Journal: Neuron

Article Title: Regulation of Thalamic and Cortical Network Synchrony by Scn8a

doi: 10.1016/j.neuron.2017.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Monoclonal mouse anti-GluR2 , Neuromab , Cat# 75-002; RRID: AB_2232661.

Techniques: Plasmid Preparation, Virus, Recombinant, Avidin-Biotin Assay, Software, Imaging

Journal: Autophagy

Article Title: Table 1.

doi: 10.1080/15548627.2025.2597463

Figure Lengend Snippet: Autophagy-deficient PVALB-interneurons exhibit an altered synaptic composition, resulting in functional adaptations. (A) Representative images of surface GRIA2/GluA2 immunoreactivity (green) in hippocampal CA1 PVALB cells (magenta) from control ( Atg5 f/f ) vs. Pvalb-atg5 mice. Scale bar: 10 µm. (B) Quantification of the mean surface GRIA2/GluA2 intensity in control versus Pvalb-atg5 PVALB cells in hippocampal CA1. Results are expressed as a percentage of control values. Statistical analysis was performed using an unpaired, two-tailed Student’s t test (** p = 0.0028, t = 3.737, df = 12). Bars represent mean values ±SEM. N = 7 mice per genotype. (C) Representative images of total GRIA2/GluA2 immunoreactivity (green) in hippocampal CA1 PVALB cells (magenta) from control ( Atg5 f/f ) vs. Pvalb-atg5 mice. Scale bar: 10 µm. (D) Quantification of the mean total GRIA2/GluA2 intensity in control versus Pvalb-atg5 PVALB cells in hippocampal CA1. Results are expressed as a percentage of control values. Statistical analysis was performed using an unpaired, two-tailed Student’s t test ( ns p = 0.4154, t = 0.9076, df = 4). Bars represent mean values ±SEM. N = 3 mice per genotype. (E-I) Patch clamp recordings of AMPA (E) and NMDA (F) currents from CA1 PVALB cells from control ( Pvalb-TdT ) and Atg5 -deficient PVALB interneurons ( Pvalb-atg5-TdT ). These data were also plotted as the AMPA:NMDA ratio for controls and Pvalb-atg5-TdT animals, where all values were normalized to the control (G). (H) represents the average AMPA (black) and NMDA (gray) traces recorded at + 40 mV for control and Pvalb-atg5-TdT cells. (I) Reduced AMPA currents over NMDA currents in Pvalb-atg5-TdT mice when compared to controls. Black line indicates R 2 = 1. Statistical analyses were performed using a Student’s t test (for AMPA currents: * p = 0.0464, t = 2.100, df = 24; for NMDA currents, ns p = 0.3955, t = 0.8651, df = 24; for AMPA:NMDA ratios: * p = 0.0348, t = 2.238, df = 24). Bars represent mean values ± SEM. N = 3 animals per genotype ( Pvalb-TdT, n = 14; Pvalb-atg5-TdT , n = 16). (J) Current-clamp sample traces (400 pA injection, top) and the action potentials evoked by current steps (bottom) from PVALB neurons, from slices of control (Pvalb-TdT , RMP = −63 mV) and mutant Pvalb-atg5-TdT (RMP = −62 mV) mice. (K) Input-output plots from control (Pvalb-TdT) ( n = 14, N = 3) and Pvalb-atg5-TdT cells ( n = 16, N = 3). Statistical analysis was performed using a two-way repeated measures ANOVA followed by Sidak post hoc test; F(5,140) = 23.85 (**** p < 0.005). Bars represent mean values ± SEM. (L) Comparison of capacitance, resting membrane potential, action potential threshold and amplitude in control (Pvalb-TdT) ( n = 14, N = 3) and Pvalb-atg5-TdT cells ( n = 16, N = 3). Statistical analyses were performed using a Student’s t test (ap threshold, * p = 0.023, t = 2.398, df = 28). Bars represent mean values ± SEM. (M) Representative traces of sIPSC from slices of control (Pvalb-TdT) and mutant Pvalb-atg5-TdT mice recorded at + 5 mV. (N-O) Bar graphs and cumulative plots related tothe frequency and amplitude of sIpscs from control (Pvalb-TdT) ( n = 14, N = 3) and mutant Pvalb-atg5-TdT mice ( n = 16, N = 3). Statistical analysis was performed using an unpaired, two-tailed Student’s t test (* p = 0.0298, t = 2.289, df = 28 and ns p = 0.8654, t = 0.1710, df = 28, respectively). Bars represent mean values ± SEM. (P) Representative traces of paired-pulse ratio (IPSC 2 :IPSC 1 ) from three-month old control (Pvalb-TdT) and mutant Pvalb-atg5-TdT mice recorded at + 5 mV. (Q) Graph showing the paired-pulse ratio (IPSC 2 :IPSC 1 ) from control (Pvalb-TdT) ( n = 8, N = 2) and mutant Pvalb-atg5-TdT mice ( n = 12, N = 2). Statistical analysis was performed using an unpaired, two-tailed Student’s t test (* p = 0.0310, t = 2.340, df = 18). Bars represent mean values ± SEM. (R) Representative traces of excitation to inhibition ratio (EPSC −60 mV :IPSC +5 mV ) from control (Pvalb-TdT) and mutant Pvalb-atg5-TdT mice. (S) Graph showing the excitation to inhibition ratio (EPSC:IPSC) from control (Pvalb-TdT) ( n = 8, N = 2) and mutant Pvalb-atg5-TdT ( n = 14, N = 3). Statistical analysis was performed using an unpaired, two-tailed Student’s t test (* p = 0.0351, t = 2.261, df = 20). Bars represent mean values ± SEM.

