AMPARs with a GluA1 antibody mixed with Atto594-conjugated anti-rabbit Fab. The postsynaptic density (PSD) was detected with Homer1c-GFP. b Representative trajectories of individual AMPARs at the neuronal surface (red) in DIV 10 neurons expressing EV, Nlg1-WT, -Y782A, or -Y782F and Homer1c-GFP (white). Scale bar, 2 µm. c Average mean square displacement (MSD) and d distributions of individual diffusion coefficients in log scale from one experiment, for the four conditions (number of cells/number of trajectories, EV: 8/5690; Nlg1-WT: 7/4340; Nlg1-Y782A: 8/18059; Nlg1-Y782F: 8/16978). Note the higher MSDs and larger peaks of mobile receptors for EV and Nlg1-Y782F, compared to Nlg1-WT and -Y782A. The fraction of slowly mobile molecules on the left of the histogram ( D < 0.01 µm² s −1 ) is gray-shaded. e The median diffusion coefficient per cell was averaged for each condition (3 independent experiments). The number of cells examined is given in the columns. f Effect of the Nlg1 mutations on the number of Homer-1c puncta per unit length of dendrite (3 independent experiments). Data in graphs e and f represent mean ± SEM and were compared by a Kruskal–Wallis test followed by Dunn’s multiple comparison test (* P < 0.05, *** P < 0.001)

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: Labeling, Expressing, Diffusion-based Assay

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Nlg1 mutants differently affect excitatory synaptic transmission and spine density. CA1 neurons of organotypic hippocampal slices from Nlg1 KO mice were single-cell electroporated at DIV 3–5 with GFP plus EV, Nlg1-WT, Nlg1-Y782A, or Nlg1-Y782F, then processed for electrophysiology or immunostaining and confocal imaging at DIV 13–15. a Dual whole-cell recording configuration (left) and corresponding image from an experiment (right). Scale bar, 10 µm. b Representative traces of evoked AMPAR- and NMDAR-mediated EPSCs recorded at −70 mV and +40 mV, respectively. Color sample traces correspond to electroporated neurons in the different conditions, and black traces correspond to control, unelectroporated neurons. c , d Average AMPAR- and NMDAR-mediated EPSC amplitudes, respectively, normalized to the control condition (the dashed line indicates 100%, dot plots corresponds to different pairs, from 4 independent experiments). e Average ratio between paired NMDAR- and AMPAR-mediated EPSCs. f Representative traces of spontaneous EPSCs recorded at −70 mV from electroporated and non-electroporated neurons. Black dots indicate synchronous events. g Average of paired spontaneous EPSC amplitudes recorded in electroporated neurons, normalized to the control condition (dashed line at 100%, dot plots corresponds to different pairs, from 3 independent experiments). h Representative confocal images of secondary apical dendritic segments from CA1 pyramidal neurons co-expressing GFP along with EV, Nlg1-WT, Nlg1-Y782A, or Nlg1-Y782F. Scale bar, 5 µm. i Average spine density for the same conditions (dot plots corresponds to different cells, from 4 independent experiments). Data from graphs c , d , and g were compared to the control condition (non-electroporated neurons) by a Wilcoxon matched-pairs signed rank test. Data in graph e were compared by a Kruskal–Wallis test followed by Dunn’s multiple comparison test. Data in graphs g and i were compared by one-way ANOVA followed by Tukey’s multiple comparison test (ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Data represent mean ± SEM

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: Transmission Assay, Immunostaining, Imaging, Expressing

