Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

Abstract

Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and stimulated emission depletion microscopy imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within ~1-30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

Figure 1. Imaging granule fusion in chromaffin cells

(a) Left: schematic drawing showing whole-cell recording of ICa and capacitance (Cm), and imaging at the cell bottom or center with a fluorescent dye (red) in the bath. Right: Confocal images at a real cell’s bottom (lower) or center (upper) with A647 (30 μM) in the bath. The dark area at the cell bottom represents a thin layer of A647 solution between the cell bottom and the coverslip. (b) Left: sampled ICa (upper) and Cm (lower) induced by depol_1s (arrow). Right: A647 confocal/cell-bottom images at 1 s before, during (0.5 s), and 10 s after depol_1s (same cell as from left). Arrows indicate A647 spots. (c) The accumulated number of A647 spots (ΣN_spot, upper) plotted versus the time at which the spots occurred in 60 cells subjected to depol_1s (arrow). The corresponding mean Cm change is also plotted (lower). (d) The A647 spot number (N_spot) plotted versus Cm from the same cell (n =60, each circle represents one cell). Data were fitted with a linear regression line (correlation coefficient: 0.71). (e) Concurrent imaging of NPY-EGFP (green) and A647 (red) at 0.5 s before (upper), and 0.3 s (middle) and 0.5 s (lower) during depol_1s. Arrows indicate NPY-EGFP release (green spots disappeared at 0.5 s) coincident with A647 spots. Circles indicate an A647 spot without an overlapping NPY-EGFP spot. (f) The percentage of A647 spots that are co-localized with NPY-EGFP release (Spot_release) plotted versus the number of NPY-EGFP granules per μm² at the cell bottom (N_NPY/μm²). Each circle represents one cell (n = 23 cells; cells with > 5 A647 spots were used).

Figure 2. Resolving the Ω-profile at the fusion instant

(a) Confocal images of a NPY-EGFP-positive granule before release and the A647 spot at the spot onset at the same location (upper). Normalized fluorescence intensity profiles (F_norm) from dotted lines are also shown (lower, applies to panel b). (b) STED images (upper) of a NPY-EGFP granule and an A488 spot at the spot onset (from different cells). Line profiles are also plotted (lower). (c) STED/cell-center images at 0.2 s before (left) and 0.3 s (right) during depol_1s (upper). F_norm are also shown for two lines across the spot center, one perpendicular to the plasma membrane, the other 45° apart (applies to d–f). The arrow indicates the typical feature of the Ω-profile: a dip in the line profile which is wider and larger for the 45° line. Bright fluorescence in the right side of each image represents extracellular A488, whereas dim fluorescence in the left side of each image means the intracellular compartment with no A488 (applies to all plots at the STED/cell-center setting). (d–e) Simulation showing side images and line profiles (solid and dotted) before (left) and after (right) the appearance of an Ω-profile (d, pore size: 50 nm, vesicle size: 300 nm) or a collapsed profile (e). The arrow in d indicates the typical feature of the Ω-profile: a dip in the line profile which is wider and larger for the 45° line. Images are taken from Supplementary Figs. 2f, 2g and 3c. Simulation methods are described in Supplementary Figs. 2–3. (f) Two STED/cell-center images and line profiles (left, right) that resemble the presumed collapsed profile. Images were obtained in resting conditions.