Article Snippet: GRIA2/GluA2 (extracellular) , Guinea pig , Alomone labs , AGC-005-GP , 1:500 (IHC).

Techniques: Functional Assay, Control, Two Tailed Test, Patch Clamp, Injection, Mutagenesis, Comparison, Membrane, Inhibition

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: Characterization of differentiated PC12 cells. ( a ) Photomicrograph depicting PC12 cells after 4 days of NGF treatment and ( b ) immunofluorescence staining of microtubule-associated protein 2 (MAP2) and β-III tubulin. Scale bars: 20 µm. ( c, d ) Representative recording of a whole-cell current-clamp step protocol obtained from PC12 neurons. ( c ) Application of increasing current steps in PC12 neuronal culture resulted in action potential generation. ( d ) Trace representing membrane potential changes in PC12 neurons. ( e, f ) Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR and mGluR1 signal. The arrows indicate single-step photobleaching. ( g, h ) Histogram showing the intensity value of every spot found in a recording superimposed with a single fitted Gaussian curve (blue line).

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Immunofluorescence, Staining, Membrane, Labeling, Patch Clamp

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: Single-molecule imaging of ATTO 488-labeled GluR2-AMPAR and mGluR1 receptors on neurites and on somas of PC12 neurons. ( a ) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar: 2 µm. ( b ) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bars: 0.1 µm. The cumulative probability functions of D values of AMPAR ( c ) and mGluR1 ( d ) on neurites and on somas are shown (n= 330-500 trajectories). **p<0.01, ***p<0.001

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Imaging, Labeling

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: Effect of E2 on the surface trafficking of GluR2-AMPAR and mGluR1. The line graphs depict changes in the diffusion coefficient (D) of GluR2-AMPAR ( a ) and mGluR1 ( b ) molecules after vehicle or 100 pM E2 administration at different time points (mean ± SEM, n=100-128 trajectories per time point). ( c ) Distributions of D (as the percentage of vehicle control) of GluR2-AMPAR (average of 20-minute measurement) in the medium (dRPMI) or after administration of E2 at different concentrations (as the percent of the vehicle treatment expressed as the median ± 25-75%, n= 426-484 trajectories per group). ( d ) Distributions of D values of AMPAR on neurites in the presence of the estrogen receptor β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1) and a GPER1 antagonist (G15) (expressed as the median ± 25-75%, n=427-468 trajectories). ( e ) Stochastic optical reconstruction microscopy images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on PC12 neurons. The dashed lines delineate the border between the neurite and soma. ( f ) Distributions of the minimal distance (greater than 500 nm) between GluR2-AMPAR and GPER1 after vehicle or E2 treatment (expressed as the median ± 25-75%, n=203-657). Scale bar: 2 µm; insert scale bar: 0.5 µm. **p<0.01, ***p<0.001

