Study of Na Deposition Formation in Mixed Ethylene: Propylene Carbonate Electrolytes by Inert/Cryoelectron Microscopy.

The sodium anode-free combines low-cost and high energy density, demonstrating a promising alternative to the Li battery counterpart. Nevertheless, the uptake of a sodium anode-free battery is greatly impeded by the uncontrollable dendrite proliferation upon the chemically active metallic Na. An insightful mechanistic understanding of Na deposition nucleation and growth behavior in ethylene carbonate and propylene carbonate (EC/PC, 1:1) is revealed via various inert and/or cryo-electron microscopy characterization techniques. The deposit morphology, size, and distribution were studied with different current densities and areal capacity. The Na deposit distribution changes from nonparametric distribution to normal distribution which can be attributed to the effect of interparticle diffusion coupling (IDP). The atomic information on the Na deposit was revealed via cryogenic transmission electron microscopy.

Cryo-ultramicrotomy and Mass Spectrometry Imaging Analysis of Nudibranch Microstructures.

In this paper, we investigate the presence of latrunculin A in the outer rim of a nudibranch Chromodoris kuiteri and show that by combining ultrathin cryosection methods with MALDI MSI we can achieve improved lateral (x and y) resolution and very high resolution in the z dimension by virtue of the ultrathin 200 nm thin cryosections. We also demonstrate that a post ionization laser increases sensitivity. Recent advances in MALDI source design have improved the lateral resolution (x and y) and sensitivity during MSI. Taken together, very high z resolution, from ultrathin sections, and improved lateral (x and y) resolution will allow for subcellular molecular imaging with the potential for subcellular 3D volume reconstruction.

Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle.

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule-associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.

Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation.

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

Combating Antibiotic-Resistant Gram-Negative Bacteria Strains with Tetracycline-Conjugated Carbon Nanoparticles.

Nontoxic carbon nanoparticle samples prepared by both bottom-up and top-down approaches do not inhibit Gram-negative bacterial growth, indicating excellent biocompatibilities. However, cell growth inhibitory efficacies increase considerably when the carbon nanoparticles are conjugated with the antibiotic tetracycline. In tetracycline-resistant bacteria, these efficacies can approach tenfold higher activities when compared to tetracycline alone. No structural abnormality such as membrane disruptions is evident in the tested bacterial strains; this is in contrast with other nanocarbon systems such as graphene oxides, carbon nanotubes, and amine-functionalized carbon nanoparticles which do exhibit membrane disruptions. In comparison, the tetracycline-conjugated carbon nanoparticles induce membrane perturbations (but not membrane disruptions), inhibiting bacterial efflux mechanisms. It is proposed that when tetracycline is conjugated to the surface of carbon nanoparticles, it functions to direct the nanoparticles to membrane-associated tetracycline efflux pumps, thereby blocking and subsequently inhibiting their function. The conjugation between biocompatible carbon nanoparticles and subtherapeutic but well-established antibiotic molecules may provide hybrid antibiotic assembly strategies resulting in effective multidrug efflux inhibition for combating antibiotic resistance.

Faceted polymersomes: a sphere-to-polyhedron shape transformation.

The creation of "soft" deformable hollow polymeric nanoparticles with complex non-spherical shapes via block copolymer self-assembly remains a challenge. In this work, we show that a perylene-bearing block copolymer can self-assemble into polymeric membrane sacs (polymersomes) that not only possess uncommonly faceted polyhedral shapes but are also intrinsically fluorescent. Here, we further reveal for the first time an experimental visualization of the entire polymersome faceting process. We uncover how our polymersomes facet through a sphere-to-polyhedron shape transformation pathway that is driven by perylene aggregation confined within a topologically spherical polymersome shell. Finally, we illustrate the importance in understanding this shape transformation process by demonstrating our ability to controllably isolate different intermediate polymersome morphologies. The findings presented herein should provide opportunities for those who utilize non-spherical polymersomes for drug delivery, nanoreactor or templating applications, and those who are interested in the fundamental aspects of polymersome self-assembly.

Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA).

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram-negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid-state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N-termini facing the extracellular surface, and a second with the N-termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.

Antimicrobial Activity of Molluscan Hemocyanins from Helix and Rapana Snails.

For the first time the antimicrobial activities of hemocyanins from the molluscs Rapana venosa (RvH) and Helix aspersa (HaH) have been tested. From the hemolymph of the garden snail H. aspersa one structural subunit (ßc-HaH ) and eight functional units (FUs, ßc-HaH-a to ßc-HaH-h) were isolated, and their N-terminal sequences and molecular weights, ranging between 45 and 65 kDa, determined. The antimicrobial test of the hemocyanins against different bacteria showed that only two FUs from Rapana, RvH1-b and RvH1-e, exhibit a low inhibition effect against Staphylococcus aureus. In contrast and surprisingly, the structural subunit ßc-HaH of H. aspersa not only shows strong antimicrobial activities against S. aureus and the likewise Gram-positive Streptococcus epidermidis, but also against the Gram-negative bacterium Escherichia coli. We suggest that this subunit therefore has the potential to become a substitute for the commonly used antibiotics against which bacterial resistance has gradually been developed.

