Figure 1.
The fusion pores of LSVs expand and constrict. (A) Schematic representation illustrating the process of LSV exocytosis by vesicle membrane crumpling. Actomyosin recruitment to LSVs after fusion mediates membrane deformation and subsequent content release. Images were obtained via confocal microscopy in multiple planes, as illustrated with the unfused vesicle (left). For fusion pore visualization, we selected LSVs where the fusion pore is aligned to the XY imaging plane. The presented view was generated by capturing a stack of XY planes and subsequently projecting them along the Z plane. (B) Time-lapse sequence of representative exocytotic events (Confocal, top; SRRF, bottom). Fusion onset is detected by LSV swelling (white dashed arrow; Fig. S1 A). LifeAct-Ruby (actomyosin) recruitment is first visible ∼50 s after fusion and followed by content release. Fusion pore diameter was defined and measured at the narrowest aperture observed at the mid-plane through the vesicle, which connects the vesicle and the lumen (white arrowheads). The fusion pore expands before actomyosin recruitment and constricts during content release. Time mm:ss, relative to fusion. Glue-GFP (green) and LifeAct-Ruby (magenta) images were generated by averaging several XY planes along the Z axis (intensity-projection). (C) Quantification of LSV swelling as % change in LSV diameter and roundness after fusion (n [LSVs] = 14), mean and SEM values are noted on the panel. (D) Mean relative pore diameter (% maximal diameter) over time. Time 0 is defined at the time of maximal pore diameter, which coincides with the onset of actomyosin assembly (n [pores] = 12; gray—SEM; see also Fig S1 B). (E) Pore diameter of fused vesicles visualized by FIB-SEM before and after crumpling (n [pores]) = 21 and 22 respectively; P: ** = 0.0026. Two-tailed, unpaired t test. (F) Red and blue circles denote the individual LSVs. Mean and SEM values are noted on the panel. (F) Representative slices from FIB-SEM volumes of the mid-plane of LSVs before (red) and after (blue) crumpling. White arrowheads show the narrowest aperture of the fusion pore where it was measured. Whiskers in C and E show mean and SEM. Scale bars in B and F, 1 µm.

The fusion pores of LSVs expand and constrict. (A) Schematic representation illustrating the process of LSV exocytosis by vesicle membrane crumpling. Actomyosin recruitment to LSVs after fusion mediates membrane deformation and subsequent content release. Images were obtained via confocal microscopy in multiple planes, as illustrated with the unfused vesicle (left). For fusion pore visualization, we selected LSVs where the fusion pore is aligned to the XY imaging plane. The presented view was generated by capturing a stack of XY planes and subsequently projecting them along the Z plane. (B) Time-lapse sequence of representative exocytotic events (Confocal, top; SRRF, bottom). Fusion onset is detected by LSV swelling (white dashed arrow; Fig. S1 A). LifeAct-Ruby (actomyosin) recruitment is first visible ∼50 s after fusion and followed by content release. Fusion pore diameter was defined and measured at the narrowest aperture observed at the mid-plane through the vesicle, which connects the vesicle and the lumen (white arrowheads). The fusion pore expands before actomyosin recruitment and constricts during content release. Time mm:ss, relative to fusion. Glue-GFP (green) and LifeAct-Ruby (magenta) images were generated by averaging several XY planes along the Z axis (intensity-projection). (C) Quantification of LSV swelling as % change in LSV diameter and roundness after fusion (n [LSVs] = 14), mean and SEM values are noted on the panel. (D) Mean relative pore diameter (% maximal diameter) over time. Time 0 is defined at the time of maximal pore diameter, which coincides with the onset of actomyosin assembly (n [pores] = 12; gray—SEM; see also Fig S1 B). (E) Pore diameter of fused vesicles visualized by FIB-SEM before and after crumpling (n [pores]) = 21 and 22 respectively; P: ** = 0.0026. Two-tailed, unpaired t test. (F) Red and blue circles denote the individual LSVs. Mean and SEM values are noted on the panel. (F) Representative slices from FIB-SEM volumes of the mid-plane of LSVs before (red) and after (blue) crumpling. White arrowheads show the narrowest aperture of the fusion pore where it was measured. Whiskers in C and E show mean and SEM. Scale bars in B and F, 1 µm.

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