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Diffusion-based Assay, Control, Microscopy, Immunolabeling, Immunofluorescence

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: Effect of estrogen receptor modulation on surface trafficking of AMPAR. ( a ) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor mRNA (with a lack of expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and G protein-coupled estrogen receptor 1 (GPER1)) in PC12 neurons. ( b ) Effect of E2 on the D value of mGluR1 (as the percent of vehicle treatment expressed as the median ± 25-75%, n= 840-860 trajectories per group). ( c ) Table showing the effect of ERβ agonist (DPN), GPER1 agonist (G1) and GPER1 antagonist (G15) treatment on D (µm 2 /s) values of GluR2-AMPAR molecules (expressed as the median ± 25-75% (in brackets), n=411-525 trajectories). ( d ) Table of the distributions of each minimal distance interaction type, including indirect (50-100 nm), microdomain (100-500 nm) and remote (500< nm), between GluR2-AMPAR and GPER1 on PC12 neurons after vehicle or E2 treatment (the minimal distance is expressed as the median ± 25-75% (in brackets)).

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Gel Electrophoresis, Expressing, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: The role of the cortical actin network in the rapid effect of E2. ( a ) The actin polymerization blocker latrunculin A (latA) decreased the dynamics of the cortical actin network in CHO cells (as % of control: 51 ± 11) (for the methods of analysis of cortical actin network dynamics, see Supplementary Methods). Neither 100 nM nor 100 pM E2 affected actin dynamics (as % of control: 100 pM E2 = 108 ± 30; 100 nM E2 = 93 ± 14) (n=5-6 cells per group) ( b ) Confocal images depicting the Alexa Fluor 488 phalloidin-labeled cortical actin network in PC12 neurons after vehicle or 10-minute 1 µM latA treatment in PC12 neurons. The bar graph shows the decreased integrated density of the cortical actin network after latA exposure (right, n=3 cells per group (3 regions of interest per cell)). Scale bar: 5 µm. ( c ). LatA treatment increased the diffusion coefficient (D) of GluR2-AMPA molecules on somas with no effect on the molecules on neurites (expressed as the median ± 25-75%, n = 307-507 trajectories). ( d ) On somas, 100 pM E2 decreased the D values of GluR2-AMPAR molecules. This effect was blocked in the presence of latA. Ineffectiveness of E2 action on neurite was not affected by latA treatment (expressed as the median ± 25-75%, n = 119-205 trajectories). **p<0.01, ***p<0.001

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Control, Analysis, Confocal, Labeling, Diffusion-based Assay

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Expression of KIF5A, GluR2 and beta 2+3 subunits of gamma aminobutyric acid receptors (Gabrb2+3) in an in vivo model of seizures. (A) EEG results: There was no epileptic discharge in the Ctl group after the injection of saline; however, epileptic discharge was observed in the Sez group after the injection of PTZ. (B) Total protein expression: the gray level of the total protein expression bands was normalized with GAPDH, and the total protein expression levels of KIF5A, GluR2 and Gabrb2+3 in the hippocampus of the Sez group did not change significantly (n=6 in each group, vs. Ctl, P>0.05). (C) Surface protein expression: The gray level of the total protein expression bands was normalized with Sodium/potassium-transporting ATPase subunit alpha-1 (ATP1A1). In the Sez group, the expression of GluR2 on the surface increased significantly to 181.74%±14.44% ( vs. Ctl, # , P<0.01). Conversely, the expression of GluR2 on the surface decreased to 19.62%±8.01% ( vs. Ctl, # , P<0.01). KIF5A, kinesin superfamily proteins 5A; GluR2, glutamate receptors subunit-2; Ctl, control; Sez, seizures; PTZ, pentylenetetrazol.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Expressing, In Vivo, Injection, Saline, Control

