Carbon-coated Grids Research Articles

Supramolecular Fluorophores for Biological Studies: Phenylene Vinylene-Amino Acid Amphiphiles

We report here on a family of self-assembling fluorescent organic amphiphiles with a biomolecular L-lysine hydrophile and a photonically active phenylene vinylene hydrophobe. Unlike conventional amphiphiles, these segmented dendrimers feature a rigid, branched hydrophobe, and have packing characteristics controlled by the ratio of cross-sectional areas of the hydrophobe and hydrophile. In dilute solution, the amphiphiles form supramolecular aggregates, which are easily taken in by cells through an endocytic pathway, and have no discernible effect on cell proliferation or morphology. An analogous pyrene-based amphiphile was cytotoxic, suggesting that cell survival may be linked either to the self-assembling nature of the amphiphiles, or to the specific properties of the phenylene vinylene segment. The combination of photonic and biological components in these amphiphiles provides great potential for applications in sensing or delivery of molecules to intracellular targets.

The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.

Size distribution of sputtered particles from Au nanoislands due to MeV self-ion bombardment

Nanoisland gold films, deposited by vacuum evaporation of gold onto Si(100) substrates, were irradiated with 1.5MeV Au2+ ions up to a fluence of 5×1014ionscm−2 and at incidence angles up to 60° with respect to the surface normal. The sputtered particles were collected on carbon-coated grids (catcher grid) during ion irradiation and were analyzed with transmission electron microscopy and Rutherford backscattering spectrometry. The average sputtered particle size and the areal coverage are determined from transmission electron microscopy measurements, whereas the amount of gold on the substrate is found by Rutherford backscattering spectrometry. The size distributions of larger particles (number of atoms/particle, n⩾1000) show an inverse power law with an exponent of ∼−1 in broad agreement with a molecular-dynamics simulation of ion impact on cluster targets.

Dodecameric Structure of the Small Heat Shock Protein Acr1 from Mycobacterium tuberculosis

Small heat shock proteins are a ubiquitous and diverse family of stress proteins that have in common an alpha-crystallin domain. Mycobacterium tuberculosis has two small heat shock proteins, Acr1 (alpha-crystallin-related protein 1, or Hsp16.3/16-kDa antigen) and Acr2 (HrpA), both of which are highly expressed under different stress conditions. Small heat shock proteins form large oligomeric assemblies and are commonly polydisperse. Nanoelectrospray mass spectrometry showed that Acr2 formed a range of oligomers composed of dimers and tetramers, whereas Acr1 was a dodecamer. Electron microscopy of Acr2 showed a variety of particle sizes. Using three-dimensional analysis of negative stain electron microscope images, we have shown that Acr1 forms a tetrahedral assembly with 12 polypeptide chains. The atomic structure of a related alpha-crystallin domain dimer was docked into the density to build a molecular structure of the dodecameric Acr1 complex. Along with the differential regulation of these two proteins, the differences in their quaternary structures demonstrated here supports their distinct functional roles.

Correlation Microscopy: Bridging the Gap between Light- and Cryo-Electron Microscopy

Cryo-electron tomography (cryo-ET) of frozen, hydrated, biological samples on carbon-coated copper grids is a powerful technique for quasi in vivo studies of cellular structures with nanometerscale resolution [1]. A major inconvenience of this method exists in the difficulty of locating and unequivocally identifying the structures of interest within a ice embedded sample. Additionally, kinetic experiments are almost impossible, due to the fact that the information about the dynamic state in vitrified samples has been lost. By establishing a direct correlation between cryofluorescence microscopy and cryo-electron microscopy (cryo-EM), we propose an effective way to overcome these limitations, opening the way for a wide spectrum of novel applications. Our concept of correlation microscopy is based on the possibility of identifying and determining the position of fluorescently-labelled structures in vitrified samples directly on the TEM grid by means of cryofluorescence microscopy, and the recovery of these positions during subsequent investigations with cryo-EM. The entire vitrified grid can be imaged and mapped with epifluorescence under cryo-conditions thanks to a new designed and homemade cryo-holder for TEM grids, adapted to a fully automated, inverted light microscope (Zeiss Axiovert 200) with long working-distance objectives (20x and 40x magnification). The cryo-holder is devised to keep the sample at liquid nitrogen temperature (lN2) and it is efficiently isolated from the external environment to ensure a thermal equilibrium and to prevent undesirable contamination during investigations. The absolute coordinates of the area of interest on the grid can be determined and recorded by cryo-light microscopy and directly recovered in the electron microscope with a MatLab-based program, integrated within the TOM toolbox [2]. Fluorescently labelled neurons grown on carbon-coated TEM grids have been embedded in vitreous ice and successfully used for first studies. In the case of thin specimens suitable for cryo-electron tomography (where the thickness of the amorphous ice does not exceed 200-400 nm), the resolution is comparable to investigations in buffer solutions. The ‘blurring’ of the fluorescence signal and the decrease in resolution is directly related to the thickness of the ice, which can be estimated from focusing and transmitted light measurements. Our current applications involve the localisation of synapses by immunolabelling of glutamate receptors with fluorescent dyes and the distribution of fluorescent markers in vitreous cryo-sections. Future applications will involve labelling of extra-cellular molecules, structures and receptors with Quantum Dots [3], which are ideal candidates for correlation microscopy due to their fluorescence and electron-dense characteristics, which allow visualisation and targeting of the label both with cryo-light microscopy and cryo-EM, especially cryo-ET.

