Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis

Abstract

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.

Keywords: STED; chromaffin cell; endocytosis; exocytosis; fission; fusion; fusion pore; pore constriction; pore expansion; release; super-resolution imaging.

Figure 1. Ω-profiles with a pore in live cells

(A) Setup drawing. cell’s membrane and bath are labelled with PH_G (green) and A532 (red, pseudo-color). ICa and Cm (capacitance) are recorded via a whole-cell pipette. (B) ICa and Cm changes induced by depol_1s. (C) STED XY/Z_fix images of PH_G and A532 before (−1 s) and after (+1 s) depol_1s. Inset: drawing of XY- focal plane, ~100–200 nm above cell-bottom. (D) XZ PH_G/A532 images of a PH-Ω along Y-axis every 50 nm (Pore_v observed). Cytosol, PM and coverslip location are labelled. XZ images are STED images (applies to Figures 1–5). (E) Upper, PH-Ω at Y=250 nm from panel D and the fluorescence profile of the dotted line across the pore with W_H labelled. Lower, XY images with a Z-focal plane at the pore (dotted line in upper panel). Images were reconstructed from panel D XZ/Y_stack images (inset: drawing of reconstructed plane in gray). (F–G) Similar to panel D–E, respectively, but for a PH-Ω with Pore_noV. (H) Sampled depol_1s-induced PH_G/A532-labelled Ω-profiles with Pore_v (left: i–v) or Pore_noV (right, vi–ix). (I) Distribution of Pore_v W_H and corresponding PH-Ω diameter (n = 67, binning width: 50 nm). Inset: drawing of Pore_v W_H and PH-Ω diameter. (J–K) Pore_v W_H (J) and Pore_v/Ω (Pore_v W_H/PH-Ω diameter, K) plotted versus PH-Ω diameter (n = 67, XZ/Y_stack imaging). Linear-fit correlation coefficient is 0.55 (J) and −0.03 (K); ANOVA test of linear fit: p < 0.001 (J), p = 0.81 (K). See also Figure S1.

Figure 2. Observing rapid fusion pore opening and release

(A) PH-mCherry (PH-mCh, red) and mCLING-A488 (green) images and their fluorescence profile (F) across the dotted line (the pore) before (−5 s) and after (+5 s) depol_1s. F traces are also enlarged (Lower). Line profiles are normalized to the peak value after depol_1s. The cell was overexpressed with PH-mCh and treated with mCLING-A488 (0.5 μM, bath, 10 min). (B) PH_G/FFN511 images at rest (pseudo-color). FFN511 was in the bath for 3 min, and then washed. (C) PH_G/FFN511 images before (−1 s) and after (+5 s) depol_1s. (D) PH_G/NPY-mT images at rest (pseudo-color). (E) PH_G/NPY-mT images before (−1 s) and after (+1 s) depol_1s. (F–G) EM images of a cell (F) and sampled Ω-profiles with different pore sizes (G). Cells were in 70 mM KCl for 90 s. (H) Sampled PH_G/A532 images immediately before (time 0) and after fusion during XZ/Y_fix imaging every 0.1 s (i), 0.3 s (ii), 46 ms (iii), or 26 ms (iv). PH-Ω with a Pore_v was induced by depol_1s applied 0.1–2 s earlier. (I) Similar to panel H, but showing Pore_noV sampled every 0.1 s (i) or 0.3 s (ii). See also Figures S2, S3 and S6.

Figure 3. Fusion pore expansion, constriction and closure at different rates

(A–E) PH-Ω fluorescence (F_PH, normalized to baseline), A532 spot fluorescence (F₅₃₂, normalized to baseline), Pore_v W_H, and sampled images at times indicated with lines showing different Pore_v dynamics: A, rapid opening and slow constriction (same PH-Ω as in Figure 2H-iv); B, delayed expansion; C, rapid constriction and closure; D, Pore_v unchanged; E, Pore_v disappearance due to Ω-profile shrinking. A and C: the rate of Pore_v W_H changes (Rate) are plotted (A, 26 ms/frame; C, 46 ms/frame); insets, W_H and Rate at larger time scale. Gray circles in A–B refer to non-visible A532-permeable pores with a W_H <60 nm. Fusion was induced by depol_1s at 0.1–2 s before PH-Ω appeared. (F) Left, number of Pore_v observed at the fusion onset or 0.5–4 s after fusion (delay). Right, number of Pore_v that subsequently constricted, constricted and closed, remained unchanged, or disappeared. Data from 51 Pore_v (49 cells, XZ/Y_fix image every 26–300 ms). See also Figures S2 and S4.