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Nlg1 C-terminal truncation affects AMPAR-dependent synaptic transmission and spine density. a Diagram of Nlg1 truncation mutants lacking the last 5 aa comprising the C-terminal PDZ domain-binding motif (Nlg1-Δ5) or the last 72 amino acids also including the gephyrin-binding motif (Nlg1-Δ72). TM: transmembrane domain. b Representative traces of evoked AMPAR- and NMDAR-mediated EPSCs recorded at −70 mV and +40 mV, respectively. Color sample traces correspond to neurons co-electroporated with GFP and Nlg1-WT, Nlg1-Δ5, or Nlg1-Δ72, respectively, while black traces correspond to control, unelectroporated neurons. c , d Average AMPAR- and NMDAR-mediated EPSC amplitudes, respectively, normalized to control (the dashed line indicates 100%, dot plots corresponds to different pairs, from 3 independent experiments). e Confocal images showing CA1 neurons from Nlg1 KO organotypic slices electroporated with GFP (green), BirA ER , and AP-tagged Nlg1-Δ5 or Nlg1-Δ72 which were labeled with streptavidin-Atto647 (red). Scale bars, 30 µm (upper panels) and 10 µm (lower panels). f Average spine density for CA1 neurons electroporated with EV, Nlg1-WT, Nlg1-Δ5, or Nlg1-Δ72 (dot plots corresponds to different cells, from 2 independent experiments). Data in graphs c and d were compared to the control condition by Wilcoxon matched-pairs signed rank test, and between themselves using one-way ANOVA followed by Tukey’s multiple comparison (ns: not significant, * P < 0.05, *** P < 0.001). Data in graph f were compared by a Kruskal–Wallis test followed by Dunn’s multiple comparison test (** P < 0.01). Data represent mean ± SEM

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: Transmission Assay, Binding Assay, Labeling

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Nlg1 replacement by Nlg1 point mutants and LTP experiments. a – e Nlg1 replacement strategy. a CA1 neurons of organotypic hippocampal slices from wild-type mice were single-cell electroporated at DIV 3–5 with either Nlg1 shRNA alone (knockdown), Nlg1 shRNA + resistant Nlg1-WT (rescue), or with resistant Nlg1-WT alone (overexpression). b Representative confocal images of a CA1 neuron co-expressing the GFP reporter from the shRNA (green) and biotinylated AP-Nlg1 stained with streptavidin-Atto647 (red). Scale bars, 40 µm (left panels) and 10 µm (right panels). c Representative traces of evoked AMPAR and NMDAR-mediated EPSCs recorded at −70 mV and +40 mV, respectively, in electroporated (black) and control (gray) neurons. d , e Average AMPAR- and NMDAR-mediated EPSCs, respectively, in the three conditions (dot plots corresponds to different pairs, from 3 independent experiments). f Average basal AMPAR-mediated EPSCs in CA1 cells co-expressing shRNA to Nlg1 plus Nlg1-WT, -Y782A, or -Y782F rescue constructs (dot plots corresponds to different pairs, from 5 independent experiments). g Average AMPAR-mediated EPSCs for the three conditions, upon LTP induction at time 0. Sample traces are shown at time 0 and 30 min after LTP induction. h Cumulative distribution of the long-term plateau of AMPAR-mediated EPSC in the three conditions, expressed as a percentage of the baseline level. Data in graphs d – g represent mean ± SEM and were compared to the control condition (unelectroporated) by Wilcoxon matched-pairs signed rank test (* P < 0.05, *** P < 0.001, ns: not significant compared to control)

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: shRNA, Over Expression, Expressing, Staining, Construct

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Expressing Nlg1-Y782F but not Nlg1-Y782A increases the number of functional inhibitory synapses. a Dendrites from DIV 14–15 dissociated neurons transfected with EV, Nlg1-WT, -Y782A, or -Y782F along with GFP (green). Neurons were live stained with an antibody against the γ2 subunit of GABA A receptors (red), then fixed and counterstained with an antibody against VGAT (blue). Scale bar, 5 µm. b , c Density of VGAT- or γ2-positive clusters, respectively, normalized to the EV condition (number of cells indicated within the bars, from 3 independent experiments). d Representative traces of mIPSCs recordings from DIV 14–15 neurons expressing EV, Nlg1-WT, -Y782A, or -Y782F clamped at +10 mV in presence of TTX and NBQX. e Cumulative distributions of the mIPSCs inter-event intervals for the corresponding conditions. f , g Mean mIPSC frequencies and amplitudes, respectively, for each condition (number of cells indicated within the bars, from 4 independent experiments). Data in graphs b , c , f , and g represent mean ± SEM and were compared by a Kruskal–Wallis test followed by Dunn’s multiple comparison test (ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: Expressing, Functional Assay, Transfection, Staining

