Exocytotic fusion pores exhibit semi-stable states

Abstract

Rapid-freezing/freeze-fracture electron microscopy and whole-cell capacitance techniques were used to study degranulation in peritoneal mast cells of the rat and the mutant beige mouse. These studies allowed us to create a time-resolved picture for fusion pore formation. After stimulation, a dimple in the plasma membrane formed a small contact area with the secretory granule membrane. Within this zone of apposition no ordered proteinaceous specializations were seen. Electrophysiological technique measured a small fusion pore which widened rapidly to 1 nS. Thereafter, the fusion pore remained at semi-stable conductances between 1 and 20 nS for a wide range of times, between 10 and 15,000 msec. These conductances correspond to pore diameters 25-36 nm. Ultrastructural data confirmed small pores of hourglass morphology, composed of biological membrane coplanar with both the plasma and granular membranes. Later, the fusion pore rapidly increased in conductance, consistent with the observed morphology of omega-figures. The hallmarks of channel-like behavior, instantaneous jumps in pore conductance between defined levels, and sharp peaks in histograms of conductance dwell-time, were not seen. Since the morphology of small pores shows contiguous fracture planes, the electrical data represent pores that contain lipid. These combined morphological and electrophysiological data are consistent with a lipid/protein complex mediating both the initial and later stages of membrane fusion.

Dynamic Regulation of the Large Exocytotic Fusion Pore in Pancreatic Acinar Cells

“…The kinetics of exocytosis are slow (Nemoto et al, 2001;Thorn et al, 2004), and we have proposed that endocytosis may occur by recovery of granule membrane in a piecemeal process . Here, we show that the fusion pore formed by the zymogen granule is one of the largest pores identified (Curran et al, 1993;Melikyan et al, 1995) (we estimate a pore diameter of between 29 and 55 nm), and yet it is capable of closing. We provide evidence that this closure is likely a prelude to endocytosis and that it occurs after the majority of granule contents are lost.…”

“…In other systems, there is evidence that the fusion pore is dynamic, changing over time and existing in one of three distinct states (Fernandez et al, 1984, Spruce et al, 1990Curran et al, 1993;Wang et al, 2001). It is first observed as a small pore with a conductance similar to that of an ion channel (Zimmerberg et al, 1987;Spruce et al, 1990).…”

“…However, these large pores are thought to occur at a terminal phase of exocytosis when the pore dilates and the granule collapses into the plasma membrane (Heuser and Reese, 1973;Curran et al, 1993). Semistable fusion pores, capable of closure, are thought to have much smaller diameters (Albillos et al, 1997;Alés et al, 1999).…”

Imaging single membrane fusion events mediated by SNARE proteins

“…However, release continues afterward for many tens to hundreds of milliseconds. The initial opening of the fusion pore has been monitored by capacitance; the pore flickers open and closed for 10-15,000 ms before it starts to expand (27)(28)(29)(30). The pore may continue to widen for hundreds of milliseconds before the vesicle is flattened into the plasma membrane.…”

Cytoplasmic Ca2+ Signals and Secretion in Exocrine and Endocrine Glands

“…The rate of fusion pore expansion of phase I was around 30 pS ms-I. In addition to this initial phase, which has been studied extensively (Breckenridge & Almers, 1987;Alvarez de Toledo & Fernandez, 1988;Curran et al 1993), we could distinguish two more stages clearly identified by their rate of expansion. An abrupt opening of the pore, phase H, had expansion rates as high as 3000 pS ms-I.…”