Morphofunctional changes at the active zone during synaptic vesicle exocytosis.

Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

Architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography.

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2-rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

Cryo-EM reconstruction of Type VI secretion system baseplate and sheath distal end.

The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath-tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath-tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non-contractile Vibrio cholerae sheaths by cryo-electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F2/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.

Structure of the T4 baseplate and its function in triggering sheath contraction.

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

Deuterium induces a distinctive Escherichia coli proteome that correlates with the reduction in growth rate.

Substitution of protium (H) for deuterium (D) strongly affects biological systems. Whereas higher eukaryotes such as plants and mammals hardly survive a deuterium content of >30%, many microorganisms can grow on fully deuterated media, albeit at reduced rates. Very little is known about how the H/D replacement influences life at the systems level. Here, we used MS-based analysis to follow the adaptation of a large part of the Escherichia coli proteome from growth on a protonated full medium, over a protonated minimal medium, to a completely deuterated minimal medium. We could quantify >1800 proteins under all conditions, several 100 of which exhibited strong regulation during both adaptation processes. The adaptation to minimal medium strongly up-regulated amino acid synthesis and sugar metabolism and down-regulated translational proteins on average by 9%, concomitant with a reduction in growth rate from 1.8 to 0.67 h-1 In contrast, deuteration caused a very wide proteomic response over many cell functional categories, together with an additional down-regulation of the translational proteins by 5%. The latter coincided with a further reduction in growth rate to 0.37 h-1, revealing a clear linear correlation between growth rate and abundance of translational proteins. No significant morphological effects are observed under light and electron microscopies. Across all protein categories, about 80% of the proteins up-regulated under deuteration are enzymes with hydrogen transfer functions. Thus, the H/D kinetic isotope effect appears as the major limiting factor of cellular functions under deuteration.

3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum.

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser.

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics".

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

Phospholipid-Block Copolymer Hybrid Vesicles with Lysosomal Escape Ability.

The success of nanoparticulate formulations in drug delivery depends on various aspects including their toxicity, internalization, and intracellular location. Vesicular assemblies consisting of phospholipids and amphiphilic block copolymers are an emerging platform, which combines the benefits from liposomes and polymersomes while overcoming their challenges. We report the synthesis of poly(cholesteryl methacrylate)- block-poly(2-(dimethylamino) ethyl methacrylate) (pCMA- b-pDMAEMA) block copolymers and their assembly with phospholipids into hybrid vesicles. Their geometry, their ζ-potential, and their ability to adsorb onto polymer-coated surfaces were assessed. Giant unilamellar vesicles were employed to confirm the presence of both the phospholipids and the block copolymer in the same membrane. Furthermore, the cytotoxicity of selected hybrid vesicles was determined in RAW 264.7 mouse macrophages, primary rat Kupffer cells, and human macrophages. The internalization and lysosomal escape ability of the hybrid vesicles were confirmed using RAW 264.7 mouse macrophages. Taken together, our findings illustrate that the reported hybrid vesicles are a promising complementary drug delivery platform for existing liposomes and polymersomes.

Rapid Structure Determination of Microcrystalline Molecular Compounds Using Electron Diffraction.

Chemists of all fields currently publish about 50 000 crystal structures per year, the vast majority of which are X-ray structures. We determined two molecular structures by employing electron rather than X-ray diffraction. For this purpose, an EIGER hybrid pixel detector was fitted to a transmission electron microscope, yielding an electron diffractometer. The structure of a new methylene blue derivative was determined at 0.9 Šresolution from a crystal smaller than 1×2 µm2 . Several thousand active pharmaceutical ingredients (APIs) are only available as submicrocrystalline powders. To illustrate the potential of electron crystallography for the pharmaceutical industry, we also determined the structure of an API from its pill. We demonstrate that electron crystallography complements X-ray crystallography and is the technique of choice for all unsolved cases in which submicrometer-sized crystals were the limiting factor.

Focus: The interface between data collection and data processing in cryo-EM.

We present a new software package called Focus that interfaces cryo-transmission electron microscopy (cryo-EM) data collection with computer image processing. Focus creates a user-friendly environment to import and manage data recorded by direct electron detectors and perform elemental image processing tasks in a high-throughput manner while new data is being acquired at the microscope. It provides the functionality required to remotely monitor the progress of data collection and data processing, which is essential now that automation in cryo-EM allows a steady flow of images of single particles, two-dimensional crystals, or electron tomography data to be recorded in overnight sessions. The rapid detection of any errors that may occur greatly increases the productivity of recording sessions at the electron microscope.

Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts.

We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3-20nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics.

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.

openBEB: open biological experiment browser for correlative measurements.

BACKGROUND: New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis. RESULTS: We have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository. CONCLUSIONS: The versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the "house keeping" abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated.

