Two separate mechanisms are involved in membrane permeabilization during lipid oxidation.

Lipid oxidation is a universal degradative process of cell membrane lipids that is induced by oxidative stress and reactive oxygen and nitrogen species (RONS) in multiple pathophysiological situations. It has been shown that certain oxidized lipids alter membrane properties, leading to a loss of membrane function. Alteration of membrane properties is thought to depend on the initial membrane lipid composition, such as the number of acyl chain unsaturations. However, it is unclear how oxidative damage is related to biophysical properties of membranes. We therefore set out to quantify lipid oxidation through various analytical methods and determine key biophysical membrane parameters using model membranes containing lipids with different degrees of lipid unsaturation. As source for RONS, we used cold plasma, which is currently developed as treatment for infections and cancer. Our data revealed complex lipid oxidation that can lead to two main permeabilization mechanisms. The first one appears upon direct contact of membranes with RONS and depends on the formation of truncated oxidized phospholipids. These lipids seem to be partly released from the bilayer, implying that they are likely to interact with other membranes and potentially act as signaling molecules. This mechanism is independent of lipid unsaturation, does not rely on large variations in lipid packing, and is most probably mediated via short-living RONS. The second mechanism takes over after longer incubation periods and probably depends on the continued formation of lipid oxygen adducts such as lipid hydroperoxides or ketones. This mechanism depends on lipid unsaturation and involves large variations in lipid packing. This study indicates that polyunsaturated lipids, which are present in mammalian membranes rather than in bacteria, do not sensitize membranes to instant permeabilization by RONS but could promote long-term damage.

Microbubble-Assisted Ultrasound for Drug Delivery to the Retina in an Ex Vivo Eye Model.

Drug delivery to the retina is one of the major challenges in ophthalmology due to the biological barriers that protect it from harmful substances in the body. Despite the advancement in ocular therapeutics, there are many unmet needs for the treatment of retinal diseases. Ultrasound combined with microbubbles (USMB) was proposed as a minimally invasive method for improving delivery of drugs in the retina from the blood circulation. This study aimed to investigate the applicability of USMB for the delivery of model drugs (molecular weight varying from 600 Da to 20 kDa) in the retina of ex vivo porcine eyes. A clinical ultrasound system, in combination with microbubbles approved for clinical ultrasound imaging, was used for the treatment. Intracellular accumulation of model drugs was observed in the cells lining blood vessels in the retina and choroid of eyes treated with USMB but not in eyes that received ultrasound only. Specifically, 25.6 ± 2.9% of cells had intracellular uptake at mechanical index (MI) 0.2 and 34.5 ± 6.0% at MI 0.4. Histological examination of retinal and choroid tissues revealed that at these USMB conditions, no irreversible alterations were induced at the USMB conditions used. These results indicate that USMB can be used as a minimally invasive targeted means to induce intracellular accumulation of drugs for the treatment of retinal diseases.

Implementation of Artificial Intelligence in Diagnostic Practice as a Next Step after Going Digital: The UMC Utrecht Perspective.

Building on a growing number of pathology labs having a full digital infrastructure for pathology diagnostics, there is a growing interest in implementing artificial intelligence (AI) algorithms for diagnostic purposes. This article provides an overview of the current status of the digital pathology infrastructure at the University Medical Center Utrecht and our roadmap for implementing AI algorithms in the next few years.

Arrangements of proteins at reconstituted synaptic vesicle fusion sites depend on membrane separation.

Synaptic vesicle proteins, including N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), Synaptotagmin-1 and Complexin, are responsible for controlling the synchronised fusion of synaptic vesicles with the presynaptic plasma membrane in response to elevated cytosolic calcium levels. A range of structures of SNAREs and their regulatory proteins have been elucidated, but the exact organisation of these proteins at synaptic junction membranes remains elusive. Here, we have used cryoelectron tomography to investigate the arrangement of synaptic proteins in an in vitro reconstituted fusion system. We found that the separation between vesicle and target membranes strongly correlates with the organisation of protein complexes at junctions. At larger membrane separations, protein complexes assume a 'clustered' distribution at the docking site, inducing a protrusion in the target membrane. As the membrane separation decreases, protein complexes become displaced radially outwards and assume a 'ring-like' arrangement. Our findings indicate that docked vesicles can possess a wide range of protein complex numbers and be heterogeneous in their protein arrangements.

Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane-Proximal Regions of VAMP2 and SNAP25.

The neuronal protein complexin contains multiple domains that exert clamping and facilitatory functions to tune spontaneous and action potential-triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that, prior to fusion, the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitters in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.

A saposin-lipoprotein nanoparticle system for membrane proteins.

A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state.

Measuring kinetic drivers of pneumolysin pore structure.

Most membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins are thought to form pores in target membranes by assembling into pre-pore oligomers before undergoing a pre-pore to pore transition. Assembly during pore formation is into both full rings of subunits and incomplete rings (arcs). The balance between arcs and full rings is determined by a mechanism dependent on protein concentration in which arc pores arise due to kinetic trapping of the pre-pore forms by the depletion of free protein subunits during oligomerization. Here we describe the use of a kinetic assay to study pore formation in red blood cells by the MACPF/CDC pneumolysin from Streptococcus pneumoniae. We show that cell lysis displays two kinds of dependence on protein concentration. At lower concentrations, it is dependent on the pre-pore to pore transition of arc oligomers, which we show to be a cooperative process. At higher concentrations, it is dependent on the amount of pneumolysin bound to the membrane and reflects the affinity of the protein for its receptor, cholesterol. A lag occurs before cell lysis begins; this is dependent on oligomerization of pneumolysin. Kinetic dissection of cell lysis by pneumolysin demonstrates the capacity of MACPF/CDCs to generate pore-forming oligomeric structures of variable size with, most likely, different functional roles in biology.

Structural biology of the membrane attack complex.

The complement system is an intricate network of serum proteins that mediates humoral innate immunity through an amplification cascade that ultimately leads to recruitment of inflammatory cells or opsonisation or killing of pathogens. One effector arm of this network is the terminal pathway of complement, which leads to the formation of the membrane attack complex (MAC) composed of complement components C5b, C6, C7, C8 and C9. Upon formation of C5 convertases via the classical or alternative pathways of complement activation, C5b is generated from C5 by proteolytic cleavage, nucleating a series of association and polymerisation reactions of the MAC-constituting complement components that culminate in pore formation of pathogenic membranes. Recent structures of MAC components and homologous proteins significantly increased our understanding of oligomerisation, membrane association and integration, shedding light onto the molecular mechanism of this important branch of the innate immune system.

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.

Role of pore-forming toxins in neonatal sepsis.

Protein toxins are important virulence factors contributing to neonatal sepsis. The major pathogens of neonatal sepsis, group B Streptococci, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus, secrete toxins of different molecular nature, which are key for defining the disease. Amongst these toxins are pore-forming exotoxins that are expressed as soluble monomers prior to engagement of the target cell membrane with subsequent formation of an aqueous membrane pore. Membrane pore formation is not only a means for immediate lysis of the targeted cell but also a general mechanism that contributes to penetration of epithelial barriers and evasion of the immune system, thus creating survival niches for the pathogens. Pore-forming toxins, however, can also contribute to the induction of inflammation and hence to the manifestation of sepsis. Clearly, pore-forming toxins are not the sole factors that drive sepsis progression, but they often act in concert with other bacterial effectors, especially in the initial stages of neonatal sepsis manifestation.

Mucociliary clearance defects in a murine in vitro model of pneumococcal airway infection.

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where--in pneumococcal infection--persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.

Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition.

Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.

Perforin activity at membranes leads to invaginations and vesicle formation.

