Incomplete pneumolysin oligomers form membrane pores

Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.     

Electroporation of a lipid bilayer as a chemical reaction

When a cell's transmembrane potential is increased from a physiological one to more than 370 mV, the transmembrane current increases more than hundredfold within a millisecond. This is due to the formation of conductive pores in the membrane. We construct a model in which we conceive of pore formation as a voltage sensitive chemical reaction. The model predicts the logarithm of the pore formation rate to increase proportionally to the square of the voltage. We measure currents through frog muscle cell membranes under 8 ms pulses of up to 440 mV. The experimental data appear consistent with the model.     

SNARE-mediated vesicle navigation,vesicle anatomy and exocytotic fusion pore

The focus of this special issue (SI) »Membrane Merger in Conventional and Unconventional Vesicle Secretion« is regulated exocytosis, a universally conserved mechanism, consisting of a merger between the vesicle and the plasma membranes. Although this process evolved with eukaryotic organisms some three billion years ago (Spang et al., 2015), the understanding of physiology and patobiology of this process, especially at elementary vesicle level, remains unclear. Exocytotic fusion consists of several stages, starting by vesicle delivery to the plasma membrane, initially establishing a very narrow and stable fusion pore, that can reversibly open and close several times before it can fully widen. This allows vesicle cargo to be completely discharged from the vesicle lumen and permits vesicle-membrane resident proteins including channels, transporters, receptors and other signalling molecules, to be incorporated into the plasma membrane. The contributions in this SI bring new insights on the complexity of vesicle–based secretion, including discussion that vesicle anatomy appears to modulate exocytotic fusion pore properties and that the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins (SNARE-proteins), not only facilitate pre- and post-fusion stages of exocytosis, but also serve in vesicle navigation within the cytoplasm.     

Creating realistic silica nanopores for molecular dynamics simulations

We present a detailed approach to create realistic silica pores for computer simulations especially molecular dynamics (MD) simulations. These pores are essential for all different kinds of simulations with liquids in silica confinements. Despite wide use of silica pores in simulations, a detailed documentation how to create these pores for simulations still lacks. This issue is of high significance because with the help of this paper every researcher can build own silica pores with desired geometries and is not stick to already existing pores. We discuss problems that might occur during the whole process and how to solve these problems. So far more than 3 different silica pores have been created with this method and used successfully as confinement material in MD simulations.     

Reversible electrical breakdown of lipid bilayers: formation and evolution of pores

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.     

On the existence of a hysteresis loop in open and closed end pores

We have studied the adsorption of argon at 87 K in slit pores of finite length with a smooth graphitic potential, open at both ends or closed at one end. Simulations were carried out using conventional GCMC (grand canonical Monte Carlo) or kMC (kinetic Monte Carlo) in the canonical ensemble with extremely long Markov chain, of at least 2 × 10⁸ configurations; selected simulations with much longer Markov chains do not show any change in the results. When the pore width is in the micropore range (0.65 nm), type I isotherms are obtained for both pore models and for both simulation methods. However, wider pores (1, 2 and 3 nm in width) all exhibit hysteresis loops in the GCMC simulations, while in the canonical ensemble simulations, the isotherms pass through a sigmoid van der Waals type loop in the transition region. This loop locates the true equilibrium transition. For the pores with one closed end, this transition is close to, or coincides with, the adsorption branch of the GCMC hysteresis loop, but for the open-ended pores, it is more closely associated with the desorption branch. In a separate study of adsorption hysteresis in an infinitely long slit pore, using both simulation techniques, the van der Waals loop follows the adsorption branch of the GCMC isotherm to the transition, then reverts to a long vertical section that falls midway between the two hysteresis branches and finally moves to the desorption transition close to the evaporation pressure. An examination of molecular distributions inside the pores reveals two coexisting phases in the canonical simulations, whereas in the grand canonical simulations, the molecules are uniformly distributed along the length of the pores.     

Antimicrobial peptides bind more strongly to membrane pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis.     