Ena/VASP Proteins Enhance Actin Polymerization in the Presence of Barbed End Capping Proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.

Obligate Heterodimerization of the Archaeal Alba2 Protein with Alba1 Provides a Mechanism for Control of DNA Packaging

Organisms growing at elevated temperatures face a particular challenge to maintain the integrity of their genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of the Alba (Sac10b) gene. Alba is an abundant, dimeric, highly basic protein that binds cooperatively and at high density to DNA. Sulfolobus solfataricus encodes a second copy of the Alba gene, and the Alba2 protein is expressed at approximately 5% of the level of Alba1. We demonstrate by NMR, ITC, and crystallography that Alba2 exists exclusively as a heterodimer with Alba1 at physiological concentrations and that heterodimerization exerts a clear effect upon the DNA packaging, as observed by EM, potentially by changing the interface between adjacent Alba dimers in DNA complexes. A functional role for Alba2 in modulation of higher order chromatin structure and DNA condensation is suggested.

Compartmentalization of Lipid Biosynthesis in Mycobacteria

The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.

Reversible Inhibition of α-Synuclein Fibrillization by Dopaminochrome-mediated Conformational Alterations

Previous studies demonstrated that alpha-synuclein (alpha-syn) fibrillization is inhibited by dopamine, and studies to understand the molecular basis of this process were conducted (Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Science 294, 1346-1349). Dopamine inhibition of alpha-syn fibrillization generated exclusively spherical oligomers that depended on dopamine autoxidation but not alpha-syn oxidation, because mutagenesis of Met, His, and Tyr residues in alpha-syn did not abrogate this inhibition. However, truncation of alpha-syn at residue 125 restored the ability of alpha-syn to fibrillize in the presence of dopamine. Mutagenesis and competition studies with specific synthetic peptides identified alpha-syn residues 125-129 (i.e. YEMPS) as an important region in the dopamine-induced inhibition of alpha-syn fibrillization. Significantly, the dopamine oxidation product dopaminochrome was identified as a specific inhibitor of alpha-syn fibrillization. Dopaminochrome promotes the formation of spherical oligomers by inducing conformational changes, as these oligomers regained the ability to fibrillize by simple denaturation/renaturation. Taken together, these data indicate that dopamine inhibits alpha-syn fibrillization by inducing structural changes in alpha-syn that can occur through the interaction of dopaminochrome with the 125YEMPS129 motif of alpha-syn. These results suggest that the dopamine autoxidation can prevent alpha-syn fibrillization in dopaminergic neurons through a novel mechanism. Thus, decreased dopamine levels in substantia nigra neurons might promote alpha-syn aggregation in Parkinson's disease.