The cytotoxic cell granule secretory pathway is essential for immune defence. How the pore-forming protein perforin (PFN) facilitates the cytosolic delivery of granule-associated proteases (granzymes) remains enigmatic. Here we show that PFN is able to induce invaginations and formation of complete internal vesicles in giant unilamellar vesicles. Formation of internal vesicles depends on native PFN and calcium and antibody labeling shows the localization of PFN at the invaginations. This vesiculation is recapitulated in large unilamellar vesicles and in this case PFN oligomers can be seen associated with the necks of the invaginations. Capacitance measurements show PFN is able to increase a planar lipid membrane surface area in the absence of pore formation, in agreement with the ability to induce invaginations. Finally, addition of PFN to Jurkat cells causes the formation of internal vesicles prior to pore formation. PFN is capable of triggering an endocytosis-like event in addition to pore formation, suggesting a new paradigm for its role in delivering apoptosis-inducing granzymes into target cells.

Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells.

Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3-5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3-4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a=43.9, b=51.5, c=102.9 Šand space group P2(1)2(1)2(1) that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.

Rigid-body ligand recognition drives cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor triggering.

The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced "triggering" of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-Å resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s).

Phase-dependent lateral diffusion of α-tocopherol in DPPC liposomes monitored by fluorescence quenching.

The temperature-dependent fluorescence quenching of an amphiphilic palmitoyl derivative of 2,3-diazabicyclo[2.2.2]oct-2-ene (Fluorazophore-L) by α-tocopherol (α-Toc) has been determined in liposomes composed of a saturated lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The mutual lateral diffusion coefficients (D(L)) were extracted according to a laterally diffusion-controlled dynamic quenching model. Three distinct temperature regimes were identified: one between 65 and 39 °C, where the lateral diffusion coefficients were in the range of 10(-7) cm(2) s(-1) and the lifetime of the probe was monoexponential in the absence of α-Toc, a second one between 39 and 30 °C, where the lateral diffusion coefficients were in the range of 10(-8) cm(2) s(-1) and the lifetime of the probe was biexponential in the absence of α-Toc, and a third one below 30 °C, in which no diffusion was detectable, suggesting D(L) < 10(-9) cm(2)s (-1). These temperature domains were assigned, supported by differential scanning calorimetry (DSC) measurements, to the liquid-crystalline, ripple, and solid-gel phases of DPPC liposomes in the presence of the two additives. The absolute values of the individual lateral diffusion coefficients (taken as (1)/(2) of the D(L) values) of the Fluorazophore-L/α-Toc (ca. 2.5 × 10(-7) cm(2) s(-1) at 52 °C) couple demonstrates that α-Toc does not diffuse at an unexpectedly high rate in comparison to the self-diffusion of DPPC (1.5 × 10(-7) cm(2) s(-1) at 52 °C). However, diffusion in DPPC liposomes is distinctly slower than that in POPC ones (e.g., D(L) = 4.9 × 10(-7) cm(2) s(-1) versus 6.4 × 10(-7) cm(2) s(-1) at 50 °C), with an activation energy of 49 ± 5 kJ mol(-1) (value for POPC: 47 ± 5 kJ mol(-1)), in the temperature range of the liquid-crystalline phase. Diffusion in the ripple phase, that is, below the main phase transition temperature, was found to be non-negligible, with an apparent activation energy of 175 ± 50 kJ mol(-1).

Domain metastability: a molecular basis for immunoglobulin deposition?

We present the crystal structure of an immunoglobulin light-chain-like domain, CTLA-4, as a strand-swapped dimer displaying cis-trans proline isomerisation and native-like hydrogen bonding. We also show that CTLA-4 can form amyloid-like fibres and amorphous deposits explainable by the same strand swapping. Our results suggest a molecular basis for the pathological aggregation of immunoglobulin domains and why amyloid-like fibres are more often composed of homologous rather than heterologous subunits.

Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1.

Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. "Stealthing" of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.

Oligomerisation of pneumolysin on cholesterol crystals: similarities to the behaviour of polyene antibiotics.

Pneumolysin is a cytolytic toxin of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. The prepore and pore states of pneumolysin have recently been investigated by cryo-electron microscopy and atomic force microscopy, confirming the existence of arc-shaped as well as ring-form oligomers. Here we provide further insights into the pneumolysin oligomer by studying the interaction of pneumolysin with cholesterol crystals, comparing the results to those obtained for polyene antibiotics, which also bind cholesterol.