The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle.

Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1(-/-) muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin-cavin system.

In vitro antiproliferative effect of Helix aspersa hemocyanin on multiple malignant cell lines.

As an extension of our studies on the antitumour properties of various hemocyanins, we sought to compare the antiproliferative effects of hemocyanins derived from two snail species: Helix lucorum (HIH) and Helix aspersa (HaH). This is the first report on the antitumour effects of HaH. We hypothesized that HaH has antitumour effects not only against bladder cancer, as previously shown with other hemocyanins, but also on other cancer cell lines. The antiproliferative properties of the mentioned hemocyanins were investigated in vitro on the following human cell lines: bladder cancer (CAL-29 and T-24), ovarian cancer (FraWü), acute monocytic leukemia (THP-1), prostate cancer (DU-145), glioma cancer (LN-18), and Burkitt's lymphoma (Daudi). The properties of HaH were compared to those of HlH, keyhole limpet hemocyanin (KLH), and two positive controls (doxorubicin and mitomycin C). An antiproliferative effect of the total molecule and one structural subunit of HaH, betac-HaH, against both bladder cancer cell lines, T-24 and CAL-29, was observed. The cytotoxic effect of HaH ranged between 15% and 60% among the other tested cell lines. The endotoxin contamination did not affect the efficacy of HaH. Therefore, HlH and HaH could be appropriate for more detailed investigations of their use as antitumour agents for the studied cancers.

Potential role of nitrite for abiotic Fe(II) oxidation and cell encrustation during nitrate reduction by denitrifying bacteria.

Microorganisms have been observed to oxidize Fe(II) at neutral pH under anoxic and microoxic conditions. While most of the mixotrophic nitrate-reducing Fe(II)-oxidizing bacteria become encrusted with Fe(III)-rich minerals, photoautotrophic and microaerophilic Fe(II) oxidizers avoid cell encrustation. The Fe(II) oxidation mechanisms and the reasons for encrustation remain largely unresolved. Here we used cultivation-based methods and electron microscopy to compare two previously described nitrate-reducing Fe(II) oxidizers ( Acidovorax sp. strain BoFeN1 and Pseudogulbenkiania sp. strain 2002) and two heterotrophic nitrate reducers (Paracoccus denitrificans ATCC 19367 and P. denitrificans Pd 1222). All four strains oxidized ∼8 mM Fe(II) within 5 days in the presence of 5 mM acetate and accumulated nitrite (maximum concentrations of 0.8 to 1.0 mM) in the culture media. Iron(III) minerals, mainly goethite, formed and precipitated extracellularly in close proximity to the cell surface. Interestingly, mineral formation was also observed within the periplasm and cytoplasm; intracellular mineralization is expected to be physiologically disadvantageous, yet acetate consumption continued to be observed even at an advanced stage of Fe(II) oxidation. Extracellular polymeric substances (EPS) were detected by lectin staining with fluorescence microscopy, particularly in the presence of Fe(II), suggesting that EPS production is a response to Fe(II) toxicity or a strategy to decrease encrustation. Based on the data presented here, we propose a nitrite-driven, indirect mechanism of cell encrustation whereby nitrite forms during heterotrophic denitrification and abiotically oxidizes Fe(II). This work adds to the known assemblage of Fe(II)-oxidizing bacteria in nature and complicates our ability to delineate microbial Fe(II) oxidation in ancient microbes preserved as fossils in the geological record.

Constitutive formation of caveolae in a bacterium.

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.

Protons released during pancreatic acinar cell secretion acidify the lumen and contribute to pancreatitis in mice.

BACKGROUND & AIMS: Secretory granules are acidic; cell secretion will therefore lead to extracellular acidification. We propose that during secretion, protons co-released with proteins from secretory granules of pancreatic acinar cells acidify the restricted extracellular space of the pancreatic lumen to regulate normal physiological and pathophysiological functions in this organ METHODS: Extracellular changes in pH were quantified in real time using 2-photon microscopy analysis of pancreatic tissue fragments from mouse models of acute pancreatitis (mice given physiological concentrations [10 -20 pM] of cholecystokinin or high concentrations of [100 nM] cerulein). The effects of extracellular changes in pH on cell behavior and structures were measured. RESULTS: With physiological stimulation, secretory granule fusion (exocytosis) caused acidification of the pancreatic lumen. Acidifications specifically affected intracellular calcium responses and accelerated the rate of recovery from agonist-evoked calcium signals. Protons therefore appear to function as negative-feedback, extracellular messengers during coupling of cell stimuli with secretion. At high concentrations of cerulein, large increases in secretory activity were associated with extreme, prolonged acidification of the luminal space. These pathological changes in pH led to disruption of intercellular junctional coupling, measured by movement of occludin and E-cadherin. CONCLUSIONS: By measuring changes in extracellular pH in pancreas of mice, we observed that luminal acidification resulted from exocytosis of zymogen granules from acinar cells. This process is part of normal organ function but could contribute to the tissue damage in cases of acute pancreatitis.