立枯丝核菌菌丝隔膜超微结构的研究*

通过生长两天的菌丝超微结构的观察,我们首次发现在生长两天的菌丝中内含体堵塞隔膜孔帽的孔口和隔膜孔通道中折叠的细胞膜挤压通道,使通道成扁半圆形。因此,推测内含体和隔膜孔通道中折叠的细胞膜在菌丝隔膜形成后的最初阶段调控相邻细胞间物质的流量和物质交流,维持细胞间的动态平衡。通过生长两天的菌核内隔膜超微结构的观察,隔膜孔通道已被完全堵塞,菌丝细胞显示出了老化的结构特征。     

Mechanism of methane adsorption on groove space in organic matter surface

Organic matter plays an important role in methane adsorption in shale. Pore surface of organic matter is usually rough and uneven, which results in a large amount of groove space on the pore surface. Thus, the influence of groove space on the adsorption capacity of methane in shale cannot be neglected. Nanoscale pore structures of the organic-rich shale in the Longmaxi Formation were investigated by low-pressure nitrogen gas adsorption as a basis for constructing models. We simplified the internal groove space into triangular prisms with different angles. The grand canonical Monte Carlo simulation and molecular dynamics simulation were used to analyse the methane molecule adsorption behaviour in pores. The results showed that the pore morphology of organic-rich shale in the Longmaxi Formation was mainly slit-shaped pores. The excess adsorption isotherms showed good agreement between experiment and simulation, indicating that the model is suitable and reliable. Methane molecules can enter into the groove space with an opening size of 0.738 nm, while they fail to enter into groove spaces with an opening size less than 0.492 nm. This understanding has important significance for the study of the adsorption characteristics of organic pores which have undergone multiple evolutions in geological history.     

Flow dynamics in bioreactors containing tissue engineering scaffolds

Bioreactors are widely used in tissue engineering as a way to distribute nutrients within porous materials and provide physical stimulus required by many tissues. However, the fluid dynamics within the large porous structure are not well understood. In this study, we explored the effect of reactor geometry by using rectangular and circular reactors with three different inlet and outlet patterns. Geometries were simulated with and without the porous structure using the computational fluid dynamics software Comsol Multiphysics 3.4 and/or ANSYS CFX 11 respectively. Residence time distribution analysis using a step change of a tracer within the reactor revealed non-ideal fluid distribution characteristics within the reactors. The Brinkman equation was used to model the permeability characteristics with in the chitosan porous structure. Pore size was varied from 10 to 200 microm and the number of pores per unit area was varied from 15 to 1,500 pores/mm(2). Effect of cellular growth and tissue remodeling on flow distribution was also assessed by changing the pore size (85-10 microm) while keeping the number of pores per unit area constant. These results showed significant increase in pressure with reduction in pore size, which could limit the fluid flow and nutrient transport. However, measured pressure drop was marginally higher than the simulation results. Maximum shear stress was similar in both reactors and ranged approximately 0.2-0.3 dynes/cm(2). The simulations were validated experimentally using both a rectangular and circular bioreactor, constructed in-house. Porous structures for the experiments were formed using 0.5% chitosan solution freeze-dried at -80 degrees C, and the pressure drop across the reactor was monitored.     

Antimicrobial peptides in toroidal and cylindrical pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ∼ 7, the net charge of alamethicin is − 1 whereas that of melittin is + 5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions.     

Thermocyclops dumonti sp. n. (Crustacea,Copepoda), from a Temporary Waterbody in China

Six species of the genus Thermocyclops have hitherto been known from Chinese freshwaters. A new species is here recorded from a sample collected from an eutrophic pool in central China. It is described and compared using classical morphology and mapping of its pore signature. Thermocyclops dumonti differs from T. kawamurai by absence of ornamentation on prominences of intercoxal plates of P₁‐P₃, by relative length of apical spines of Enp₃P₄ and caudal ramus. It differs from T. dybowskii by ornamentation of P₄ intercoxal plate, relative length of Enp₃P₄ and caudal ramus, by shape of Tmi. As in other species of Thermocyclops, the perforations are bilaterally symmetrical and, species‐specific patterns occur on the cephalosome, metasome, and urosome. Conserved patterns are found elsewhere on the rostrum, cephalosome, metasome, and furcal branches. Based on pore pattern, Thermocyclops dumonti is separated from two close relatives, T. schmeili and T. dybowskii.     