Investigation of the morphology of intermediate filaments adsorbed to different solid supports

Morphologically, glutaraldehyde-fixed and -dried intermediate filaments (IFs) appear flexible, and with a width of 8–12 nm when observed by electron microscopy. Sometimes, the filaments are even unraveled on the carbon-coated grid and reveal a protofilamentous architecture. In this study, we have used atomic force microscopy to further investigate the morphology of IFs in a more physiological environment. First, we have imaged hydrated glutaraldehyde-fixed IFs adsorbed to a graphite support. In such conditions, human vimentin and desmin IFs appeared compact with a height of 5–8 nm and revealed either a beading repeat or a helical morphology. Second, we have analyzed the architecture of hydrated vimentin, desmin, and neurofilament IFs adsorbed to mica, graphite, and hydrophilic glass without the presence of fixative. On mica, vimentin IFs had a height of only 3–5 nm, whereas desmin IFs appeared as 8–10 nm height filaments with a helical twist. Neurofilaments were 10–12 nm in height with a pronounced 30–50 nm beading along their length. On graphite, the different IFs were either not adsorbing properly or their architecture was modified yielding, for example, broad, flattened filaments. Finally, hydrophilic glass was the surface which seemed to best preserve the architecture of the three IFs, even if, in some cases, unraveled vimentin filaments were observed on this support. These results are straightening the idea that mature IFs are dynamic polymers in vitro and that IFs can be distinguished from each others by their physicochemical properties.

Bacteriophage P22 Tail Accessory Factor GP26 Is a Long Triple-stranded Coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.

Novel Mechanism of Inhibition of HIV-1 Reverse Transcriptase by a New Non-nucleoside Analog, KM-1

2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1)) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) that was designed to bind at an unconventional site on human immunodeficiency virus type 1 reverse transcriptase (RT) (Skillman, A. G., Maurer, K. W., Roe, D. C., Stauber, M. J., Eargle, D., Ewing, T. J., Muscate, A., Davioud-Charvet, E., Medaglia, M. V., Fisher, R. J., Arnold, E., Gao, H. Q., Buckheit, R., Boyer, P. L., Hughes, S. H., Kuntz, I. D., and Kenyon, G. L. (2002) Bioorg. Chem. 30, 443-458). We have investigated the mechanism by which KM-1 inhibits wild-type human immunodeficiency virus type 1 RT by using pre-steady state kinetic methods to examine the effect of KM-1 on the parameters governing the single nucleotide incorporation catalyzed by RT. Analysis of the pre-steady-state burst phase of dATP incorporation showed that KM-1 decreased the amplitude of the reaction as previously shown for other NNRTIs, because of the slow equilibration of the inhibitor with RT. In the ternary enzyme-DNA-KM-1 complex (E-DNA-I), incorporation of the next nucleotide onto the primer is blocked. However, unlike conventional NNRTIs, the inhibitory effect was caused primarily by weakening the DNA binding affinity and displacing DNA from the enzyme. Wild-type RT binds a 25/45-mer DNA duplex with an apparent K(d) of 3 nm, which was increased to 400 nm upon saturation with KM-1. Likewise, the apparent K(d) for KM-1 binding to RT increased at higher DNA concentrations. We therefore conclude that KM-1 represents a new class of inhibitor distinct from nevirapine and related NNRTIs. KM-1 can bind to RT in both the absence and presence of DNA but weakens the affinity for DNA 140-fold so that it favors DNA dissociation. The data suggest that KM-1 distorts RT conformation and misaligns DNA at the active site.

Beet necrotic yellow vein benyvirus

Beet necrotic yellow vein benyvirus

Cryoelectron microscopy of refrozen cryosections

Cryoelectron microscopy makes it possible to record high-resolution detail from large and complex structures. However, its application to understanding cellular structure is limited by the requirement that samples should be no thicker than ∼0.5–1 μm. Therefore it is important to develop the ability to section biological material so that it can be imaged in its native frozen state. Here we have adapted standard methods of preparing cryosections so that they can be imaged by cryoelectron microscopy. As used for immunolabeling, cryosections of chemically fixed, cryoprotected frozen rat cardiac muscle were thawed, applied to carbon-coated grids, and rinsed on a drop of buffer. The special step here is that the cryosections were then refrozen by being plunged into liquid ethane and imaged at approximately −180 °C in a 200-kV field-emission gun electron microscope. The unstained cryosections have good contrast, allowing the identification of optimum regions of the sample. Considerable fine detail is observed within the substructure of the sarcomere A-band and I-band. Fourier transform analysis of the micrographs shows that this method preserves high structural order, hence these sections are well-suited to 3D reconstruction. We conclude that this approach has considerable potential for obtaining intermediate- and high-resolution structural detail from bulk tissue.