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Receptor recycling assay (IF). The recycling ratio of GluR2 was 0.30±0.05 in the Ctl group and 0.60±0.07 in the Mg 2+ -free group (7 cells per group, vs. Ctl, # , P<0.01), and the recycling ratio of Gabrb2+3 was 0.49±0.04 in Ctl group and 0.32±0.05 in the Mg 2+ -free group (7 cells per group, vs. Ctl, # , P<0.01) (×600). Scale Bar: 50 µm. IF, immunofluorescence; Ctl, control.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Immunofluorescence, Control

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Interaction between KIF5A and GluR2 and Gabrb2+3 in the seizure model. (A) Co-ip results: The protein bands showed the co-ip levels of KIF5A, GluR2, and Gabrb2+3, and normalized with the protein levels of KIF5A. The GluR2 level of KIF5A pull-down in the hippocampi of the rats in the Sez group increased to 130.42%±53.24% (n=6 per, vs. Ctl, *, P<0.05). However, the Gabrb2+3 level of KIF5A decreased to 50.86%±5.33% in the Sez group (n=6 per group, vs. Ctl, # , P<0.01). (B) IF results: the Pearson’s correlation coefficients (PCC) of KIF5A/GluR2 was 0.40±0.19 in the Ctl group and 0.87±0.11 in the Mg 2+ -free solution group (n=6 per group, vs. Ctl, # , P<0.01) (×400). Scale Bar: 100 µm. (C) IF results: the PCC of KIF5A/Gabrb2+3 was 0.97±0.02 in the Ctl group and 0.32±0.11 in the Mg 2+ -free solution group (n=6 per group, vs. Ctl, # , P<0.01) (×400). Scale Bar: 100 µm. IF, immunofluorescence.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence

Journal: Scientific Reports

Article Title: New Alzheimer’s disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid β oligomers

doi: 10.1038/s41598-019-53415-8

Figure Lengend Snippet: Immunoblot analysis of expression ratios of the pre and postsynaptic proteins in Aβ-GFP Tg and non-Tg mice synapse. ( A,B ) Synaptosome fractions from hippocampus (20 μg/lane) were immunoblotted against presynaptic markers ( A ; synaptophysin, VAMP-2, syntaxin-1, and Munc13–1) and postsynaptic markers ( B ; PSD-95, GluN1, GluN2A, GluN2B, GluA2, and neuroligin). We prepared 4 sets of Aβ-GFP Tg/non-Tg samples. Band intensities of Aβ-GFP Tg mice were normalized by those of actin and the expression were calculated ratios with the values of non-Tg mice as 1. There was no difference in expression ratios of the presynaptic proteins we examined. However, expression of the postsynaptic proteins GluN2B, Glu2A, and neuroligin were significantly decreased in Aβ-GFP Tg mice at 3-month-old (*p < 0.05 by Mann-Whitney’s U test, n = 4 that included hippocampi from 3 animals each, data are presented as means ± SEM). ( C ) The expression ratios of GluN2B in homogenate. There was no significant difference in the expression ratios of GluN2B in homogenates of Aβ-GFP Tg and non-Tg mice (Mann-Whitney’s U test, n = 5 animals each, data are presented as means ± SEM).

Article Snippet: VAMP-2 (1:5000, Abcam, Cambridge, UK), Munc13-1(1:1000, Synaptic Systems, Gottingen, Germany), syntaxin-1 (1:1000, Merck), synaptophysin (1:2000, Sigma-Aldrich), PSD-95 (1:200, Merck), GluN1 (1:200, Merck), GluN2A (1:500, Merck), GluN2B (1:200, Merck), GluA2 (1:200, Merck), neuroligin 1(1:500, Synaptic Systems), and actin (1:2000, Merck) antibodies were used to probe the corresponding proteins and ECL was used for detection (Immunostar Zeta and Immunostar LD, FUJIFILM Wako Pure Chemical Corporation).