Figure 3. Ω-stay and Ω-close fusion

a–c, Ω-stay. (a) F₆₄₇ (red), F₄₈₈ (green), W_H, and sampled images (average of 4) at times indicated (lines) are plotted versus time for a spot at the confocalA647/A488 setting (cell-bottom). F₆₄₇ and F₄₈₈ were normalized to the mean value before spot appeared (applies to all plots of F₆₄₇, F₄₈₈, and F_STED). Images were collected every 15 ms. (b) F_STED (STED fluorescence intensity), W_H, and sampled images (average of 2) at times indicated are plotted versus time for a spot at the STED/cell-bottom setting (60 μM A488 in bath). Images were collected every 26 ms. (c) F_STED, W_H, sampled images (average of 8, side images of the Ω-profile) and their line profiles (normalized to peak, F_norm) are plotted versus time for a spot at the STED/cell-center setting. Images were collected every 36 ms. W_H was measured from the profile of a vertical line (not shown, parallel to cell membrane) across the spot center. Solid and dotted line profiles correspond to solid and dotted lines, respectively. The arrangements in panels a, b and c apply to all plots in Figs. 3 – 6 at confocal/A647/A488, STED/cell-bottom, and STED/cell-center setting, respectively. (d–f) Ω-close at confocal/A647/A488 (d–e) and STED/cell-bottom setting (f). Arrows indicate pore closure (apply to ‘close’ fusion in Figs. 3–6). Panel e shows two spots (upper, lower) with different pore closing time (W_H and sampled images not shown).

Figure 4. Ω-enlarge-stay and Ω-enlarge-close

(a–b) Ω-enlarge-stay at confocal/A647/A488 (a) and STED/cell-center setting (b). (c) Ω-enlarge-close at confocal/A647/A488 setting.

Figure 5. Ω-shrink-stay and Ω-shrink-close

(a–c) Ω-shrink-stay at confocal/A647/A488 (a), STED/cell-bottom (b), and STED/cell-center setting (c). In a, the scale was set to see dim red images, but partly saturate the brightest red image. In b, F_STED (peak normalized, F_{STED_n}) in the inner circle (red) and the outer ring (between red and blue circles, blue) are also plotted, showing faster decay of blue trace and thus the spot shrinkage. c: left 2 images, average of 2 single images; right 2 images, average of 8 single images. (d–f) Ω-shrink-close at confocal/A647/A488 (d), STED/cell-bottom (e), and STED/cell-center setting (f). In e, F_{STED_n} in the inner circle (red) and the outer ring (between red and blue circles, blue) are also plotted to show spot shrinkage. f: left 2 images, average of 2 single images; right 3 images, average of 8 single images.

Figure 6. Ω-shrink fusion

(a) Ω-shrink at confocal/A647/A488 setting. (b) Distribution of the F₆₄₇ decay τ during Ω-shrink fusion at confocal/A647/A488 setting (115 spots, 60 cells, data binned every 0.5 s). (c–d) Ω-shrink at STED/cell-bottom (c) and STED/cell-center setting (d). In c, F_{STED_n} in the inner circle (red) and the outer ring (blue) are also plotted to show spot shrinkage. d: left 2 images, average of 2 single images; right 3 images, average of 8 single images.

Figure 7. Seven fusion modes confirmed by imaging with Atto 655 and Atto 488

(a–b) Schematic drawings of our model called Ω-exo-endocytosis (a, 7 modes) and the classical FC and KR model (b). Dotted arrows mean that the transition may or may not take place. (c) Atto 655 fluorescence intensity (F_Atto655, red), Atto 488 fluorescence intensity (F_Atto488, green) and sampled images (average of 5–10 frames) at times indicated (lines) are plotted versus time for a spot undergoing Ω-stay fusion. F_Atto655 and F_Atto488 were normalized to the mean value before spot appeared. Images were collected every 17–34 ms at the confocal cell-bottom setting with Atto 655 (strong excitation) and Atto 488 (weak excitation) in the bath. (d–i) Similar to panel c, but for spots undergoing Ω-close (d), Ω-enlarge-stay (e), Ω-enlarge-close (f), Ω-shrink-stay (g), Ω-shrink-close (h), and Ω-shrink (i).