Borg/Septin Interactions and the Assembly of Mammalian Septin Heterodimers, Trimers, and Filaments

Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.

Electron emission suppression characteristics of molybdenum grids coated with carbon film by ion beam assisted deposition

Carbon was coated on the surface of molybdenum grids by ion beam assisted deposition. The electron emission characteristics from the grids with and without carbon, which were contaminated by an active electron emission substance of the cathode, was measured using an analogous diode method. The results show that electron emission from the carbon-coated Mo grid was much less than that from the Mo grid without carbon. The cathode emission substance deposited on the carbon surface of the grid was analyzed by x-ray photoelectron spectroscopy and electron probes. Analysis results revealed that the carbon-coated Mo grid could effectively reduce the accumulation of the cathode emitting substances Ba and BaO on the grid surface. The reason for the suppression of electron emission of the carbon-coated Mo grid is discussed. The carbon-coated Mo grids were used in pulsed-controlled grid traveling wave tubes and the working lifetime of the tube was increased from tens of hours to over 1000 h.

Langmuir films of thiolated gold nanoparticles transferred onto functionalized substrate: 2-D local organization

The aim of this work is to fabricate ordered arrays of thiolated gold nanoparticles grafted through electrically conductive molecular tethers onto gold electrodes. Dodecanethiol-capped gold nanoparticles were synthesized in a two-phase liquid–liquid system. Cluster size selection by the vapor transfer technique was repeatedly performed in order to obtain a narrow nanoparticle core diameter distribution that peaked around 2 nm. Transmission electron microscopy (TEM) imaging of drop cast films prepared onto carbon-coated grids allowed us to assess the performance of each fractionalization process step and the corresponding particle aggregation behavior. A 1,4-benzenedimethanethiol self-assembled film onto hetero-epitaxial gold on mica has been chosen as a grafting substrate for size-selected gold nanocluster films. Using an air, room temperature scanning tunneling microscopy (STM), the behavior of drop cast and Langmuir nanocluster films onto this substrate was investigated. Better results in terms of adhesion and 2-D aggregation were obtained in the case of Langmuir films. This performance has been further enhanced by application of the multi-step creep method during the Langmuir film making.

Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Pα and Pβ) and two identical regulatory subunits (Pγs). Pγ regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Pαβγγ, and compare its average image with those of Pγ-released PDE. The structure of Pαβγγ appears to be a flattened bell-shape, with dimensions of 150×108×60 Å, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Pαβγγ is determined by hetero-dimerization of Pα and Pβ, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Pαβγγ with those of Pγ-released PDE suggests that Pγ release does not affect the overall structure of Pαβ, and that the Pαβ C-terminus, but not Pγ, is a determinant for the Pαβ orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Pαβ is regulated by its regional interaction with Pγ.

Synthesis, Self-Assembly, and Magnetic Properties of FexCoyPt100-x-y Nanoparticles

FexCoyPt100-x-y alloy nanoparticles were prepared by the simultaneous reduction of cobalt acetylacetonate and platinum acetylacetonate and the thermal decomposition of iron pentacarbonyl. The relative amounts of iron, cobalt, and platinum in the particles depended on the amount of iron, cobalt and platinum charged to the reaction. As prepared, the particles were superparamagnetic and had a distorted fcc structure. The average particle diameter was 3.5 nm and the size distribution was very narrow. The particles could be dispersed in hydrocarbon solvents and formed films consisting of hexagonal close-packed particles on carbon-coated copper TEM grids. The films were sputter coated with amorphous carbon and then annealed at temperatures ranging from 550 to 700 °C to transform the particles to the tetragonal (L10) phase. The coercivity of the annealed films increased with increasing annealing temperature. For films with a similar degree of transformation to the tetragonal phase, increasing the cobalt content ...

Controlled Synthesis of Carbon-Encapsulated Co Nanoparticles by CVD

Carbon-encapsulated cobalt nanoparticles are prepared over a range of temperaturesand partial pressures by CVD from cyclohexane on prepared substrates consisting of ground powders of SiO2 impregnated with Co(NO3)2 and (NH4)2Mo2O7. The encapsulated cobalt nanoparticles are freed from the incorporated SiO2 and molybdenum by treating the deposition product with aqueous hydrofluoric acid. Extracted material is dispersed in ethanol and deposited on a carbon-coated grid prior to investigation by high-resolution TEM.