GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi‐protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill‐characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N‐terminal fragment of caspase‐1‐cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N‐terminal fragment of caspase‐1‐cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome‐inserted GSDMD at nanometer resolution by cryo‐electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.     

Single molecule atomic force microscopy of aerolysin pore complexes reveals unexpected star‐shaped topography

Aerolysin is the paradigmatic member of a large family of toxins that convert from a water‐soluble monomer/dimer into a membrane‐spanning oligomeric pore. While there is x‐ray crystallographic data of its water‐soluble conformation, the most recent structural model of the membrane‐inserted pore is based primarily on data of water‐soluble tetradecamers of mutant protein, together with computational modeling ultimately performed in vacuum. Here we examine this pore model with atomic force microscopy (AFM) of membrane‐associated wild‐type complexes and all‐atom molecular dynamics (MD) simulations in water. In striking contrast to a disc‐shaped cap region predicted by the present model, the AFM images reveal a star‐shaped complex, with a central ring surrounded by seven radial projections. Further, the MD simulations suggest that the locations of the receptor‐binding (D1) domains in the present model are not correct. However, a modified model in which the D1 domains, rather than localized at fixed positions, adopt a wide range of configurations through fluctuations of an intervening linker is compatible with existing data. Thus our work not only demonstrates the importance of directly resolving such complexes in their native environment but also points to a dynamic receptor binding region, which may be critical for toxin assembly on the cell surface. Copyright © 2015 John Wiley & Sons, Ltd.     

Single organelle dynamics linked to 3D structure by correlative live‐cell imaging and 3D electron microscopy

Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB‐SEM, which significantly reduces time required for image acquisition and data processing.      

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin‐like protein

Various aerolysin‐like pore‐forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin‐like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a β‐prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high‐mannose glycans triggers drastic conformational changes of the aerolysin module in a pH‐dependent manner, ultimately resulting in the formation of a membrane‐bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore‐forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish‐specific defense molecule.     

Direct evidence for membrane pore formation by the apoptotic protein Bax

Direct imaging of the interaction of the apoptotic protein, Bax, with membrane bilayers shows the presence of toroidal-shaped pores using atomic force microscopy. These pores are sufficiently large to allow passage of proteins from the intermitochondrial space. Both the perturbation of the membrane and the amount of protein bound to the bilayer are increased in the presence of calcium. The results from the imaging are consistent with leakage studies from liposomes of the same composition. The work shows that Bax by itself can form pores in membrane bilayers.     

Membrane‐directed molecular assembly of the neuronal SNARE complex

Since the discovery and implication of N‐ethylmaleimide‐sensitive factor (NSF)‐attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full‐length recombinant t‐SNAREs and v‐SNARE present in opposing liposomes, leading to the assembly of a t‐/v‐SNARE ring complex. Using high‐resolution electron microscopy, the electron density maps and 3D topography of the membrane‐directed SNARE ring complex was determined at nanometre resolution. Similar to the t‐/v‐SNARE ring complex formed when 50 nm v‐SNARE liposomes meet a t‐SNARE‐reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t‐SNARE‐reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane‐directed t‐/v‐SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome‐reconstituted recombinant t‐/v‐SNARE proteins, and native v‐SNARE present in isolated SV membrane, the membrane‐directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.     

Anchorage‐dependent binding of integrin I‐domain to adhesion ligands

The dynamic interactions between leukocyte integrin receptors and ligands in the vascular endothelium, extracellular matrix, or invading pathogens result in leukocyte adhesion, extravasation, and phagocytosis. This work examined the mechanical strength of the connection between iC3b, a complement component that stimulates phagocytosis, and the ligand‐binding domain, the I‐domain, of integrin α_Mβ₂. Single‐molecule force measurements of α_M I‐domain–iC3b complexes were conducted by atomic force microscope. Strikingly, depending on loading rates, immobilization of the I‐domain via its C‐terminus resulted in a 1.3‐fold to 1.5‐fold increase in unbinding force compared with I‐domains immobilized via the N‐terminus. The force spectra (unbinding force versus loading rate) of the I‐domain–iC3b complexes revealed that the enhanced mechanical strength is due to a 2.4‐fold increase in the lifetime of the I‐domain–iC3b bond. Given the structural and functional similarity of all integrin I‐domains, our result supports the existing allosteric regulatory model by which the ligand binding strength of integrin can be increased rapidly when a force is allowed to stretch the C‐terminus of the I‐domain. This type of mechanism may account for the rapid ligand affinity adjustment during leukocyte migration. Copyright © 2015 John Wiley & Sons, Ltd.     

