Fusion Pore Diameter Research Articles

Control of membrane fusion pore diameter constriction and widening

Eukaryotic endo- and exocytotic vesicles merge with the plasmalemma, thus forming an intermediate, the membrane fusion-pore. Current hypothesis holds that the fusion-pore, once established, enters a stable, dynamically regulated state, with diameters from subnanometers to several tens of nanometers. These pores can then undergo either reversible constriction-closure to limit vesicle discharge, or full widening (full fusion exocytosis), to facilitate vesicle discharge. Fusion-pore closure and widening was shown to involve proteins, consistent with the first proposal that the fusion-pore is a proteinaceous structure in 1990 by Almers and Tse.

Vesicle cholesterol controls exocytotic fusion pore

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

Vesicle Cholesterol Controls Exocytotic Fusion Pore

In lysosomal storage diseases (LSD) cholesterol accumulates in vesicles, which may affect vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes evokes vesicle hormonal secretion. A new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In fibroblasts lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

Local electrostatic interactions determine the diameter of fusion pores

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.

Fusion Properties of Gliotransmitter Vesicles in Cultured Astrocytes

Astrocytes actively participate in brain signalling by releasing a plethora of gliotransmitters which can modulate synaptic transmission. Vesicle-based mechanisms mediate the release of gliotransmitters. However, the anatomy and nature of exocytotic vesicle interaction with the plasma membrane is unclear. Using STED and SIM super-resolution microscopies, we studied the morphology of distinct gliotransmitter vesicles, whereas the interaction between a single vesicle with the plasma membrane was monitored by measurements of membrane capacitance (Cm), a parameter linearly related to the surface area of the plasma membrane. Immunolabelling of vesicles containing D-serine, glutamate, atrial natriuretic peptide (ANP) and brain derived neurotrophic factor (BDNF) yielded their diameter to be ∼70 nm, whereas ATP was found in larger vesicles (∼200 nm diameter). Cell-attached measurements have shown the predominant reversible unitary exocytotic events exhibiting transient fusion were found in two different populations, the first corresponding to smaller vesicles with diameters around 70 nm and the second corresponding to bigger vesicles with the median at 200 nm, consistent with the STED and SIM measurements. Upon stimulation with ATP, which increases intracellular Ca2+ concentration, the smaller vesicles persisted in transient fusion, whereas the bigger vesicles proceeded to full fusion. This was interpreted previously to be due to different SNARE protein densities in different sized vesicles and was tested here by astrocyte treatment with botulinum neurotoxin D (BotD), which significantly reduced the occurrence of unitary exocytotic events for both vesicle types. Additionally, when we expressed dominant-negative SNARE in astrocytes, the fusion-pore diameter was narrower in both vesicle types. Taken together, this work shows that vesicle content discharge is modulated by vesicle size, however independently of the functional integrity of the SNARE proteins, indicating that SNARE-dependent mechanisms determining vesicle merger with the plasmalemma are not strictly part of those modulating fusion-pore geometry.

Erratum to “Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy”

1 Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, 1000 Ljubljana, Slovenia 2 Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia 3 Departamento de Biologia e CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal 4 Celica Biomedical Center, Tehnoloski Park 24, 1000 Ljubljana, Slovenia 5 Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia

Munc18–1, exocytotic fusion pore regulation and local membrane anisotropy

The release of hormones and neurotransmitters from vesicles can be modified by the regulation of the fusion pore, an aqueous channel that forms upon the fusion of the vesicle membrane with the plasma membrane. However, the mechanisms are unclear. Munc18–1 protein interacts with Syntaxin1 (Synt 1), a member of the SNARE proteins, which plays an important role in exocytosis. It has been shown that Munc18–1 has multiple roles, both in pre- and post-fusion stages of exocytosis. It regulates the traffic of Synt1 to the plasma membrane. By inhibiting the tethering of the vesicle SNARE protein Synaptobrevin 2 (Syb2) solely to Synt1 at the plasma membrane, but favoring the vesicular tethering to the preformed binary cis SNARE complex of Synt1A-SNAP25B, Munc18–1 is tuning vesicle docking and the membrane merger process. Additionally, Munc18–1 affects exocytosis at the post-fusion stage by regulating the fusion pore properties (i.e., dwell-time and fusion pore diameter). Among many possible mechanisms that may regulate the fusion pore, but have never been considered previously, is the influence of Munc18–1 on the membrane anisotropy, which determines the local spontaneous membrane curvature and the architecture of the fusion pore. We here propose that Munc18–1 affects the fusion pore by modulating the dynamic local (re)arrangement of anisotropic membrane components within the highly curved fusion pore nanostructure, to which proteins, lipids or their complexes can participate.

Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy

The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion) or widen completely (full fusion) to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration) likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore.