Poly(N-isopropylacrylamide)/poly(dopamine) capsules.

Polymer capsules are an interesting concept considered in nanobiotechnology. Approaches that facilitate their assembly remain sought after. Poly(dopamine) (PDA) has been considered and successfully applied in this context. We recently demonstrated that PDA could be copolymerized with different types of poly(N-isopropylacrylamide) (pNiPAAm) to assemble mixed films on planar substrates. Herein, we transferred this approach onto colloidal substrates and characterized the film thickness depending on the film composition and template particles size. While the membrane of capsules assembled using 5 µm template particles exhibited strong dependency on the film composition, smaller templates led to capsules with similar membrane thickness. We then compared the permeability of different capsules using fluorescently labeled dextran and fluorescein. We found that the permeability of capsules was heavily dependent on the polymer composition and the template particle size. These fundamental findings contribute to the potential of these capsules, assembled in one-step, for biomedical applications.

Poly(vinyl alcohol) physical hydrogel nanoparticles, not polymer solutions, exert inhibition of nitric oxide synthesis in cultured macrophages.

Hydrogel nanoparticles (HNP) are an emerging tool of biomedicine with unique materials characteristics, scope, and utility. These hydrated, soft colloidal carriers can penetrate through voids with dimensions narrower than the size of the particle, provide stabilization for fragile biological cargo and allow diffusion and exchange of solutes with external phase. However, techniques to assemble HNP are few; solitary examples exist of biocompatible polymers being formulated into HNP; and knowledge on the biomedical properties of HNP remains rather cursory. In this work, we investigate assembly of HNP based on a polymer with decades of prominence in the biomedical field, poly(vinyl alcohol), PVA. We develop a novel method for production of PVA HNP through nanoprecipitation-based assembly of polymer nanoparticles and subsequent physical hydrogelation of the polymer. Polymer nanoparticles and HNP were visualized using scanning electron microscopy and fluorescence imaging, and characterized using dynamic light scattering and zeta potential measurements. Interaction of PVA HNP with mammalian cells was investigated using flow cytometry, viability screening, and measurements of nitric oxide production by cultured macrophages. The latter analyses revealed that PVA administered as a polymer solution or in the form of HNP resulted in no measurable increase in production of the inflammation marker. Unexpectedly, PVA HNP exerted a pronounced inhibition of NO synthesis by stimulated macrophages, that is, had an anti-inflammatory activity. This effect was accomplished with a negligible change in the cell viability and was not observed when PVA was administered as a polymer solution. To the best of our knowledge, this is the first observation of inhibition of NO synthesis in macrophages by administered nanoparticles and specifically hydrogel nanoparticles. Taken together, our results present PVA HNP as promising colloidal hydrogel nanocarriers for biomedical applications, specifically drug delivery and assembly of intracellular biosensors.

Connecting µ-fluidics to electron microscopy.

A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned.

The outer membrane protein LptO is essential for the O-deacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis.

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) essential for secretion and attachment to the cell surface. Inactivation of lptO (PG0027) or porT produced mutants that lacked surface protease activity and an electron-dense surface layer. Both mutants showed co-accumulation of A-LPS and unmodified CTD proteins in the periplasm. Lipid profiling by mass spectrometry showed the presence of both tetra- and penta-acylated forms of mono-phosphorylated lipid A in the wild-type and porT mutant, while only the penta-acylated forms of mono-phosphorylated lipid A were found in the lptO mutant, indicating a specific role of LptO in the O-deacylation of mono-phosphorylated lipid A. Increased levels of non-phosphorylated lipid A and the presence of novel phospholipids in the lptO mutant were also observed that may compensate for the missing mono-phosphorylated tetra-acylated lipid A in the outer membrane (OM). Molecular modelling predicted LptO to adopt a ß-barrel structure characteristic of an OM protein, supported by the enrichment of LptO in OM vesicles. The results suggest that LPS deacylation by LptO is linked to the co-ordinated secretion of A-LPS and CTD proteins by a novel secretion and attachment system to form a structured surface layer.

Assembly of poly(dopamine) films mixed with a nonionic polymer.

Poly(dopamine) (PDA) coatings have recently attracted considerable interest for a variety of applications. Here, we investigate the film deposition of dopamine mixed with a nonionic polymer (i.e., poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), and poly(N-vinyl pyrrolidone) (PVP)) onto silica substrates using X-ray photoelectron spectroscopy and quartz crystal microbalance. Furthermore, we assess the possibility of coating silica colloids to yield polymer capsules and liposomes with these mixtures. We found that mixed PDA/PEG and PDA/PVA films are deposited without the need for a covalent linker such as an amine or thiol. We also discovered the first material, namely, PVP, that can suppress PDA film assembly. These fundamental findings give further insight into PDA film properties and contribute to establish PDA as a widely applicable coating.