‘Porosome’ discovered nearly 20 years ago provides molecular insights into the kiss‐and‐run mechanism of cell secretion

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950s, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of an additional mechanism that allows partial discharge of intra‐vesicular contents during secretion. This proposed mechanism requires the involvement of a plasma membrane structure called ‘porosome’, which serves to prevent the collapse of secretory vesicles, and to transiently fuse with the plasma membrane (Kiss‐and‐run), expel a portion of its contents and disengage. Porosomes are cup‐shaped supramolecular lipoprotein structures at the cell plasma membrane ranging in size from 15 nm in neurons and astrocytes to 100–180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins. In comparison, the 120 nm nuclear pore complex is composed of >500 protein molecules. Elucidation of the porosome structure, its chemical composition and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex resulting in the establishment of membrane continuity to form a fusion pore at the porosome base, has been demonstrated. Additionally, the molecular mechanism of secretory vesicle swelling, and its requirement for intra‐vesicular content release during cell secretion has also been elucidated. Collectively, these observations provide a molecular understanding of cell secretion, resulting in a paradigm shift in our understanding of the secretory process.     

AFM‐ and NSOM‐based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane

Although CD69 is well known as an early T cell‐activation marker, the possibility that CD69 are distributed as nano‐structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force‐binding nanotechnology as well as near‐field scanning optical microscopy (NSOM)‐/fluorescence quantum dot (QD)‐based nanosacle imaging. Unstimulated CD4⁺ T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab‐functinalized AFM tip, and no detectable QD‐bound CD69 as examined by NSOM/QD‐based imaging. In contrast, Phytohemagglutinin (PHA)‐activated CD4⁺ T cells expressed CD69, and displayed many force‐binding spots binding to the CD69 Ab‐functionalized AFM tip on about 45% of cell membrane, with mean binding‐rupture forces 276 ± 71 pN. Most CD69 molecules appeared to be expressed as 100–200 nm nanoclusters on the membrane of PHA‐activated CD4⁺ T cells. Meanwhile, NSOM/QD‐based nanoscale imaging showed that CD69 were non‐uniformly distributed as 80–200 nm nanoclusters on cell‐membrane of PHA‐activated CD4⁺ T cells. This study represents the first demonstration of the nano‐biology of CD69 expression during T cell activation. Copyright © 2009 John Wiley & Sons, Ltd.     

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

Self‐assembly of regenerated silk fibroin from random coil nanostructures to antiparallel β‐sheet nanostructures

In this work, we studied the effects of incubation concentration and time on the self‐assembly behaviors of regenerated silk fibroin (RSF). Our results showed the assembly ways of RSF were concentration‐dependent and there were four self‐assembly ways of RSF: (i) At relatively low concentration (≤0.015%), RSF molecules assembled into protofilaments (random coil), and then the thickness decreased and the secondary conformation changed to antiparallel β‐sheet; (ii) at the concentration of 0.015%, RSF molecules assembled into protofilaments (random coil), and then assembled into protofibrils (antiparallel β‐sheet). The protofibrils experienced the appearance and disappearance of phase periodic intervals in turn; (iii) at the concentration of 0.03%, RSF molecules assembled into bead‐like oligomers (random coil), and then assembled into protofibrils (antiparallel β‐sheet), and finally the height and phase periodic intervals of RSF protofibrils disappeared in turn; and (iv) at the relatively high concentration (≥0.15%), RSF molecules assembled into protofilaments (random coil), then aggregated into blurry cuboid‐like micelles (random coil), and finally self‐arranged to form smooth and clear cuboid‐like micelles (antiparallel β‐sheet). These results provide useful insights into the process by which the RSF molecules self‐assemble into protofilaments, protofibrils and micelles. Furthermore, our work will be beneficial to basic understanding of the nanoscale structure formations in different silk‐based biomaterials. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1181–1192, 2014.     

Ocellatin‐PT antimicrobial peptides: High‐resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin‐PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin‐PT peptides have an α‐helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria‐mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin‐PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.     

Operando Observations of SEI Film Evolution by Mass‐Sensitive Scanning Transmission Electron Microscopy

The solid electrolyte interphase (SEI) spontaneously formed on anode surfaces as a passivation layer plays a critical role in the lithium dissolution and deposition upon discharge/charge in lithium ion batteries and lithium‐metal batteries. The formation kinetics and failure of the SEI films are the key factors determining the safety, power capability, and cycle life of lithium ion and lithium‐metal batteries. Since SEI films evolve with the volumetric and interfacial changes of anodes, it is technically challenging in experimental study of SEI kinetics. Here operando observations are reported of SEI formation, growth, and failure at a high current density by utilizing a mass‐sensitive Cs‐corrected scanning transmission electron microscopy. The sub‐nano‐scale observations reveal a bilayer hybrid structure of SEI films and demonstrate the radical assisted SEI growth after the SEI thickness beyond the electron tunneling regime. The failure of SEI films is associated with rapid dissolution of inorganic layers when they directly contact with the electrolyte in broken SEI films. The initiation of cracks in SEI films is caused by heterogeneous volume changes of the electrodes during delithiation. These microscopic insights have important implications in understanding SEI kinetics and in developing high‐performance anodes with the formation of robust SEI films.     

Plasma membrane and nuclear envelope integrity during the blebbing stage of apoptosis: a time‐lapse study

Background information. The execution phase of apoptosis is characterized by extensive blebbing of the plasma membrane, which usually results in secondary lysis in vitro. To analyse the permeability of cellular membranes during this process, we induced apoptosis in human melanoma A375 cells that had been transfected with fluorescently tagged proteins which were targeted to different subcellular locations. Results. The dual treatment of resveratrol and butyrate produced a synergistic induction of apoptosis by blocking different phases of the cell cycle. Changes in the plasma membrane, nuclear envelope and nucleoli were monitored by time‐lapse confocal microscopy. Fluorescently labelled proteins were not mis‐localized from their original locations in any of the cells undergoing blebbing for several hours. Thus the maintenance of karyophilic and nucleolar proteins within the nucleus during the blebbing stage and the accessibility of vital selective chromatin dyes confirmed a functional preservation of the nuclear compartment until the final necrotic blister. The translocation of phosphatidylserine to the outer leaflet of the plasma membrane was not detected during the blebbing period. Conclusion. These results show that the functional integrity of the nuclear envelope and plasma membrane may be conserved until the end of the execution phase of apoptosis.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.     

Magnetic and structural properties of magnetite in radular teeth of chiton Acanthochiton rubrolinestus

The teeth of the Polyplacophora Chiton Acanthochiton Rubrolinestus contain biomineralized magnetite crystallites whose biological functions in relation to structure and magnetic properties are not well understood. Here, using superconducting quantum interference device (SQUID) magnetometry, we find that the saturation magnetization (σ(s)) and the Verwey transition temperature (T(v)) of tooth particles are 78.4 emu/g and 105 K, respectively. These values are below those of the stoichiometric magnetite. An in situ examination of the structure of the magnetite-bearing region within an individual tooth using high-resolution transmission electron microscopy indicates magnetite microcrystals form electron dense polycrystalline sheets with typical lengths of about 800 nm and widths of about 150 nm. These polycrystalline sheets are arranged regularly along the longitudinal direction of the tooth cutting surface. In addition, the crystallites in polycrystalline sheets take on generally good crystallinity. The magnetic microstructures of in situ magnetic force microscopy demonstrate that the [111] easy direction of magnetite microcrystals are aligned along the length of the tooth, whereas the [111] direction is parallel to the thickness of the tooth. Both M?ssbauer spectra and magnetization versus temperature measurements under field cooled and zero-field cooled conditions do not detect superparamagnetic magnetite crystallites in the mature major lateral tooth particles of this chiton.     

Comparative Cryo‐SEM and AFM studies of hylid and rhacophorid tree frog toe pads

Cryo‐scanning electron microscopy (cryo‐SEM) and atomic force microscopy (AFM) offer new avenues for the study of the morphology of tree frog adhesive toe pads. Using these techniques, we compare toe pad microstructure in two distantly related species of tree frog, Litoria caerulea, White (Hylidae) and Rhacophorus prominanus, Smith (Rhacophoridae), in which the toe pads are considered to be convergent. AFM demonstrates the extraordinary similarity of both surface microstructures (largely hexagonal epithelial cells surrounded by deep channels) and nanostructures (an array of nanopillars, ca. 350 nm in diameter, all with a small dimple at the apex). The cryo‐SEM studies examined the distribution of the fibrillar cytoskeleton within the different layers of the stratified toe pad epithelium, demonstrating that the cytoskeletal elements (keratin tonofilaments) that lie at an angle to the surface are relatively poorly developed in L. caerulea, clearly so in comparison to R. prominanus. Cryo‐SEM also enabled the visualization of the fluid layer that is critical to a toe pad's adhesive function. This was achieved by examination of the frozen fluid residues left behind after removal of a toe within the cryo‐SEM's experimental chamber. Such ‘toeprints’ demonstrated the presence of a wedge of fluid surrounding each toe pad, as well as fluid filling the channels that surround each epithelial cell. Cryo‐SEM was used to examine epithelial cell shape. In a sample of 582 cells, 59.5% were hexagonal, the remainder being mainly pentagonal (23.1%) or heptagonal (16.1%). The distribution of differently‐shaped cells was not random, but was not associated with either pad curvature or the distribution of mucous pores that provide fluid for the frogs' wet adhesion mechanism. Our main finding, the great similarity of toe pad structure in these two species, has important implications for biomimetics, for such convergent evolution suggests a good starting point for attempts to develop adhesives that will function in wet conditions. J. Morphol. 274:1384–1396, 2013. © 2013 Wiley Periodicals, Inc.     

