Journal: bioRxiv

Article Title: Expansion microscopy at one nanometer resolution

doi: 10.1101/2022.08.03.502284

Figure Lengend Snippet: a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .

Article Snippet: The primary antibodies used were anti synaptotagmin1 (SYT1, #105011 Synaptic Systems), anti Homer1 (#160 003, Synpatic Systems), anti Shank2 (#162204 Synaptic Systems), anti GluR2 (Alomone Labs, #AGC-005, Jerusalem, Israel), anti GluN2b (Neuromab 75-101, California, USA), anti MAP2 (Novus Biologicals #NB300-213), anti vGluT1 (#135304, Synaptic Systems), anti Bassoon (#ADI-VAM-PS003-F, Enzo, New York, USA).

Techniques: Labeling, MANN-WHITNEY, Immunostaining

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: 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: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: ( A - C ) Surface biotinylation was used to isolate endogenous surface (s) GluA1 and sGluA2 in acute striatal slices derived from wildtype (WT) and Lrrk2 G2019S (GS) mice. A ) Representative Western blot images of sGluA1, sGluA2, and actin from dataset quantified in B and C . Scatterplot/bar graphs plot values normalized to WT ± SEM (n = 7 - 8 mice/genotype, 3 slices/mouse). Unpaired t test *p= 0.0492, compared to WT. D ) Confocal image of DARPP32-labeled (white) co-cultured SPN (left) and super-resolution, STED images (right) of dendritic processes. Punctate sGluA1 (magenta), sGluA2 (green) labeling (tagged prior to permeabilization) associates largely, but not completed with PSD95 labeling (blue). E and F ) Violin plots compare surface AMPAR area ( E ) and intensity ( F ) within masks defined by PSD95 labeling in SPNs. Unpaired t test, ****p<0.0001; ***p<0.0001; Mann Whitney test *p=0.04. n= 3 preps and 15 ROIs/genotype.

Article Snippet: After blocking, coverslips were incubated with primary antibodies targeting surface epitopes (GluA1;1:500, Alomone AGP-009, RRID:AB_2340961 or GluA2; 1:500, Alomone AGC-005-GP, RRID:AB_2756617) at 4°C overnight.

Techniques: Derivative Assay, Western Blot, Labeling, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: A ) Schematic outlines antibody feeding assay used to monitor GluA1 internalization in wildtype (WT) and Lrrk2 G2019S (GS) corticostriatal co-cultures (DIV16-18) and serves as a key for the colors used to show data. B and C ) Overlay images show labeled surface (s) GluA1 (green mask + red mask) and internalized (i) GluA1 (red mask only) signal contained within DARPP-32 labeled SPNs (shown at a reduced intensity to permit visualization of puncta. Masks were generated in Image J and magnification is shown in B. D - G ) Intensity distribution of green and red labeling along a 60 µm line scan. H ) Quantification of the internalization index of GluA1 receptors in WT and GS SPNs at 0 and 60 min (n = 12 - 16 cells, 3 preps/genotype). Two-way ANOVA (F ( , ) = 9.809, p = 0.0029), post hoc Šidák test **p = 0.0049.

Article Snippet: After blocking, coverslips were incubated with primary antibodies targeting surface epitopes (GluA1;1:500, Alomone AGP-009, RRID:AB_2340961 or GluA2; 1:500, Alomone AGC-005-GP, RRID:AB_2756617) at 4°C overnight.

Techniques: Feeding Assay, Labeling, Generated

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: A, C ) Bar graph/scatterplots comparing functional contribution of CP-AMPARs using ratios of EPSCs evoked in the presence/absence of NASPM in D 1 R ( A; Drd1tdTom+ ) and D 2 R ( C; Drd1tdTom- ) SPNs in WT and GS mice (P70 - P90) in acute slices through dorsal striatum. Bars are mean ± SEM (n = 11 cells, 5 - 6 mice/group, unpaired t test *p = 0.0122). Example traces ( B ) show AMPAR currents before (black) and after (lavender) bath application of NASPM (200µM, 10 min). D - G , Superresolution (tau-STED) images ( D, E ) and quantification ( F, G ) of synaptic and extrasynaptic receptors in 21 DIV WT and GS D 1 R SPNs expressing tdTomato (white, Drd1 Cre/+; Ai14 , in D, E, and used to segment D1R SPNs for F, G) and co-cultured with unlabeled cortical neurons of the same genotype. sGluA1 ( D , magenta, STED) and sGluA2 ( E , green, STED) puncta in relation to PSD95 labeled postsynaptic sites (blue, confocal). Circled zones are enlarged in the center panels. F, G ) Bar graph/scatterplots show synaptic ( F , within a mask defined by PSD95) and extrasynaptic ( G , outside a PSD95 mask) sGluA1 (lavender) and sGluA2 (green) nanoclusters in D 1 R SPNs. F : Unpaired t test **p = 0.0369; n = 16 ROIs/genotype). G : Unpaired t test ****p<0.0001; n = 16 ROIs/genotype. H - K ) Examples ( H ) and quantification ( I - K ) of FRAP experiments. (H) Time lapse confocal images pre- and postphotobleaching (dotted circles approximate ROIs) in WT and GS D 1 R SPNs labeled and co-cultured as above. Table ( I ) compares time constant, diffusion (D) and % mobile receptors. D was calculated using: D = 0.25 (r 2 /τ 1/2 ), where r refers to the bleach radius and τ 1/2 to the time constant 46 . Graph ( J ) plots normalized SEP-GluA1 fluorescence recov-ery in WT and GS neurons imaged every 2.5 s. Lighter shading is ± SEM. Two-way RM-ANOVA (F(89, 3115) = 17.79, **p < 0.001, n = 19-20 spines/genotype. Scatterplot ( K ) compares recovery of SEP-GluA1 intensity at T222.5 s time point relative to post-bleach in WT and GS D 1 R SPNs. One-way ANOVA (F(3, 70) = 24.15, p < 0.0001), post hoc Tukey’s multiple comparison test ****p<0.0001.

Article Snippet: After blocking, coverslips were incubated with primary antibodies targeting surface epitopes (GluA1;1:500, Alomone AGP-009, RRID:AB_2340961 or GluA2; 1:500, Alomone AGC-005-GP, RRID:AB_2756617) at 4°C overnight.

Techniques: Functional Assay, Expressing, Cell Culture, Labeling, Diffusion-based Assay, Fluorescence, Comparison

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