Techniques: Western Blot, Expressing

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: GluA1 monomers form metastable homooligomers with exponential lifetimes of <200 ms, and dissociate into monomers. Typical single-molecule fluorescence image sequences of two, three, and four diffusing ACP-GluA1 molecules undergoing transient dimerization, trimerization, and tetramerization in the HEK293-cell PM (from 236, 54, and 90 independent movies; a , b , and c , respectively). In the image sequence in a , two ACP-GluA1 molecules (green and magenta arrowheads) became colocalized in frame 23 (33 ms per frame, normal video rate) and diffused together for the next eight frames (for a 0.30-s total colocalization time; light blue arrowheads), until they became separated and diffused independently (blue and purple arrowheads). Colors of the subtrajectories are the same as those for the arrowheads (the circle and arrow show the locations at the start and at the time in the frame number on top, respectively). Since these experiments were conducted with a single color (ATTO594), after the dimerization event, which molecule corresponds to each one before dimer formation could not be determined (hence, different colors in the trajectory). b , c The spots with the intensities of monomers, dimers, trimers, and tetramers are indicated by magenta, blue, green, and orange arrowheads. d The distributions of the homodimer durations for ACP-GluA1, ACP-GluA1ΔNTD, and ACP-TM, obtained by measuring the durations of all of the observed homo-dimerization events. The distribution for ACP-TM was obtained at 22 °C, a temperature at which ACP-TM’s diffusion coefficient becomes practically the same as those of ACP-GluA1 and ACP-GluA1ΔNTD observed at 37 °C. Each histogram could be fitted well with a single exponential decay function (dotted curves), providing the dimer lifetime (given in parentheses after correction for the photobleaching lifetime of the fluorescent probe). Related data for Halo7-GluA1 and Halo7-TM are shown in Supplementary Fig. . e The distributions of the homotetramer durations of ACP-GluA1 and ACP-GluA1ΔNTD. Each histogram could be fitted well with a single exponential decay function (solid curves), providing the tetramer lifetime (dotted curves for GluA1 homodimers). For statistical parameters, see Supplementary Table . Related GluA2 results are shown in Supplementary Fig.

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques: Fluorescence, Sequencing, Diffusion-based Assay

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: Heteromer lifetimes are longer than those of homomers. Typical single fluorescent-molecule image sequences, showing that ACP-GluA1 (magenta spots) and Halo7-GluA2 (green spots) form transient heterodimers ( a ), heterotrimers ( b ), and heterotetramers ( c ) in the HEK293-PM (typical results from 128 or more independent movies, respectively). Magenta and green arrowheads indicate ACP-GluA1 and Halo7-GluA2 monomers, respectively, cyan arrowheads indicate heterodimers, yellow arrowheads show heterotrimers ( b, c ), and orange arrowheads indicate heterotetramers ( c ). d The distribution of the durations of GluA1–GluA2-heterodimers (magenta), shown together with those of homodimers of GluA1–GluA1, GluA2–GluA2, and TM–TM, obtained by simultaneous two-color imaging using ACP- and Halo7-tags. Here, the colocalizations of a protein of interest using different tag proteins (ACP and Halo7 proteins) are called homo (rather than hetero)-dimers, trimers, and tetramers because ACP and Halo7 by themselves did not exhibit any sign of homo and hetero interactions , and in two-color experiments, only homodimers of different colors were included in the colocaization duration histograms. Each histogram could be fitted as those in Fig. . Since virtually the same lifetimes were obtained from the histograms for GluA1–GluA2 heterodimers (and heterotetramers in e ) when the tag proteins were exchanged (Supplementary Fig. ), the two histograms were averaged in this figure (and also in e ). To avoid excessive complexity of the figure, only the best-fit functions are shown without histograms for the homodimers of GluA1–GluA1 and GluA2–GluA2 and for the incidental colocalization of ACP-TM and Halo7-TM (see Supplementary Fig. for the histograms). The homodimer lifetimes obtained by two-color experiments were virtually the same as those obtained by single-color experiments (Fig. ; Supplementary Figs. and ; Table ). e The distribution of the durations of heterotetramers, including 1:3, 2:2, and 3:1 GluA1/A2 heterotetramers (any heterotetramers detected with two colors), shown together with those of the homotetramers of GluA1 and GluA2. Note that the distributions for the homotetramers were obtained by single-color experiments. For the duration distributions of homotetramers of GluA1 and GluA2, see Fig. and Supplementary Fig. . For statistical parameters, see Supplementary Table

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques: Imaging

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: TARP2 dynamically forms metastable complexes with GluA1. a The distributions of homodimer durations of ACP-GluA1 in the HEK293-PM with (magenta) and without (gray, see Fig. ) TARP2-mGFP overexpression (expression levels 47 (±30) times higher than those of ACP-GluA1; n = 13 cells). b Typical single fluorescent-molecule image sequences, showing that a TARP2-mGFP monomer (green arrowheads) formed a transient heteromer with an ACP-GluA1 monomer (magenta arrowheads) in the HEK293-PM (a typical result from 358 independent movies). Cyan arrowheads indicate the heteromer. c The duration distributions of the heteromers of TARP2-mGFP with ACP-GluA1 monomers (left) and dimers (right) in the HEK293-PM. The histograms could be fitted well with single exponential decay functions, providing the heteromer lifetimes, which are shown in parentheses (after correction for the probe photobleaching lifetimes). Related ACP-GluA2 results are shown in Supplementary Fig. . For statistical parameters, see Supplementary Table

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques: Over Expression, Expressing

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: GluA1 existed as monomers, homodimers, and homotrimers as well as homotetramers when expressed in the HEK293-PM. Note that most of the data about GluA2 are shown in the Supplementary Information. a Schematic figure showing the structures of ACP-GluA1, ACP-GluA1ΔNTD, ACP-GluA2, and ACP-TM. LBD represents the ligand-binding domain. Representative snapshot images and distributions (histograms) of the signal intensities of individual fluorescent spots of ATTO594-labeled ACP-TM ( b ) and ACP-GluA1 ( c ) expressed in HEK293-PMs. Distributions of the signal intensities of individual fluorescent spots (histograms) were obtained at various expression levels (number densities). The numbers of examined fluorescent spots: 779, 1729, and 2192 spots for ACP-TM (from left to right in b and 4305, 7730, and 7107 spots for ACP-GluA1 (from left to right in c ). Each distribution was fitted with the sum of two (for ACP-TM, b ) or four (for ACP-GluA1, c ) lognormal functions, representing the fractions of monomers (magenta), homodimers (blue), homotrimers (green), and homotetramers (orange). Numbers in the figure indicate molecular fractions. Magenta arrowheads and yellow arrows in the images indicate spots with fluorescence intensities of <3.6 and >3.6 arbitrary units (A.U.). This threshold intensity was determined as that at which the ratio of the copy numbers of ACP-TM with higher vs. lower intensities in the experimental histogram became the same as the ratio of apparent dimers vs. monomers determined by the lognormal fitting. Related ACP-GluA2 results are shown in Supplementary Fig.

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques: Ligand Binding Assay, Labeling, Expressing, Fluorescence