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Identification of tyrosine kinases that phosphorylate Nlg1. a , b In vitro kinase assay. Purified tyrosine kinases (GST-FGFR1, -TrkB, or -TrkC) were incubated in the presence of ATP with GST-Nlg1, GST-Nlg1-Y782A, or a positive substrate (NTRK), and run on a polyacrylamide gel. a pTyr immunoblot. Note the phosphorylation of NTRK (blue arrow) and GST-Nlg1 (red arrow) but not GST-Nlg1-Y782A by the three kinases. Note the autophosphorylation of the three kinases (green arrow). b Corresponding Coomassie gel. GST-Nlg1 shows several bands, corresponding to partial degradation. The strongest band at 40 kD contains the gephyrin-binding motif. The NTRK and kinases are present in low amounts and barely appear on the gel. c Protein extracts from COS cells expressing Nlg1-WT, Nlg1-Y782A, with or without FGFR1, TrkB, or TrkC, were immunoprecipitated with Nlg1 antibodies. Some cells were pretreated with FGFR1 or pan-Trk inhibitors. pTyr and Nlg1 immunoblots are shown. d pTyr signals normalized to Nlg1 levels in the different conditions (number of experiments within bars). e , f Protein extracts from cortical cultures at DIV 10–14 pretreated with the kinase inhibitors for 24 h were immunoprecipitated with Nlg1, Nlg2, or Nlg3 antibodies. pTyr and Nlg1/2/3 immunoblots are shown, respectively. g Average pTyr signals for the different inhibitors, normalized to the control, untreated condition (number of experiments within bars). h CA1 neurons from Nlg1 KO slices were electroporated with Nlg1-WT or Nlg1-Y782A, and AMPAR-mediated EPSCs were recorded upon stimulation of Schaffer’s collaterals, in comparison to unelectroporated neighboring neurons (black traces). Organotypic cultures were treated or not with 1 µM GNF5837 for 7 days before the recordings. i Average AMPAR-mediated EPSCs amplitude in the 3 conditions, normalized to non-electroporated controls (number of pairs indicated within the bars, from 3 independent experiments). Data in graphs d and g were compared by one-way ANOVA followed by Bonferroni post hoc test (ns: not significant, * P < 0.05, ** P < 0.01, **** P < 0.001). Data in graph i were compared to the control condition by Wilcoxon matched-pairs signed rank test, and between themselves using one-way ANOVA followed by Tukey’s multiple comparison (ns: not significant, * P < 0.05, ** P < 0.01). Data represent mean ± SEM

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: In Vitro, Kinase Assay, Purification, Incubation, Western Blot, Binding Assay, Expressing, Immunoprecipitation

Journal: Nature Communications

Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

doi: 10.1038/s41467-018-06220-2

Figure Lengend Snippet: Working model for the role of Nlg1 tyrosine phosphorylation in synapse differentiation and potentiation. Phosphorylated Nlg1 (mimicked by Nlg1-Y782A) preferentially recruits PSD-95 and AMPARs and favors dendritic spines. In contrast, non-phosphorylated Nlg1 (mimicked by Nlg1-Y782F) diffuses out of dendritic spines and interacts preferentially with gephyrin, thus preventing PSD-95 and AMPAR recruitment and resulting in silent synapses. NMDARs are recruited independently of mutations in the Nlg1 intracellular domain, consistent with a direct extracellular coupling to Nlg1. A fraction of Nlg1-Y782F associates with gephyrin scaffolds and GABA A receptors to make inhibitory synapses

Article Snippet: Neurons expressing Homer1c-GFP + Nlg1-WT, Y782A, or Y782F were labeled using a pre-mix containing low concentrations of rabbit anti-GluA1 (Agrobio, clone G02141, 0.2 mg ml −1 , 1:2000) or rabbit anti-γ2 antibody (Alomone, AGA-005, 1:2000), and Atto594-conjugated anti-rabbit Fab (0.1 mg ml −1 , 1:2000) to label individual AMPA and GABA A receptors, respectively.

Techniques: