synapto iatpsnfr2 mirfp670nano3  (Addgene inc)

Journal: Nature Communications

Article Title: Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis

doi: 10.1038/s41467-023-38595-2

Figure Lengend Snippet: a (top) Example airyscan micrographs showing the localization of Epsin1 relative to F-actin in wild-type neurons. Epsin1 stained with Epsin1-antibody and its secondary antibody conjugated with Atto647N. F-actin is labeled by neuronal expression of EGFP-UtrCH, which is stained by a GFP-antibody and its secondary antibody conjugated to Atto488. (bottom) Plot showing the Pearson’s correlation coefficient. Each dot = synapse. n = 94 synapses. N = 3 cultures. b Example 2D STED micrographs showing the localization of Epsin 1 relative to the active zone in wild-type neurons. Active zone is marked by anti-Bassoon antibody and its secondary antibody conjugated with Alexa594. Epsin1 stained with Epsin1-antibody and its secondary antibody conjugated with Atto647N. c The distribution of Epsin1 signals against the active zone boundary. The active zone boundary was defined by Bassoon signals. See Methods for the analysis method. Error bars are SEM. N = 2 cultures, n = 30–60 per culture. d Example confocal fluorescence micrographs showing F-actin signals in neurons expressing scramble shRNA and Epsin1 knock-down (KD) shRNA. F-actin is stained by expressing EGFP-UtrCH. e The normalized intensity of F-actin signals from neurons expressing scramble (scr) shRNA, or Epsin1 shRNA, measured by Airyscan. Signals are normalized to the fluorescence signals in axons. Kruskal-Wallis test, with Dunn’s multiple comparisons test. *** p < 0.001. See Supplementary Table for the detailed numbers for each sample. f Example electron micrographs showing wild-type and Epsin1 KD synapses unstimulated or stimulated with a single electrical pulse (1 ms) and frozen 100 ms or 1 s later. Black arrow: endocytic pit. Black arrowhead: ferritin-positive endosomes. g , h Number of endocytic pits at 100 ms after stimulation ( g ) or ferritin-positive structures at 1 s after stimulation ( h ) in neurons expressing scramble shRNA (scrRNA) or Epsin1 shRNA. Mean and 95% confidential interval are shown. Kruskal-Wallis test, with Dunn’s multiple comparisons test. **** p < 0.0001. p-values are only shown for direct comparison between unstimulated and stimulated neurons treated with the same drug. See Supplementary Table for the detailed numbers for each sample. Source data are provided as a Source Data file.

Article Snippet: For visualizing Epsin1, we used a plasmid expressing Epsin1-EGFP, obtained from Addgene (pEGFPC1-Epsin1, #22228).

Techniques: Staining, Labeling, Expressing, Fluorescence, shRNA, Knockdown, Comparison

Journal: Scientific reports

Article Title: Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses.

doi: 10.1038/srep31997

Figure Lengend Snippet: Figure 4. Defects in endocytic phenotype are restored by expressing shRNA-insensitive Epsin1 in Epsin1 KD neurons. (a) Representative vG-pH trace responses to 100 AP from control (black), Epsin1-KD1 (red), and rescue (green) neurons. For rescue experiments, neurons were transfected with shRNA1-Epsin1, shRNA1- insensitive Epsin1, and vG-pH. (b) Mean values of post-stimulus endocytic time constants from control, Epsin1-KD1, and rescue neurons. (c) Endocytosis during stimulation is restored upon shRNA-insensitive Epsin1 expression in Epsin1-KD1 neurons, calculated as for Fig. 3. (d) The amount of endocytosis during stimulation (22.83 ± 2.9%) is similar to that of the control. (e,f) Surface accumulation of vG-pH and total vesicle pool size (TVS) are restored to control levels in rescue neurons. Surface fraction rescue 5.0 ± 1.2% (n = 7), TVS rescue = 2056 ± 292 a.u (n = 7). (g) Representative images of rescue neurons. Neurons expressing shRNA1- Epsin1, shRNA-insensitive Epsin1 and vG-pH were fixed and stained with anti-Epsin1 (red). (h) Expression of Epsin1 is almost completely restored (99.2 ± 5.7%) to levels in non-transfected neurons by re-expressing shRNA-insensitive Epsin1 in Epsin1-KD1 neurons. *p < 0.05, **p < 0.01. One-way ANOVA; ns means non- significant.

Article Snippet: Rat Epsin1 cDNA was obtained from Addgene (ID#21066).

Techniques: Expressing, shRNA, Control, Transfection, Staining