Journal: Cell

Article Title: Visualization of membrane pore in live cells reveals a dynamic-pore theory governing fusion and endocytosis

doi: 10.1016/j.cell.2018.02.062

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Both PH-EGFP and PH-mNeonGreen are abbreviated as PH G . Dynamin 2-mTurquoise2 construct was created by replacing the EGFP tag of dynamin 2-EGFP (Addgene) with mTurquoise2 (Addgene).

Techniques: Recombinant, Cell Culture, Plasmid Preparation, Software, Imaging

Journal: bioRxiv

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

doi: 10.1101/2022.06.12.495801

Figure Lengend Snippet: a. A diagram showing the effect of Latrunculin A (Lat A) and Jasplakinolide and example STED micrographs showing the localization of filamentous actin (F-actin) relative to the active zone in neurons treated with DMSO (control), Lat A, and Jasplakinolide. Active zone is marked by anti-Bassoon antibody and its secondary antibody conjugated with Alexa594. F-actin binding EGFP-UtrCH is expressed in neurons and stained with GFP-antibody and its secondary antibody conjugated with Atto646. b. Cumulative plots showing distribution of F-actin signals against the active zone boundary and intensity of F-actin signals. The active zone boundary was defined by Bassoon signals. See Supplementary Table 2 for detailed statistical analysis. c. A schematic showing the lateral membrane compression model for ultrafast endocytosis and basic values used for simulations. The lateral membrane pressure exerted by exocytosis is predicted to compress the plasma membrane against the stiff periactive zone membrane and induce pit formation at the interface between actin-free and actin-enriched regions, or at the endocytic zone. d. Schematics showing the initial conditions of simulations from top-down view (left) and orthogonal view (right). The initial length of active zone (blue) is set at 500 nm. The width of F-actin band (purple) is set at 50 nm. Here, the active zone refers to the actin-free membrane area that includes not only the vesicle fusing area but also the endocytic zone. In contrast, the periactive zone is represented by the F-actin band where actin cortex impinges upon the membrane. The center of the active zone is set as (x, y) = (0, 0). One fusing vesicle (light blue circle) is placed at (D, 0), while two other vesicles are placed at (C, -C) and (-C, -C) such that three vesicles would form an isosceles triangle. As the initial condition, we set C = D = 60 nm. e. Snapshots from simulations, showing the evolution of membrane curvature within the active zone over time. Three fusing vesicles are organized with C = D = 60 nm. At 58 ms, simulations reach the steady state, with 2 endocytic pits forming at the boundary between active zone and actin-enriched region. f. Plot showing the depth of exocytic pits and endocytic pits as a function of time. g. Plot showing the resulting membrane curvature as a function of the spatial arrangement of fused vesicles. Distances among vesicles are modulated by changing C and D, depicted in d. h. Plot showing the dependence of successful endocytic pit formation on bending moduli of active zone and periactive zone membranes. The colored areas indicate successful formation of endocytic pits. i. Snapshots from simulations, showing the evolution of membrane shape within the active zone as a function of tension. As shown in the plot, the membrane area conservation was relaxed at 2.5 ms; consequently, the membrane tension decreases from 0.6 pN/nm to 0.3 pN/nm. This reduction in tension mimicks the expected tension change that occurs with exocytosis. Simulations reach the steady sate at 5 ms without inducing any curvature.

Article Snippet: To label the endocytic zone, we used a plasmid expressing Dynamin1xA C-terminally tagged to GFP, purchased from addgene (phsDyn1xA-EGFP-N1, #120313).

Techniques: Control, Binding Assay, Staining, Membrane, Clinical Proteomics

Journal: bioRxiv

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

doi: 10.1101/2022.06.12.495801

Figure Lengend Snippet: a. A schematic showing the protein structural elements of Epsin1. Epsin1 contains ENTH domain, NPF and DPW. The C-terminal domain, marked light red, interacts with F-actin, while the ENTH domain, marked light blue, interacts with plasma membrane. b. Example 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 Atto646. 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. d. Example confocal fluorescence micrographs showing F-actin signals at wild-type or Epsin1 knock-down (KD) neurons. F-actin binding EGFP-UtrCH is expressed in neurons and stained with GFP-antibody and its secondary antibody conjugated with Atto646. False-colored images of the bottom panels show relative fluorescence intensity of 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 the fluorescence signals in axons. 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. Brown-Forsythe and Welch ANOVA analysis, with Games-Howell 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 2 for the detailed numbers for each sample.

Article Snippet: To label the endocytic zone, we used a plasmid expressing Dynamin1xA C-terminally tagged to GFP, purchased from addgene (phsDyn1xA-EGFP-N1, #120313).

Techniques: Clinical Proteomics, Membrane, Staining, Fluorescence, Knockdown, Binding Assay, Expressing, shRNA, Comparison