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: With an increase of the GluA1 expression level, the monomer fraction is decreased whereas the homotetramer fraction is increased. a The fractions of ACP-GluA1 molecules that exist as monomers, homodimers, homotrimers, and homotetramers (magenta, blue, green, and orange circles and lines, respectively, which are the same for all of the panels) are plotted as a function of the number density of ACP-GluA1 molecules expressed in the HEK293-PM. Curves are to help the eye (curve fitting with quadratic functions). Error bars in all panels represent standard errors. The numbers of independent experiments conducted to obtain the results shown in this figure are summarized in Supplementary Tables and . Related ACP-GluA2 results and Halo7-GluA1 data are shown in Supplementary Figs. and , respectively. b The same as a , but for ACP-GluA1ΔNTD. c The effects of the agonists (0.1 mM AMPA [+AMPA, n = 5 cells] and 10 mM l -glutamate [+Glu, n = 5 cells]; No addition, n = 7 cells) on the fractions of ACP-GluA1 molecules that exist as monomers, homodimers, homotrimers, and homotetramers (ACP-GluA1 expressed at 0.50 ± 0.09 copies μm −2 ). Related ACP-GluA2 results are shown in Supplementary Fig. . d Tetramer number densities of ACP-GluA1, ACP-GluA2, Halo7-GluA1, and ACP-GluA1ΔNTD, plotted as a function of the number density of respective molecules expressed in the HEK293-PM. Curves are to help the eye (curve fitting with quadratic functions). The ACP-GluA2 data shown here are based on the data shown in Supplementary Fig.

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques: Expressing

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: Summary of colocalization durations of fluorescently-labeled, tagged molecules of GluA1, GluA1ΔNTD, GluA2, and TM in the HEK293-PM

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques:

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: Transient GluA1 homotetramers form Ca 2+ channels. a Typical time series of images of the Ca 2+ -sensitive dye Fluo-8 incorporated in the cytoplasm of HEK293 cells after l -glutamate stimulation (green images), with the images of ATTO594-labeled Halo7-GluA1 and ACP-GluA1ΔNTD expressed in the same cells (magenta images). (Top row) Two cells expressing Halo7-GluA1 at high (1.10 copies μm −2 ) and low (0.33 copies μm −2 ) number densities exhibited large or virtually no Ca 2+ responses, respectively (typical results from 36 independent time series). (Bottom row) A cell expressing ACP-GluA1ΔNTD at a high number density (0.92 copies μm −2 ), exhibiting no changes in the cytoplasmic Ca 2+ -concentration (among 38 independent time series). Since we were unable to express ACP-GluA1 at number densities of 1.10 copies μm −2 or higher, for these experiments, we employed Halo7-GluA1, which could be expressed up to 2.44 copies μm −2 . Dashed lines indicate the cell peripheries. b Time courses of the %increases in the Fluo-8 fluorescence intensity ([ F - F 0 ] F 0 −1 , where F and F 0 represent the fluorescence intensities in the cytoplasm after background subtraction, measured at times t and 0, respectively) after stimulation (time 0, vertical dotted line). The values in the graph represent the number densities of molecules expressed in the PM (copies μm −2 ). (left) The time courses for the cells on the top (orange) and bottom (cyan) shown in a . (middle) The time course for cells expressing Halo7-GluA2 at a number density of 1.18 copies μm −2 (among 3 independent time series). (right) The time course of the cells expressing ACP-GluA1ΔNTD, shown in the bottom row in a . c [( F - F 0 ) F 0 −1 ] Max (see the graph in b ; mean ± SEM) plotted as a function of the number density of Halo7-GluA1 and ACP-GluA1ΔNTD expressed in the PM (graph with a bin size; 0.333 copies μm −2 ). Curves are to help the eye (fitting with quadratic functions). For the numbers of observed cells, see the circles (Supplementary Table ). d [( F - F 0 ) F 0 −1 ] Max plotted as a function of the number density of homotetramers in the PM for Halo7-GluA1 and ACP-GluA1ΔNTD. Each graph could be fitted with a linear function

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques: Labeling, Expressing, Concentration Assay, Fluorescence