Alpha-actinin-3 deficiency results in reduced glycogen phosphorylase activity and altered calcium handling in skeletal muscle.

Approximately one billion people worldwide are homozygous for a stop codon polymorphism in the ACTN3 gene (R577X) which results in complete deficiency of the fast fibre muscle protein alpha-actinin-3. ACTN3 genotype is associated with human athletic performance and alpha-actinin-3 deficient mice [Actn3 knockout (KO) mice] have a shift in the properties of fast muscle fibres towards slower fibre properties, with increased activity of multiple enzymes in the aerobic metabolic pathway and slower contractile properties. alpha-Actinins have been shown to interact with a number of muscle proteins including the key metabolic regulator glycogen phosphorylase (GPh). In this study, we demonstrated a link between alpha-actinin-3 and glycogen metabolism which may underlie the metabolic changes seen in the KO mouse. Actn3 KO mice have higher muscle glycogen content and a 50% reduction in the activity of GPh. The reduction in enzyme activity is accompanied by altered post-translational modification of GPh, suggesting that alpha-actinin-3 regulates GPh activity by altering its level of phosphorylation. We propose that the changes in glycogen metabolism underlie the downstream metabolic consequences of alpha-actinin-3 deficiency. Finally, as GPh has been shown to regulate calcium handling, we examined calcium handling in KO mouse primary mouse myoblasts and find changes that may explain the slower contractile properties previously observed in these mice. We propose that the alteration in GPh activity in the absence of alpha-actinin-3 is a fundamental mechanistic link in the association between ACTN3 genotype and human performance.

Supramolecular organization of phycobiliproteins in the chlorophyll d-containing cyanobacterium Acaryochloris marina.

Here we report the high-resolution detail of the organization of phycobiliprotein structures associated with photosynthetic membranes of the chlorophyll d-containing cyanobacterium Acaryochloris marina. Cryo-electron transmission-microscopy on native cell sections show extensive patches of near-crystalline phycobiliprotein rods that are associated with the stromal side of photosynthetic membranes. This supramolecular photosynthetic structure represents a novel mechanism of organizing the photosynthetic light-harvesting machinery. In addition, the specific location of phycobiliprotein patches suggests a physical separation of photosystem I and photosystem II reaction centres. Based on this finding and the known photosystem's structure in Acaryochloris, we discuss possible membrane arrangements of photosynthetic membrane complexes in this species.

A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos.

The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples.

High-resolution 3D quantitative analysis of caveolar ultrastructure and caveola-cytoskeleton interactions.

Caveolae are characteristic invaginations of the mammalian plasma membrane (PM) implicated in lipid regulation, signal transduction and endocytosis. We have employed electron microscope tomography (ET) to quantify caveolae structure-function relationships in three-dimension (3D) at high resolution both in conventionally fixed and in fast-frozen/freeze-substituted (intact) cells as well as immunolabelled PM lawns. Our findings provide a detailed quantitative comparison of the average caveola dimensions for different cell types including tissue endothelial cells and cultured 3T3-L1 adipocytes. These studies revealed the presence of a spiked caveolar coat and a wide caveolar neck open to the extracellular milieu that is sensitive to conventional fixation; the neck region appeared to form a specialized microdomain with associated cytoplasmic material. In endothelial cells in situ in pancreatic islets of Langerhans, the diaphragm spanning the caveolar opening was clearly resolved by ET, and the involuted 3D topology of the cell surface mapped to measure the contribution of caveolar membranes to local increases in the surface area of the PM. The complexity of connections among caveolae and to the actin cytoskeleton and microtubules suggests that individual caveolae may be interconnected through a complex filamentous network to form a single functional unit.

Caveolin-1 is required for lateral line neuromast and notochord development.

Caveolae have been linked to diverse cellular functions and to many disease states. In this study we have used zebrafish to examine the role of caveolin-1 and caveolae during early embryonic development. During development, expression is apparent in a number of tissues including Kupffer's vesicle, tailbud, intersomite boundaries, heart, branchial arches, pronephric ducts and periderm. Particularly strong expression is observed in the sensory organs of the lateral line, the neuromasts and in the notochord where it overlaps with expression of caveolin-3. Morpholino-mediated downregulation of Cav1alpha caused a dramatic inhibition of neuromast formation. Detailed ultrastructural analysis, including electron tomography of the notochord, revealed that the central regions of the notochord has the highest density of caveolae of any embryonic tissue comparable to the highest density observed in any vertebrate tissue. In addition, Cav1alpha downregulation caused disruption of the notochord, an effect that was enhanced further by Cav3 knockdown. These results indicate an essential role for caveolin and caveolae in this vital structural and signalling component of the embryo.

Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles.

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37 degrees C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.

Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes.

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol. Chem. 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed "caves," using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.