Stabilization of the SNARE Core by Complexin-1 Facilitates Fusion Pore Expansion

In the neuron, neurotransmitter release is an essential function that must be both consistent and tightly regulated. The continuity of neurotransmitter release is dependent in large part on vesicle recycling. However, the protein factors that dictate the vesicle recycling pathway are elusive. Here, we use a single vesicle-to-supported bilayer fusion assay to investigate complexin-1 (cpx1)’s influence on SNARE-dependent fusion pore expansion. With total internal reflection (TIR) microscopy using a 10 kDa polymer fluorescence probe, we are able to detect the presence of large fusion pores. With cpx1, however, we observe a significant increase of the probability of the formation of large fusion pores. The domain deletion analysis reveals that the SNARE-binding core domain of cpx1 is mainly responsible for its ability to promote the fusion pore expansion. In addition, the results show that cpx1 helps the pore to expand larger, which results in faster release of the polymer probe. Thus, the results demonstrate a reciprocal relationship between event duration and the size of the fusion pore. Based on the data, a hypothetical mechanistic model can be deduced. In this mechanistic model, the cpx1 binding stabilizes the four-helix bundle structure of the SNARE core throughout the fusion pore expansion, whereby the highly curved bilayer within the fusion pore is stabilized by the SNARE pins.

Study on the Hydro-Thermal-Mechanical Characteristics of the Lime-Soil Compaction Pile in the Forming Process

To study the effect of the lime-soil compaction pile on the collapsible loess foundation and the influence of water-heat-force characteristics on the soil during compaction, the field test of lime-soil compaction pile treatment of collapsible loess foundation is carried out. This paper monitors the process of soil temperature, water, and soil pressure change in the process of pile forming. According to the macroprocess and micromechanism of lime-soil compaction pile treatment of collapsible loess foundation, the qualitative law of water-heat-force change in the process of pile forming is obtained. Meanwhile, the influence of soil damage is introduced in the process of compaction expansion. The model of pore expansion under linear damage conditions in the plastic zone is established. The formula of radial stress distribution under damage conditions is given. The influence of different damage factors on the expansion process is analyzed.

Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered LAMP (lysosome associated membrane protein)-carrier vesicles around multivesicular bodies, as well as the appearance of ‘hourglass’ profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of CHMP6 (charged multi-vesicular body protein 6) and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

Mechanical Characteristics and Mesostructural Damage of Saturated Limestone under Different Load and Unload Paths

To study the evolutionary characteristics of mesostructural damage to saturated limestone under different loading and unloading paths, three types of loading and unloading tests involving three different loading rates and initial peak stresses were performed. Nuclear magnetic resonance technology and scanning electron microscopy were used to investigate the evolutionary characteristics of pore water during the loading and unloading of the limestone. The results indicated that, with an increase in the initial peak stress, the rock viscoplasticity gradually decreased, and the variation of pore radius and the reduction of bound water decreased. With an increasing loading rate, the mesostructure evolution law under disturbance-increasing amplitude (DIA) cycling was opposite to those under increasing amplitude (IA) and repeated-increasing amplitude (RIA) cycling. With the increasing loading stress level, the porosity decreased and then increased. Under increasing amplitude cycling, a larger initial porosity resulted in higher pore compaction and expansion limits. Reducing the initial peak stress inhibited the pore expansion, whereas it had the opposite effect under RIA and DIA cycling. During loading and unloading, bound water exists in pores of organic matter and mesopores, and free water exists in macropores of intergranular and transgranular fractures. These changes indicate certain laws under different loading and unloading paths. The results of this study indicate that the mesostructure characteristics of rock depend on the loading and unloading paths.

Construction of Isostructural Hydrogen-bonded Organic Frameworks: Limitations and Possibilities of Pore Expansion

Library of isostructural porous frameworks enables systematic survey to optimize the structure and functionality of porous materials. Contrary to metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), a handful of...

Dynamic Evolution of Nanoscale Pores of Different Rank Coals Under Solvent Extraction

During the coalification process, coalbed methane (CBM) is formed and mainly adsorbed in the pores of coal. Pore structure evolution is critical to CBM adsorption/desorption and extraction. This paper puts forward two parameters, namely the variety degree x and variety gene σ, for characterizing pore structure through mercury injection tests. Then, under extraction with different solvents, the dynamic evolution characteristics of nanoscale pores are addressed and quantified by taking four different rank coals (lignite, medium-volatile bituminous coal, low-rank anthracite and mediumrank anthracite) from different coal mines of China as the study object. The results indicate that the content of meso- and macropores after solvent extraction is much larger, but that there is no obvious law with the content of transition pores and micropores in the size range of 50–7.2 nm, according to the basic data sets of specific surface area (SSA) and pore volume (PV) of all coal samples. This phenomenon can be explained by the pore increase and expansion effects in nanoscale pores during solvent extraction. Generally, with the increasing of the solvent extraction degree, the difference in variety degree x with respect to the total PV and total SSA of different coals shows a significant decreasing trend, which expresses a homogeneous development in the change in pore structure. In regard to different solvents, benzene mainly causes pore expansion in meso- and macropores, and CS2 has a great effect on micropores. Whereas acetone plays an important role in mesopores and transition pores with pore expansion, THF has various effects on different size pores. Further study with higher variety gene σ values shows that the total PV mainly depends on the change in the absolute content of meso- and macropores. While the change in the absolute content of transition pores and micropores (less than 50 nm) has a great influence on the total SSA. As the extraction degree increases, the influence of the transition pores and micropores on the total PV is increased, and then, the content of meso- and macropores also plays an important role on the total SSA. However, this effect is highly different for raw coals of different ranks.