Contacts of pore tubules and sensory dendrites in antennal chemosensilla of a silkmoth: Demonstration of a possible pathway for olfactory molecules

Antennal olfactory hairs of Antheraea polyphemus were investigated by means of transmission electron microscopy. Adequate preservation of dendrites and extracellular pore tubules is obtained by mechanical opening of the hair lumen and subsequent chemical fixation. The dendritic membrane has a cell coat. The dendrites contain microfilamentous structures in addition to their cytoplasmatic microtubules. The extracellular pore tubules traverse the hair cuticle and reach into the hair lumen for maximally 350 nm. Their diameter varies between 20 and 40 nm, depending on the preparation method. They consist of an electron-dense wall and an electron-lucent core. The wall has a helical substructure and is covered with a fuzzy coat. Contacts of pore tubules and dendritic membranes occur wherever dendrites are near the inner surface of the hair cuticle. Some of the pore tubules terminate approximately at right angles on the dendritic membrane, others lie against the membrane. The contact seems to be made via the surface coats of the tubules and the membrane. The structure of pore tubules which had been negatively stained with uranyl acetate is similar to the conventionally thin-sectioned material. The observations provide support for earlier assumptions that pore tubules are the pathways by which odor molecules reach the dendritic membrane.     

Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli

Abstract

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs.     

State-Dependent Accessibility of the P-S6 Linker of Pacemaker (HCN) Channels Supports a Dynamic Pore-to-Gate Coupling Model

The hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles K_v channels, much less is known about their structure-function correlation. Previously, we identified several pore residues in the S5-P linker and P-loop that are externally accessible and/or influence HCN gating, and proposed an evolutionarily conserved pore-to-gate mechanism. Here we sought dynamic evidence by assessing the functional consequences of Cys-scanning substitutions in the unexplored P-S6 linker (residues 352–359), the HCN1-R background (that is, resistant to sulfhydryl-reactive agents). None of A352C, Q353C, A354C, P355C, V356C, S357C, M358C, or S359C produced functional currents; the loss-of-function of Q353C, A354C, S357C, and M358C could be rescued by the reducing agent dithiothreitol. Q353C, A354C, and S357C, but not M358C and HCN1-R, were sensitive to Cd²⁺ blockade (IC₅₀ = 3–12 μM vs. >1 mM). External application of the positively charged covalent sulfhydryl modifier MTSET irreversibly reduced I _−140mV of Q353C and A354C to 27.9 ± 3.4% and 58.2 ± 13.1% of the control, respectively, and caused significant steady-state activation shifts (∆V _1/2 = –21.1 ± 1.6 for Q353C and −10.0 ± 2.9 mV for A354C). Interestingly, MTSET reactivity was also state dependent. MTSET, however, affected neither S357C nor M358C, indicating site specificity. Collectively, we have identified novel P-S6 residues whose extracellular accessibility was sterically and state dependent and have provided the first functional evidence consistent with a dynamic HCN pore-to-gate model.     

Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane

Perfringolysin O (PFO) is a prototype of the large family of pore-forming cholesterol-dependent cytolysins (CDCs). A central enigma of the cytolytic mechanism of the CDCs is that their membrane-spanning beta-hairpins (the transmembrane amphipathic beta-hairpins (TMHs)) appear to be approximately 40 A too far above the membrane surface to cross the bilayer and form the pore. We now present evidence, using atomic force microscopy (AFM), of a significant difference in the height by which the prepore and pore protrude from the membrane surface: 113+/-5 A for the prepore but only 73+/-5 A for the pore. Time-lapse AFM micrographs show this change in height in real time. Moreover, the monomers in both complexes exhibit nearly identical surface features and these results in combination with those of spectrofluorimetric analyses indicate that the monomers remain in a perpendicular orientation to the bilayer plane during this transition. Therefore, the PFO undergoes a vertical collapse that brings its TMHs to the membrane surface so that they can extend across the bilayer to form the beta-barrel pore.     