Aluminium-induced changes of fusion pore properties attenuate prolactin secretion in rat pituitary lactotrophs

Hormone secretion is mediated by Ca 2+-regulated exocytosis. The key step of this process consists of the merger of the vesicle and the plasma membranes, leading to the formation of a fusion pore. This is an aqueous channel through which molecules stored in the vesicle lumen exit into the extracellular space on stimulation. Here we studied the effect of sub-lethal dose of aluminium on prolactin secretion in isolated rat pituitary lactotrophs with an enzyme immunoassay and by monitoring electrophysiologically the interaction of a single vesicle with the plasma membrane in real time, by monitoring membrane capacitance. After 24-h exposure to sub-lethal AlCl 3 (30 μM), the secretion of prolactin was reduced by 14±8% and 46±11% under spontaneous and K +-stimulated conditions, respectively. The frequency of unitary exocytotic events, recorded by the high-resolution patch-clamp monitoring of membrane capacitance, a parameter linearly related to the membrane area, under spontaneous and stimulated conditions, was decreased in aluminium-treated cells. Moreover, while the fusion pore dwell-time was increased in the presence of aluminium, the fusion pore conductance, a measure of fusion pore diameter, was reduced, both under spontaneous and stimulated conditions. These results suggest that sub-lethal aluminium concentrations reduce prolactin secretion downstream of the stimulus secretion coupling by decreasing the frequency of unitary exocytotic events and by stabilizing the fusion pore diameter to a value smaller than prolactin molecule, thus preventing its discharge into the extracellular space.

Fusion Pore: An Evolutionary Invention of Nucleated Cells

This article outlines the lecture presented by Robert Zorec at the Academia Europea meeting in Liverpool on 19 September 2008, four decades after the Sherrington Lecture of Bernard Katz who, together with his colleagues, developed a number of paradigms addressing vesicles in chemical synapses. Vesicles are subcellular organelles that evolved in eukaryotic cells 1000 to 2000 million years ago. They store signalling molecules such as chemical messengers, which are essential for the function of neurons and endocrine cells in supporting the communication between tissues and organs in the human body. Upon a stimulus, the vesicle-stored signalling molecules (neurotransmitters or hormones) are released from cells. This event involves exocytosis, a fundamental biological process, consisting of the merger of the vesicle membrane with the plasma membrane. The two fusing membranes lead to the formation of an aqueous channel – the fusion pore – through which signalling molecules exit into the extracellular space or blood stream. The work of Bernard Katz and colleagues considered that vesicle cargo discharge initially requires the delivery of vesicles to the plasma membrane, where vesicles dock and get primed for fusion with the plasma membrane, and that stimulation initiates the formation of the transient fusion pore through which cargo molecules leave the vesicle lumen in an all-or-none-fashion. However, recent studies indicate that this may not be so simple. Here we highlight the novel findings which indicate that fusion pores are subject to regulations, which affect the release competence of a single vesicle. At least in pituitary lactotrophs, which are the subject of research in our laboratories, single vesicle release of peptide signalling molecules involves modulation of fusion pore diameter and fusion pore kinetics.

Subnanometer Fusion Pores in Spontaneous Exocytosis of Peptidergic Vesicles

Kiss-and-run exocytosis, consisting of reversible fusion between the vesicle membrane and the plasma membrane, is considered to lead to full fusion after stimulation of vesicles containing classical transmitters. However, whether this is also the case in the fusion of peptidergic vesicles is unknown. Previously, we have observed that spontaneous neuropeptide discharge from a single vesicle is slower than stimulated release, because of the kinetic constraints of fusion pore opening. To explore whether slow spontaneous release also reflects a relatively narrow fusion pore, we analyzed the permeation of FM 4-64 dye and HEPES molecules through spontaneously forming fusion pores in lactotroph vesicles expressing synaptopHluorin, a pH-dependent fluorescent fusion marker. Confocal imaging showed that half of the spontaneous exocytotic events exhibited fusion pore openings associated with a change in synaptopHluorin fluorescence but were impermeable to FM 4-64 and HEPES. Together with membrane capacitance measurements, these findings indicate an open fusion pore diameter <0.5 nm, much smaller than the neuropeptides. In stimulated cells, >70% of exocytotic events exhibited a larger, FM 4-64-permeable pore (>1 nm). Interestingly, capacitance measurements showed that the majority of exocytotic events in spontaneous and stimulated conditions were transient. Stimulation increased the frequency of transient events and the fusion pore dwell time but decreased the fraction of events with lowest measurable fusion pore. Kiss-and-run is the predominant mode of exocytosis in resting and in stimulated peptidergic vesicles. Stimulation prolongs the effective opening of the fusion pore and expands its primary subnanometer diameter to enable hormone secretion without full fusion.

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.