Figure 8. Close modes mediate rapid and slow endocytosis of the cell

(a) Examples showing net exo- and endocytosis (lower: Exo-endo, see also Methods) reconstructed from fluorescence changes (upper: F₆₄₇, F₄₈₈) during Ω-close, Ω-shrink-close, Ω-enlarge-close, Ω-stay, Ω-shrink-stay, Ω-enlarge-stay and Ω-shrink fusion (left to right). (b) Pore closure time (from open to close, not from stimulation time to closure) distribution for three ‘close’ modes (312 spots). (c) The mean Cm (± s.e.m., every 1 s, baseline subtracted, upper), N_exo-endo per cell (middle upper) and ICa (lower) induced by depol_1s (to +10 mV). Cm (black) and N_exo-endo (red) traces are also normalized and superimposed for comparison (middle lower). Data were from 636 spots in 60 cells. (d) Similar to panel c, except that data in panel c were divided into four groups based on Cm decay: 1) decay to baseline in 15 s (165 spots, 11 cells), 2) decay by >80% in 30 s except group 1 (101 spots, 10 cells), 3) decay by 30–80% in 30 s (173 spots, 18 cells), and 4) decay by <30% in 30 s (197 spots, 21 cells). (e) The percentage of N_exo-endo at 30 s after depol_1s (compared to the peak N_exo-endo) plotted versus the corresponding un-decayed Cm percentage at 30 s after stimulation from four groups described in panel d (left to right, group 1 to 4). A line (red) with a slope of 1 is also plotted. Error bars are s.e.m. The spot number and cell number are described in panel d. (f) The mean Cm change (upper, ΔCm) and the percentage of close fusion (Close_sum, lower, including Ω-close, Ω-shrink-close and Ω-enlarge-close) induced by depol_1s within 1 min (left, 53/68 spots are ‘close’ fusion) and > 6 min (right, 1/31 spots is close fusion) after whole-cell break-in from the same cell (n = 4 cells).

Figure 9. Strong calcium influx triggers dynamin-dependent close fusion modes and low calcium promotes stay modes and Ω-shrink

(a) The percentage of Close_sum (including all three close modes), Stay_sum (including three stay modes), and Ω-shrink plotted versus the mean ICa in four groups described in Fig. 8d (stimulation: depol_1s to +10 mV). The percentage was calculated within each group. (b) Sample ICa and Cm induced by a 1 s depolarization to +50 mV. This cell showed an ICa of ~500 pA during a 10 ms depolarization to +10 mV (not shown). (c) Re-plotting panel a (solid symbols), but including data similar to those shown in panel b (open symbols), where 1 s depolarization to +50 mV induced the smallest ICa as compared to the mean ICa induced by depol_1s to +10 mV in groups 1–4. (d) The mean Cm change (upper, ΔCm) and the percentage of close fusion (Close_sum, lower, including Ω-close, Ω-shrink-close and Ω-enlarge-close) induced by depol_1s in control (10 cells, 66 spots, left) and in cells bathed with 80 μM dynasore (14 cells, 63 spots, right). In both groups, cells with an ICa > 350 pA were selected for analysis.

Figure 10. Ω-profile retains vesicle membrane protein VAMP2

(a–g) F₆₄₇ (red), F_VAMP2 (green), W_H of A647 (red) and VAMP2-EGFP (green) spot, and sampled A647 (red) and VAMP2-EGFP (green) images (at times indicated with lines) for spots undergoing Ω-stay (a), Ω-close (b), Ω-enlarge-stay (c), Ω-shrink-stay (d), Ω-shrink-close (e), and Ω-shrink (f: rapid shrinking, diffusion cloud; g: slow shrinking, size reduction observed). Cells were expressed with VAMP2-EGFP and stimulated by depol_1s with A647 in the bath. W_H is not measured in panel f, because VAMP2-EGFP rapidly diffused into a cloud, which did not reflect the Ω-shaped membrane profile size. VAMP2-EGFP spots appeared slightly (~50–100 nm in W_H) larger than corresponding A647 spots (e.g., Fig. 10a–b), because VAMP2-EGFP was located at the membrane, whereas A647 was inside the Ω-shaped structure. (h) The F₆₄₇ and F_VAMP2 changes in response to a bath pH change from 7.4 to 5.5 (upper) for spots undergoing Ω-stay (left) and Ω-close (right).