Discovery of the 'porosome'; the universal secretory machinery in cells

The release of neurotransmitters at the nerve terminal for neurotransmission, release of insulin from beta-cells of the endocrine pancreas for regulating blood glucose levels, the release of growth hormone from GH cells of the pituitary gland to regulate body growth, or the expulsion of zymogen from exocrine pancreas to digest food, are only a few examples of key physiological processes made possible by cell secretion. It comes as no surprise that defects in cell secretion are the cause for numerous diseases, and have been under intense investigation for over half century. Only in the last decade, the molecular machinery and mechanism of cell secretion has become clear. Cell secretion involves the docking and transient fusion of membrane-bound secretory vesicles at the base of plasma membrane structures called porosomes, and the regulated expulsion of intravesicular contents to the outside, by vesicle swelling. The discovery of the porosome in live cells, its morphology and dynamics at nanometer resolution and in real time, its isolation, its composition, and its structural and functional reconstitution in lipid membrane, are complete. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and the regulated expulsion of intravesicular contents during cell secretion, are also resolved. In this minireview, the monumental discovery of the porosome, a new cellular structure at the cell plasma membrane, is briefly discussed.     

Vanadium compounds induced mitochondria permeability transition pore (PTP) opening related to oxidative stress

Vanadium compounds have been regarded as promising in therapeutic treatment of diabetes and in cancer prevention. In the present work, we studied the effects of vanadium compounds on mitochondria to investigate the mechanisms of toxicity. Mitochondria were isolated from rat liver and incubated with a variety of vanadium compounds, i.e. VOSO₄, NaVO₃, and vanadyl complexes with organic ligands. Our studies indicated that VO²⁺, , VO(acac)₂ and VOcit (1-100 μM) could induce mitochondrial swelling in a concentration dependent manner and disrupt mitochondrial membrane potential (Δψ_m) in a time dependent manner, which is quite different from the rapid Δψ_m collapse caused by Ca²⁺ or CCCP (carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupling reagent). Release of cytochrome c (Cyt c) was observed and could be inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore (PTP). Interestingly, VOdipic caused release of Cyt c without mitochondrial swelling and Δψ_m disruption, an action previously only observed on the Bax protein, suggesting a potentially role of VOdipic in regulating PTP opening. In addition, all the vanadium compounds tested stimulated mitochondrial production of reactive oxygen species (ROS). Antioxidants, i.e. vitamin C and E, significantly delayed the Δψ_m disruption. Overall, our experimental evidence indicated vanadium compounds exhibited multiple actions on mitochondria. Vanadium compounds did induce oxidative stress on mitochondrial and thus caused PTP opening, which led to collapse of Δψ_m and Cyt c release as the initiation of cell apoptosis.     

Fusion pore stability of peptidergic vesicles

Abstract

It is believed that in regulated exocytosis the vesicle membrane fuses with the plasma membrane in response to a physiological stimulus. However, in the absence of stimulation, repetitive transient fusion events are also observed, reflecting a stable state. The mechanisms by which the initial fusion pore attains stability are poorly understood. We modelled energetic stability of the fusion pore by taking into account the anisotropic, intrinsic shape of the membrane constituents and their in-plane ordering in the local curvature of the membrane. We used cell-attached membrane capacitance techniques to monitor the appearance and conductance of single fusion pore events in cultured rat lactotrophs. The results revealed a bell-shaped distribution of the fusion pore conductance with a modal value of 25 pS. The experimentally observed increase of the fusion pore stability with decreasing fusion pore radius agrees well with the theoretical predictions. Moreover, the results revealed a correlation between the amplitude of transient capacitance increases and the fusion pore conductance, indicating that larger vesicles may attain a stable fusion pore with larger fusion pore diameters.