Journal: eLife

Article Title: The tetraspanin TSPAN5 regulates AMPAR exocytosis by interacting with the AP4 complex

doi: 10.7554/eLife.76425

Figure Lengend Snippet: ( A ) Schematic of the FRAP-FLIP experiment presented in panel B . SEP-GluA2 in pre-bleaching condition is either fluorescent (green) if exposed to the extracellular media or quenched (light green) if in intracellular compartments. A region of the dendrite is bleached (black box). SEP-GluA2 that was fluorescent (and so extracellularly exposed) at the time of bleaching becomes bleached (light blue). Quenched SEP-GluA2 is not affected by the bleaching. During imaging, the ROI flanking regions are continuously bleached (black lateral boxes), thus lateral diffusing SEP-GluA2 will be bleached. Receptors that have been internalised and directed for recycling are exocytosed and become fluorescent. Newly synthesised receptors would not be present due to the application of cycloheximide (CHX) (crossed out receptors). Controls for pH sensitivity of the SEP signal are shown in . ( B ) Live confocal images of individual dendritic spines from DIV20 cultured rat hippocampal neurons transfected at DIV12 with SEP-GluA2 and either scrambled, Sh-TSPAN5, or rescue construct co-expressing mCherry. Neurons were treated for 2 hr with 200 μg/ml of cycloheximide to inhibit protein synthesis and then imaged under a FRAP-FLIP protocol for 5 min to isolate the recycling receptors. mCherry (magenta) and SEP-GluA2 (white) images (time points: prebleach, postbleach, 10, 20, 30, 60, 120, 180, 240, and 300 s) are shown. The dendritic spine mask is depicted with white dashed line. Scale bar =1 µm. ( C ) Quantification of the ΔF/F pre for SEP-GluA2 over time for scrambled-, Sh-TSPAN5-, and rescue-transfected neurons. ( D ) Quantification of the area under the curve relative to panel B (area under the curve [A.U.]: scrambled 15.56±0.74, Sh-TSPAN5 11.99±2.51, rescue 11.77±1.31). n = scrambled, 56; Sh-TSPAN5, 53; rescue, 53 dendritic spines. One Way ANOVA, Newman-Kulspost hoc multiple comparison test.( E ) Schematic of the FRAP-FLIP experiment presented in panel F. SEP-GluA2 at basal condition is either fluorescent (green) if exposed to the extracellular media or quenched (light green) if in intracellular compartments. A region of the dendrite is bleached (black box). SEP-GluA2 that was fluorescent (and so extracellularly exposed) at the time of bleaching becomes bleached (light blue). Quenched SEP-GluA2 is not affected by the bleaching. During imaging the ROI flanking regions are continuously bleached (black box), thus lateral diffusing SEP-GluA2 will be bleached. Receptors that have been internalised and directed for recycling are exocytosed and become fluorescent. Newly synthesised receptors could also travel in intracellular vesicles to be exocytosed and become fluorescent. ( F ) Confocal images of individual dendritic spines from DIV20 cultured rat hippocampal neurons transfected at DIV12 with SEP-GluA2 and either scrambled, Sh-TSPAN5, or rescue construct co-expressing mCherry. Neurons were imaged under a FRAP-FLIP protocol for 5 min to analyse receptor exocytosis. mCherry (magenta) and SEP-GluA2 (white) images (time points: prebleach, postbleach, 10, 20, 30, 60, 120, 180, 240, and 300 s) are shown. The dendritic spine mask is depicted with white dashed line. Scale bar =1 µm. ( G ) Quantification of the ΔF/F pre for SEP-GluA2 over time for scrambled-, Sh-TSPAN5-, and rescue-transfected neurons. ( H ) Quantification of the area under the curve relative to panel E (area under the curve [A.U.]: scrambled 14.85±0.89, Sh-TSPAN5 7.49±1.77, rescue 18.5±2.18). One Way ANOVA, Newman-Kulspost hoc multiple comparison test. ( I ) Quantification of the parameters A and τ, representative of the steady state ΔF/F pre and of the time constant of the exocytosis kinetics, based on the fitting of the first eight time points with the exponential function ( Δ F / F p r e = A ( 1 − e − t τ ) a (ΔF/F pre ): scrambled, 0.0522±0.0002; Sh-TSPAN5, 0.0388±0.0008; rescue, 0.0832±0.0003). (τ (s): scrambled, 4.5±0.2; Sh-TSPAN5, 12.2±1.2; rescue, 7.4±0.2). n = scrambled, 56; Sh-TSPAN5, 35; rescue, 29 dendritic spines. One Way ANOVA, Newman-Kulspost hoc multiple comparison test. Values represent the mean ± SEM. *=p < 0.05, **=p < 0.01, ***=p < 0.001. Figure 6—source data 1. Individual data values for the graphs presented in panels C and G.

Article Snippet: For recycling only experiments, neurons were previously incubated with 200 μg/ml cycloheximide (Life Technologies) for 2 hr with cycloheximide also present in the recording Tyrode’s buffer.

Techniques: Imaging, Cell Culture, Transfection, Construct, Expressing, Comparison