Role of the synaptobrevin C terminus in fusion pore formation.
Ontology highlight
ABSTRACT:
SUBMITTER: Ngatchou AN
PROVIDER: S-EPMC2972926 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Although the importance of a SNARE complex in neurotransmitter release is widely accepted, there exist different views on how the complex promotes fusion. One hypothesis is that the SNARE complex's ability to bring membranes into contact is sufficient for fusion, another points to possible roles of juxtamembrane regions (JMRs) and transmembrane domains (TMDs) in catalyzing lipid rearrangement, and another notes the complex's presumed ability to bend membranes near the point of contact. Here, we performed experiments with highly curved vesicles brought into contact using low concentrations of polyethylene glycol (PEG) to investigate the influence of the synaptobrevin (SB) TMD with an attached JMR (SB-JMR-TMD) on the rates of stalk and pore formation during vesicle fusion. SB-JMR-TMD enhanced the rates of stalk and fusion pore (FP) formation in a sharply sigmoidal fashion. We observed an optimal influence at an average of three peptides per vesicle, but only with phosphatidylserine (PS)-containing vesicles. Approximately three SB-JMR-TMDs per vesicle optimally ordered the bilayer interior and excluded water in a similar sigmoidal fashion. The catalytic influences of hexadecane and SB-JMR-TMD on fusion kinetics showed little in common, suggesting different mechanisms. Both kinetic and membrane structure measurements support the hypotheses that SB-JMR-TMD 1) catalyzes initial intermediate formation as a result of its basic JMR disrupting ordered interbilayer water and permitting closer interbilayer approach, and 2) catalyzes pore formation by forming a membrane-spanning complex that increases curvature stress at the circumference of the hemifused diaphragm of the prepore intermediate state.
| S-EPMC4643208 | biostudies-literature
Project description:Planar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve elementary steps of the neuronal fusion process. However, in previous studies, we monitored only lipid mixing between fusing large unilamellar vesicles and PSMs and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into large unilamellar vesicles containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex ΔN49 prepared on gold-coated porous silicon substrates. By dual-color spinning disk fluorescence microscopy with a time resolution of ∼20 ms, we could unambiguously distinguish between bursting vesicles, which was only rarely observed (<0.01%), and fusion pore formation. From the time-resolved dual-color fluorescence time traces, we were able to identify different fusion pathways, including remaining three-dimensional postfusion structures with released content and transient openings and closings of the fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation after the merger of the two lipid membranes occurs almost simultaneously.
| S-EPMC7335915 | biostudies-literature
Project description:Bax protein plays a key role in mitochondrial membrane permeabilization and cytochrome c release upon apoptosis. Our recent data have indicated that the 20-residue C-terminal peptide of Bax (BaxC-KK; VTIFVAGVLTASLTIWKKMG), when expressed intracellularly, translocates to the mitochondria and exerts lethal effect on cancer cells. Moreover, the BaxC-KK peptide, as well as two mutants where the two lysines are replaced with glutamate (BaxC-EE) or leucine (BaxC-LL), have been shown to form relatively large pores in lipid membranes, composed of up to eight peptide molecules per pore. Here the pore structure is analyzed by polarized Fourier transform infrared, circular dichroism, and fluorescence experiments on the peptides reconstituted in phospholipid membranes. The peptides assume an ?/?-type secondary structure within membranes. Both ?-strands and ?-helices are significantly (by 30-60 deg) tilted relative to the membrane normal. The tryptophan residue embeds into zwitterionic membranes at 8-9 Å from the membrane center. The membrane anionic charge causes a deeper insertion of tryptophan for BaxC-KK and BaxC-LL but not for BaxC-EE. Combined with the pore stoichiometry determined earlier, these structural constraints allow construction of a model of the pore where eight peptide molecules form an "?/?-ring" structure within the membrane. These results identify a strong membranotropic activity of Bax C-terminus and propose a new mechanism by which peptides can efficiently perforate cell membranes. Knowledge on the pore forming mechanism of the peptide may facilitate development of peptide-based therapies to kill cancer or other detrimental cells such as bacteria or fungi.