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: Many GluA1 molecules exist as monomers and undergo intermittent transient dimerization/oligomerization in the dendritic-shaft PM. a Representative image sequence of two diffusing Halo7-GluA1 fluorescent spots (labeled with ATTO594; magenta arrowheads) in a dendritic-shaft PM (13 DIV), exhibiting transient colocalization–codiffusion (cyan arrowheads; lasting for 132 ms) (representative results from 205 independent video clips). b The distributions of the colocalization durations of Halo7-GluA1 and Cy3-DOPE ( n = 205 and 19 events, respectively) in the dendritic-shaft PM. Each histogram could be fitted as those in Fig. . c The distributions of the diffusion coefficients ( D 200ms ) of ACP-GluA1 monomers, homodimers, and homotetramers and ACP-GluA1ΔNTD monomers in the HEK293-PM (left), those of Halo7-GluA1 and Halo7-GluA1ΔNTD in the dendritic-shaft PM (middle), and those of presumed GluA1 monomers, dimers, and tetramers in the dendritic-shaft PM (right; Supplementary Fig. ). For the breaks at a D 200ms of 0.0016 μm 2 s −1 , see the caption to Supplementary Fig. . Bars, circles, boxes, and whiskers indicate the median values, mean values, interquartile range (25–75%), and 10–90% range, respectively. For whiskers exhibiting diffusion coefficients smaller than 0.0016 μm 2 s −1 , the 10% values are shown in parentheses. Asterisks and n.s. indicate p < and > 0.05, respectively, using the Brunner–Munzel test ( p values: n.s. 1, 0.23; n.s. 2, 0.23; n.s. 3, 0.54; *1, 6.7 × 10 −12 ; *2, <2.2 × 10 −16 ; *3, 1.6 × 10 −3 ; *4, 8.6 × 10 −14 ; *5, 2.9 × 10 −12 ; *6, 1.7 × 10 −3 ; *7, 6.2 × 10 −13 ; *8, 4.4 × 10 −7 ). d The ensemble-averaged MSD plotted against time Δ t (see the caption to Supplementary Fig. ). The plot is linear showing that Halo7-GluA1 underwent simple-Brownian diffusion in the dendritic-shaft PM at a mean diffusion rate between those of hypothetical ACP-GluA1 monomers and dimers (left). Meanwhile it exhibited a saturation, indicating confined diffusion within a confinement domain of 109 nm in the Homer1b-EGFP region (right; see the caption to Supplementary Fig. ). For the statistical parameters, see Supplementary Table . Related GluA2 data are shown in Supplementary Fig.

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques: Sequencing, Labeling, Diffusion-based Assay

Journal: Nature Communications

Article Title: AMPA receptors in the synapse turnover by monomer diffusion

doi: 10.1038/s41467-019-13229-8

Figure Lengend Snippet: AMPAR subunits diffuse as monomers and dimers between the dendritic shaft and the synaptic region. Schematic figure showing that the AMPAR subunits GluA1 and GluA2 predominantly exist as monomers, and occasionally form transient dimers (and less frequently transient trimers and tetramers) in their reserve pools in the dendritic-shaft PM, where the subunit concentrations are low. Monomers and dimers diffuse quite freely in the dendritic-shaft PM, and enter the spines. In the synaptic regions in the spines, where the AMPAR subunit concentrations are high due to their binding to (plus their transient associations with) scaffolding protein complexes, including PSD95, GRIP, and SAP97 , by way of TARP2, tetramers would be the predominant species, which work as l -glutamate-evoked channels. However, the tetramer lifetimes are quite short (a lifetime of 208 ms for the heterotetramers of GluA1 and GluA2, which could be prolonged due to their binding to scaffolding protein complexes). Namely, even in the synaptic regions, tetramers become dissociated readily, whereas monomers turn into tetramers extremely quickly. In this manner, AMPARs with different compositions could be formed readily. Meanwhile, the AMPAR subunit monomers can exit from the synaptic regions quickly, further enhancing the AMPAR composition changes when they are required. This would be useful after LTP induction. As such, the associations and dissociations of AMPAR subunits would play critical roles in regulating the AMPAR functions in the synaptic membrane

Article Snippet: The proteins attached to the streptavidin agarose resin were analyzed by western blotting using anti GluA1 or anti GluA2 polyclonal antibodies (Enzo Life Sciences) .

Techniques: Binding Assay, Scaffolding