Two modes of exocytosis in an artificial cell

Abstract

The details of exocytosis, the vital cell process of neuronal communication, are still under debate with two generally accepted scenarios. The first mode of release involves secretory vesicles distending into the cell membrane to release the complete vesicle contents. The second involves partial release of the vesicle content through an intermittent fusion pore, or an opened or partially distended fusion pore. Here we show that both full and partial release can be mimicked with a single large-scale cell model for exocytosis composed of material from blebbing cell plasma membrane. The apparent switching mechanism for determining the mode of release is demonstrated to be related to membrane tension that can be differentially induced during artificial exocytosis. These results suggest that the partial distension mode might correspond to an extended kiss-and-run mechanism of release from secretory cells, which has been proposed as a major pathway of exocytosis in neurons and neuroendocrine cells.

Figure 1. Experimental protocol.

(a). Schematic of the experimental setup. The red stars symbolize the dopamine molecules. (b). Picture of the experimental setup with a micropipette inflating a daughter vesicle from the PC12 cell plasma membrane vesicle. Scale bar, 2 μm.

Figure 2. Schematic and micrographs of the two release modes.

(a). The full distension mode, where the vesicle opens up to a frustum, which then collapses into a nanotube resulting in the complete release of the vesicle content. Scale bars, 1 μm. (b). The partial distension mode where the nanotube opens up to a larger pore followed by its re-closing to a nanotube. The partial distension results in incomplete release of the vesicle content. Scale bars, 1 μm.

Figure 3. Representative traces and average peaks from the two modes of release.

The average amperometric peaks for release though full (red) and partial (blue) distension plotted together with SEM. The inset shows representative amperometric traces for the two modes of release. Data were collected from 11 plasma membrane vesicles where recordings from 2 of these vesicles were defined as full release and 9 of them as partial release.

Figure 4. Distribution of release kinetics for all events and reassigned average peaks.

(a). The full width at half max plotted against the number of molecules released for all detected peaks. The two distributions appear to result from partial and full distension modes of release. (b). Average peaks of the two modes with assignments based on amount and kinetics of release are plotted with SEM Data were collected from 11 plasma membrane vesicles where 5 traces were defined as full release and 6 as partial release.

Figure 5. Vesicle opening dynamic parameters vs. charge for all events.

The full distension events are labeled red while the partial distension events are blue. The inset of each graph is a blow up of the events with low released amounts.

Figure 6. Switching between full and partial release in the same bleb.

Peak characteristics of release events from two sizes of daughter vesicles formed inside the same bleb. The small vesicle displays a larger peak current and faster release kinetics compared to the larger daughter vesicle. The presented averages are from 4 separate blebs where both a small and a large vesicle were studied per bleb. *** p < 0.001 using t-test assuming equal variances.

Figure 7. Proposed model for the tension dependence in partial release mode.

The blue lipids symbolize a membrane at low tension while the red lipid bilayers symbolize a membrane under tension stress. (a). The inflation of the daughter vesicle creates tension in the vesicle membrane, inducing lipid flow from the mother to the daughter vesicle. (b). At some critical size the tension between the daughter and mother vesicles equalize, stopping flow. (c). The daughter vesicle inflation continues, which requires the mother vesicle to reduce surface area to accommodate the growing daughter vesicle (d). The pore formed reduces the pressure in the daughter vesicle stopping it from growing which causes a reverse in lipid flow. (e). The surface area is minimized as the toroid collapses to a lipid nanotube (f). The closing of the pore initiates the daughter vesicle inflation and the lipid flow starts again.