Direct Visualization of Wide Fusion-Fission Pores and Their Highly Varied Dynamics

Abstract

In this issue of Cell, Shin et al. report the first live-cell imaging of a fusion pore. Directly visualized pores in neuroendocrine cells can be much larger than expected yet not require vesicular full-collapse. These fusion-fission pores have diverse fates arising from opposing dynamin-driven pore constriction and F-actin-mediated pore expansion.

The fusion and fission of lipid membranes are biophysical marvels of great cellular importance. In one well-studied example, secretory vesicles fuse with the surface membrane of neuroendocrine cells, form an exocytotic fusion pore, and release neurotransmitters. Activity-dependent regulation of such fusion pores can support differential release of these substances (Fulop et al., 2005). Dynamic properties of the fusion pore and release of vesicular cargoes have been inferred from measurements of capacitance (reflecting membrane area) along with am- perometry of released amine transmitters and postsynaptic responses (reviewed in Alabi and Tsien [2013]). Fluorescent labeling of vesicular membrane or vesicular contents has provided further perspective on fusion pore properties and dynamics (reviewed in Alabi and Tsien [2013] and Ryan [2003]). What has remained elusive, however, is a direct visualization of the fusion pore and its dynamics in living cells.

Classic electron microscopy (EM) experiments at the neuromuscular junction (Heuser and Reese, 1981; Ceccarelli et al., 1973) obtained compelling images of an Ω-shape, comprised of vesicle membrane and connecting pore and plasma membrane, but left ambiguous whether the vesicle is destined for full collapse and dispersion into the plasmalemma or is able to pinch off from the surface, retaining its identity for later reuse (what Ceccarelli and colleagues (Ceccarelli et al., 1973) dubbed “kiss-and-run”). EM images give the best spatial resolution but only provide one snapshot of a secretory event. Now, in this issue of Cell, Shin et al. present the first live-cell dynamic imaging of a fusion pore (Shin et al., 2018). Using super-resolution microscopy, they show that a fusion pore can be significantly larger and longer-lived than previously thought and that its varied dynamics confer at least four types of post-fusion responses. These depend on a tug of war between F-actin-driven pore expansion and dynamin/calcium-mediated constriction.

Shin and colleagues imaged vesicle fusion in adrenal chromaffin cells, a classic secretory system. The chromaffin cells were directly excited using a 1 s depolarizing pulse (Figures 1A and1B), resulting in a large calcium current and a concomitant increase in vesicle fusion, monitored as increased cell area (membrane capacitance). Imaging showed that a green fluorescent marker of the plasma membrane diffused into the fused vesicle and formed an Ω-profile when imaged in the XZ plane (Figure 1B). A red fluorescent dye (Atto 532) in the extracellular medium passed through the open fusion pore to fill the vesicle, thus reporting the fusion event. In ~25% of the observed Ω-profiles, the pore itself was plainly seen as a green collar of membrane surrounding a red neck—the pore lumen (Figure 1B; Pore_V). Following detection of the Ω-profile, the authors observed four classes of pore dynamics, as schematized in Figure 1B₂: (1) constriction until the pore was smaller than the detection limit but still open (not shown), (2) closure of the pore (“close”), (3) maintenance of the open pore (“stay”), and (4) rapid shrinking of the vesicle (“shrink”). The close-fusion events, in which Atto 532 diffuses into the Ω-profile and then dims by photobleaching, may reflect kiss-and- run (K&R) events reported previously (reviewed in Alabi and Tsien [2013]). In a further series of experiments, Shin and colleagues imaged the release of neuro-peptide Y fused to GFP, with uptake of a red fluorophore from the bath. Similar types of fusion-fission were evident in these data.

Figure 1.

Live-Cell Imaging of the Chromaffin Cell Fusion-Fission Pore (A) Schematic of the experimental paradigm used in Shin et al. (2018). Adrenal chromaffin cells were Imaged with STED microscopy. Vesicle release was stimulated by a 1 s depolarizing step to give large Ca²⁺ influx. (B) The plasma membrane was labeled with GFP; the cells were superfused with a red fluorescent protein (Atto 532). (B₁) Cells were imaged in the XZ (top) and XY (bottom) planes. After stimulation, vesicular and plasma membranes fused, resulting in Ω-shaped GFP rings surrounding an Atto 532-filled lumen. The fusion pore itself was directly visualized in some cases (Pore_V) but not resolved in others (Pore_NoV). (B₂) After stimulation, Δt = 26 ms to 3 s, different types of fusion events occurred, with the fusion pore staying open (“stay”), decreasing in size (“shrink”), or shutting (“close”). (C) Competition between pore expansion, driven by F-actin mediated membrane tension, and pore constriction, supported by Ca²⁺ and dynamin, are shown to regulate fusion pore dynamics.

The authors next turned their attention toward the molecular mechanisms underlying pore dynamics. Shin et al. found that calcium current density was positively correlated with fusion pore constriction. Similarly, replacement of extracellular calcium with strontium increased the proportion of detectable pores and decelerated their closing, as if Sr²⁺ were a less potent effector than Ca²⁺ itself. Similar changes were caused by pharmacologically or genetically interfering with dynamin function, consistent with Ca²⁺- and dynamin- dependent pore constriction. Shin et al. went on to investigate the opposite process, membrane expansion, which has been associated with actin-dependent membrane tension. Addition of Latruncu- lin A, which hampers filamentous actin polymerization and thus generation of membrane tension, limited expansion of the fusion pore (Shin et al., 2018).

Overall, this paper provides the latest and most compelling evidence to date that secretory vesicles have potent mechanisms to avert full collapse fusion. Pores that transition directly from fusion to fission are directly visualized, along with the bidirectional passage of probes between vesicle lumen and extracellular space. The elegant experiments of Shin et al. complement pioneering studies in the 2000s, describing dynamin- dependent retrieval of secretory granules without loss of identity (Holroyd et al., 2002; Graham et al., 2002; Taraska et al., 2003). The new work shows that dynamin is not merely a pinchase, recruited at the finale of fission, but engages in ongoing interplay with actin to govern the pore’s variable aperture. When and how dynamin is recruited remains a mystery (Ryan, 2003), and what other forces might restrain vesicles needs further study (Alabi and Tsien, 2013).

In addition to the dynamin-dependent fusion-fission pores directly visualized here, earlier amperometry (Graham et al., 2002) suggested their co-existence with dynamin-independent processes that affected quantal size via (PKC- mediated) modulation of pore dynamics. Shin et al. stood clear of small pores, below the 60 nm resolution of their stimulated emission depletion (STED) microscopy, that undergo closure as the reversal of a metastable open pore but did not exclude their possibility. This idea, which dates back to even earlier observations of flickering cell capacitance and amperometric feet, aligns with recent studies of SNARE dynamics (Shi et al., 2012) but lacks the confirmation of direct imaging. Diverse approaches may be stitched together to encompass fusion pores too narrow for super-resolution imaging and wide pores too high in conductance for impedance measure-ments. Extension to the synaptic vesicles that support neurotransmission will not be trivial, given their small diameter (40-50 nm). Extrapolating from granules in neuroendocrine cells, Shin et al. envision synaptic vesicles with metastable fusion pores up to ~20 nm, roughly the size of quantum dots. Accordingly, they speculate that previous Qdot-based experiments (reviewed in Alabi and Tsien [2013]) may have underestimated the extent of kiss-and-run. Regardless, fusion with full flattening is looking less likely, for now at least, while the variety and extent of phenomena under the broad tent of kiss-and-run is on the rise.