Figure 4. Mechanisms of pore constriction, expansion and their competition

(A) Percentage of Pore_v undergoing constriction within 20 s after fusion (Constrict%, mean ± s.e.m.) is plotted versus ICa density (mean ± s.e.m.) of four cell groups with ICa density <15 pA/pF, 15–35 pA/pF, 35–50 pA/pF, and >50 pA/pF in control conditions (35 cells, 37 Pore_v). Pore_v closure was counted as constriction; Ω-profiles undergoing shrinking were excluded, because shrinking obscured Pore_v detection (also applies to C). Fusion was induced by depol_1s (applies to all figures). P<0.01, ANOVA test. (B) Sampled PH_G/A532 images before and after fusion in Ctrl, in the presence of Sr²⁺ (5 mM/bath, replacing Ca²⁺) or dynasore (DnS, 80 μM/bath, XZ/Y_fix imaging). (C)Constrict% of Pore_v in control (35 cells, 37 Pore_v), Sr²⁺ (7 cells, 8 Pore_v) or DnS (12 cells, 13 Pore_v). *, P<0.05; **, P<0.01; ***, P<0.001 (t test, compared to control, applies to all bar graphs). (D–E) Sampled PH-Ω (D) and the percentage of PH-Ω with a Pore_v (Pore_v%, E, mean + s.e.m.) in control (Ctrl, 21 cells, 267 PH-Ω), Sr²⁺ (5 mM, replacing bath Ca²⁺, 8 cells, 119 PH-Ω), dynasore (DnS, 80 μM/bath, 7 cells, 88 PH-Ω), or overexpressed dynamin 1-K44A (7 cells, 81 PH-Ω). Pore_v was detected with XZ/Y_stack imaging every 5–23 s. (F) Pore_v% at the beginning of fusion in control (202 cells, 236 PH-Ω), Sr²⁺ (10 cells, 12 PH-Ωs) or dynasore (DnS, 19 cells, 22 PH-Ωs). Pore_v was detected with XZ/Y_fix imaging every 26–300 ms. (G) Pore_v% (mean ± s.e.m.) at the beginning of fusion plotted versus ICa density (mean ± s.e.m.) in four groups with different ICa densities described in panel A (202 cells, 236 PH-Ω). *, P<0.05; ***, P<0.001; t test. Pore_v was detected with XZ/Y_fix imaging. (H) Pore_v% at 310 or 650 mOsm in Ca²⁺ (21 cells/267 PH-Ω or 7 cells/42 PH-Ω), Sr²⁺ (8 cells/119 PH-Ω or 13 cells/85 PH-Ω), or DnS (with 5 mM Ca²⁺; 7 cells/88 PH-Ω or 7 cells/64 PH-Ω). Pore_v was detected with XZ/Y_stack imaging. (I) Pore_v% at the beginning of fusion in the absence (−) or presence (+) of 3 μM Lat A in a bath containing Ca²⁺ (202 cells/236 Ω or 13 cells/15 PH-Ω), Sr²⁺ (10 cells/12 PH-Ω or 9 cells/11 PH-Ω) or DnS (10 cells/12 PH-Ω or 14 cells/17 PH-Ω). Pore_v was detected with XZ/Y_fix imaging. (J) Model: F-actin/tension-dependent pore expansion competes with calcium/dynamin-dependent pore constriction to determine pore dynamics. (K) Left, dynamin 2-EGFP (Dyn2), NPY-mT (granules) and A532 (bath) images. Right, lifeact-TagGFP2, NPY-mT and A532 images. (L) Left, PH_G and dynamin 2-mTurquoise2 (Dyn2) XZ-images at 10 s after depol_1s (XZ/Y_stack with Y-axis labelled). Right, XY-reconstructed images across the pore (dotted line). See also Figure S5.

Figure 5. Pore-dynamics control cargo release and endocytosis

(A–B) F_PH, F₅₃₂, and PH_G/A532 images (XZ/Y_fix) for depol_1s-induced Ω-profiles with Pore_v (A) or Pore_noV (B). (C) 20–80% rise time of F₅₃₂ and F_PH for depol_1s-induced PH-Ωs with Pore_v (51 Pore_v, 49 cells) or Pore_noV (185 Pore_noV, 153 cells, XZ/Y_fix). (D–E) F_PH, F_FFN (FFN511 fluorescence), and PH_G/FFN511 images for depol_1s-induced PH-Ω with Pore_v (D) or Pore_noV (E). (F) 20–80% F_FFN (FFN511 fluorescence) decay time for PH-Ωs with Pore_v (8 Pore_v, 7 cells) or Pore_noV (24 Pore_noV, 18 cells). (G–I) 20–80% rise time of F₅₃₂ and F_PH for depol_1s-induced PH-Ωs (G), close-fusion percentage (H), and Cm changes induced by depol_1s (I) in Ctrl (202 cells) or in the presence of Sr²⁺ (10 cells) or dynasore (DnS, 19 cells, XZ/Y_fix).

Figure 6. Most close-fusions release NPY-EGFP rapidly and completely as non-close-fusions

(A–D) F₆₄₇ (A647 spot fluorescence), F_NPY (NPY-EGFP spot fluorescence), A647 spot W_H, and A647/NPY-EGFP confocal XY-images at times indicated with lines for various fusion modes and rates of releasing NPY-EGFP. A, close-fusion with rapid release; B, non-close-fusion, including stay-fusion (left) and shrink-fusion (right) with rapid release; C, two stay-fusion spots with slow release; D, close-fusion (left) and stay-fusion (right) with no or partial release of NPY-EGFP. (E) Cumulative frequency (normalized) of 20–80% F_NPY decay time for close-fusion (95 spots, 28 cells) and non-close-fusion (123 spots, 28 cells, confocal XY/Z_fix imaging). ‘>30 s’: partial or no NPY-EGFP release.