Essential role of proline isomerization in stefin B tetramer formation

Here we present the tetrameric structure of stefin B, which is the result of a process by which two domain-swapped dimers of stefin B are transformed into tetramers. The transformation involves a previously unidentified process of extensive intermolecular contacts, termed hand shaking, which occurs concurrently with trans to cis isomerization of proline 74. This proline residue is widely conserved throughout the cystatin superfamily, a member of which, human cystatin C, is the key protein in cerebral amyloid angiopathy. These results are consistent with the hypothesis that isomerization of proline residues can play a decisive role in amyloidogenesis.     

Facile and Efficient Atomic Hydrogenation Enabled Black TiO2 with Enhanced Photo‐Electrochemical Activity via a Favorably Low‐Energy‐Barrier Pathway

Black TiO₂ has demonstrated a great potential for a variety of renewable energy technologies. However, its practical application is heavily hindered due to lack of efficient hydrogenation methods and a deeper understanding of hydrogenation mechanisms. Here, a simple and straightforward hot wire annealing (HWA) method is presented to prepare black TiO₂ (H–TiO₂) nanorods with enhanced photo‐electrochemical (PEC) activity by means of atomic hydrogen [H]. Compared to conventional molecular hydrogen approaches, the HWA shows remarkable effectiveness without any detrimental side effects on the device structure, and simultaneously the photocurrent density of H–TiO₂ reaches 2.5 mA cm^?2 (at 1.23 V vs reversible hydrogen electrode (RHE)). Due to the controllable and reproducible [H] flux, the HWA can be developed as a standard hydrogenation method for black TiO₂. Meanwhile, the relationships between the wire temperatures, structural, optical, and photo‐electrochemical properties are systematically investigated to verify the improved PEC activity. Furthermore, the density functional theory (DFT) study provides a comprehensive insight not only into the highly efficient mechanism of the HWA approach but also its favorably low‐energy‐barrier hydrogenation pathway. The findings will have a profound impact on the broad energy applications of H–TiO₂ and contribute to the fundamental understanding of its hydrogenation.     

Direct visualization of KirBac3.1 potassium channel gating by atomic force microscopy

KirBac3.1 belongs to a family of transmembrane potassium (K⁺) channels that permit the selective flow of K-ions across biological membranes and thereby regulate cell excitability. They are crucial for a wide range of biological processes and mutations in their genes cause multiple human diseases. Opening and closing (gating) of Kir channels may occur spontaneously but is modulated by numerous intracellular ligands that bind to the channel itself. These include lipids (such as PIP₂), G-proteins, nucleotides (such as ATP) and ions (e.g. H⁺, Mg²⁺, Ca²⁺). We have used high-resolution atomic force microscopy (AFM) to examine KirBac3.1 in two different configurations. AFM imaging of the cytoplasmic surface of KirBac3.1 embedded in a lipid bilayer has allowed visualization of the tetrameric assembly of the ligand-binding domain. In the absence of Mg²⁺, the four subunits appeared as four protrusions surrounding a central depression corresponding to the cytoplasmic pore. They did not display 4-fold symmetry, but formed a dimer-of-dimers with 2-fold symmetry. Upon addition of Mg²⁺, a marked rearrangement of the intracellular ligand-binding domains was observed: the four protrusions condensed into a single protrusion per tetramer, and there was an accompanying increase in protrusion height. The central cavity within the four intracellular domains also disappeared on addition of Mg²⁺, indicating constriction of the cytoplasmic pore. These structural changes are likely transduced to the transmembrane helices, which gate the K⁺ channel. This is the first time AFM has been used as an interactive tool to study K⁺ channels. It has enabled us to directly measure the conformational changes in the protein surface produced by ligand binding.