| S-EPMC4537061 | biostudies-literature
Project description:Exocytosis is a highly regulated intercellular communication process involving various membrane proteins, lipids, and cytoskeleton restructuring. These components help control granule fusion with the cell membrane, creating a pore through which granular contents are released into the extracellular environment. Platelets are an ideal model system for studying exocytosis due to their lack of a nucleus, resulting in decreased membrane regulation in response to cellular changes. In addition, platelets contain fewer granules than most other exocytosing cells, allowing straightforward measurement of individual granule release with carbon-fiber microelectrode amperometry. This technique monitors the concentration of serotonin, an electroactive molecule found in the dense-body granules of platelets, released as a function of time, with 50 ?s time resolution, revealing biophysical characteristics of the fundamental exocytotic process. Variations in fusion pore formation and closure cause deviations from the classic current versus time spike profile and may influence diffusion of serotonin molecules from the site of granule fusion. Physiologically, the delivery of smaller packets of chemical messengers or the prolonged delivery of chemical messengers may represent how cells/organisms tune biological response. The goals of this work are twofold: 1) to categorize secretion features that deviate from the traditional mode of secretion and 2) to examine how changing the cholesterol composition of the platelet membrane results in changes in the pore formation process. Results herein indicate that the expected traditional mode of release is actually in the minority of granule content release events. In addition, results indicate that as the cholesterol content of the plasma membrane is increased, pore opening is less continuous.
| S-EPMC4776030 | biostudies-literature
Project description:Ca(2+)-triggered release of neurotransmitters and hormones depends on soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to drive the fusion of the vesicle and plasma membranes. The formation of the SNARE complex by the vesicle SNARE synaptobrevin 2 (syb2) and the two plasma membrane SNAREs syntaxin (syx) and SNAP-25 draws the two membranes together, but the events that follow membrane juxtaposition, and the ways that SNAREs remodel lipid membranes remain poorly understood. The SNAREs syx and syb2 have transmembrane domains (TMDs) that can exert force directly on the lipid bilayers. The TMD of syx influences fusion pore flux in a manner that suggests it lines the nascent fusion pore through the plasma membrane. The TMD of syb2 traverses the vesicle membrane and is the most likely partner to syx in completing a proteinaceous fusion pore through the vesicle membrane, but the role of this vesicle SNARE in fusion pores has yet to be tested. Here amperometry and conductance measurements were performed to probe the function of the syb2 TMD in fusion pores formed during catecholamine exocytosis in mouse chromaffin cells. Fusion pore flux was sensitive to the size and charge of TMD residues near the N terminus; fusion pore conductance was altered by substitutions at these sites. Unlike syx, the syb2 residues that influence fusion pore permeation fell along two α-helical faces of its TMD, rather than one. These results indicate a role for the syb2 TMD in nascent fusion pores, but in a very different structural arrangement from that of the syx TMD.
| S-EPMC4388931 | biostudies-literature
Project description:Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca(2+). Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kiss-and-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling.
| S-EPMC3557091 | biostudies-literature
Project description:Laser powder bed fusion additive manufacturing is an emerging 3D printing technique for the fabrication of advanced metal components. Widespread adoption of it and similar additive technologies is hampered by poor understanding of laser-metal interactions under such extreme thermal regimes. Here, we elucidate the mechanism of pore formation and liquid-solid interface dynamics during typical laser powder bed fusion conditions using in situ X-ray imaging and multi-physics simulations. Pores are revealed to form during changes in laser scan velocity due to the rapid formation then collapse of deep keyhole depressions in the surface which traps inert shielding gas in the solidifying metal. We develop a universal mitigation strategy which eliminates this pore formation process and improves the geometric quality of melt tracks. Our results provide insight into the physics of laser-metal interaction and demonstrate the potential for science-based approaches to improve confidence in components produced by laser powder bed fusion.
| S-EPMC6491446 | biostudies-literature
Project description:Cell-cell fusion in animal development and in pathophysiology involves expansion of nascent fusion pores formed by protein fusogens to yield an open lumen of cell-size diameter. Here we explored the enlargement of micron-scale pores in syncytium formation, which was initiated by a well-characterized fusogen baculovirus gp64. Radial expansion of a single or, more often, of multiple fusion pores proceeds without loss of membrane material in the tight contact zone. Pore growth requires cell metabolism and is accompanied by a local disassembly of the actin cortex under the pores. Effects of actin-modifying agents indicate that the actin cortex slows down pore expansion. We propose that the growth of the strongly bent fusion-pore rim is restricted by a dynamic resistance of the actin network and driven by membrane-bending proteins that are involved in the generation of highly curved intracellular membrane compartments.
| S-EPMC3552434 | biostudies-literature
Project description:Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarse-grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion.
| S-EPMC6294955 | biostudies-literature
Project description:SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules. Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25Δ9) showed changed fusion pore properties (Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events.
| S-EPMC4331636 | biostudies-literature