Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Diffusion-based Assay, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of estrogen receptor modulation on the surface movement of GluR2-AMPAR. (A) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor beta (ERβ) and G protein-coupled estrogen receptor 1 (GPER1) mRNA in dPC12. Estrogen receptor alpha (ERα) mRNA was not detected. (B) Histograms demonstrate the mean D AMPAR as a percentage of vehicle control on somas and neurites in the presence of the estrogen receptor, β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1), G1+DPN together, a GPER1 antagonist (G15) and G15+E2 (with 100 pM of E2 on the somas and 100 nM of E2 on the neurites) (mean ± SEM; n = 215–641 trajectories). *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Nucleic Acid Electrophoresis, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 μm; inset Scale bar = 0.5 μm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas ( n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 μm. (C2) One 2 μm 2 (between parallel white bars) and one 10 μm 2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 μm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 μm wide). Scale bar = 0.5 μm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm ( n = 15 cells were evaluated in each group). * p < 0.05.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Immunolabeling, Microscopy, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: The role of the cortical actin in the rapid effect of E2. (A) Left, confocal images depict Alexa Fluor 488 phalloidin-labeled cortical actin network in dPC12 after treatment with vehicle, 1 μM of latA, 1 μM of SP600125 or 1 μM of GSK429286. Scale bar = 5 μm; insert Scale bar = 0.5 μm. Right, the bar graph shows the effect of LatA, GSK429286, and SP600125 on the integrated density of the fluorescently labeled cortical actin network [ n = 3 cells per group (3 ROIs per cell)]. (B1,B2) Effect of LatA, GSK429286, and SP600125 treatment on D AMPAR (% of vehicle treatment as the mean ± SEM; n = 215–544 trajectories). (C1,C2) Effect of 100 pM of E2 on somas and 100 nM of E2 on neurites with or without LatA, GSK429286, and SP600125 (% of vehicle treatment as the mean ± SEM; n = 184–277 trajectories). *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPA on primary hippocampal neurons. (A) Photomicrograph shows a primary hippocampal neuron labeled with homer-1 (synapse) and β-III tubulin (neuron). Scale bar = 10 μm, insert Scale bar = 2 μm. (B) Dual color STED image of a hippocampal neuron overlayed to differential interference contrast microscopy image depicts live-cell synapse labeling MitoTracker Deep Red (red) and presynaptic protein bassoon (green). Scale bar = 1 μm. (C) Distribution of D values of extrasynaptic and synaptic GluR2-AMPAR under control conditions (median ± IQR, n = 754 extrasynaptic trajectories and n = 104 synaptic trajectories). (D) Effect of E2 (100 pM and 100 nM) on D of extrasynaptic and synaptic GluR2-AMPA with or without chemical LTP (cLTP) induced by glycine/picrotoxin (gly/pic) (% of vehicle treatment as the mean ± SEM; n = 742–928 extrasynaptic trajectories and n = 104–155 synaptic trajectories). (E,F) Effect of vehicle, E2 (100 n, 100 pM) with or without cLTP (gly/pic) on synaptic dwell time (mean ± SEM (s); n = 104–155) (E) and relative surface distribution of synaptic GluR2-AMPAR content (synaptic/total GluR2-AMPA molecule trajectories) (mean ± SEM, n = 8–18 recordings) (F) . * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Labeling, Microscopy