EM Micrographs Research Articles

Self-Assembly of Switchable Protein Nanocages via Allosteric Effect

Protein cages have promising applications in highly efficient gene transportation. Many methods of engineering protein–interface interactions have been developed to build such viral mimics. Missing...

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

High-Speed Atomic Force Microscopy Shows Conformational Dynamics of Ca2+/Calmodulin-Dependent Protein Kinase II

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of Ca2+ signaling in eukaryotic cells. An elevation in intracellular Ca2+ leads to binding of Ca2+ to calmodulin (CaM), which binds to and activates CaMKII. Upon activation, CaMKII has the ability of autophosphorylation, then CaMKII phosphorylates numerous target proteins and is involved in many cellular functions, including synaptic plasticity. Previous EM micrographs show that CaMKII forms a 12-meric holoenzyme that could respond to the frequency of the activating signal of Ca2+. However, the relationship between structural dynamics of CaMKII oligomers and its function is still unknown. Here we used high-speed atomic force microscopy (HS-AFM) to directly visualize conformational dynamics of CaMKII oligomers during Ca2+/CaM binding and phosphorylation. HS-AFM is a powerful technique to visualize conformational changes of proteins at a single molecular level in action under near physiological conditions [1, 2]. At a basal state, HS-AFM images of CaMKII showed a highly fluctuated structure of kinase domains, indicating that a fluctuation of kinase domains generates a space to access Ca2+/CaM to the binding site of CaMKII. Upon binding of Ca2+/CaM, neighbor kinase domains contact each other, and leads to generate more space to bind additional Ca2+/CaM. These conformational dynamics probably causes a positive cooperativity of Ca2+/CaM binding. In the phosphorylated state, kinase domains in CaMKII form a disordered structure, suggesting that a disordered structure phosphorylates other subunits within a CaMKII oligomer as a non-specific manner. Thus, a sequential HS-AFM images shows importance of structural dynamics of kinase domains in CaMKII oligomers, such as fluctuation, conformational dynamics and a transition to a disordered structure. [1] M. Shibata et al. Nat. Commun. 8, 1430 (2017). M. Shibata et al. Sci. Rep. 8, 8262 (2018).

The Structure of the Infectious Prion Protein Constrains Potential Prion Replication Mechanisms

X-ray fiber diffraction and cryo electron microscopy revealed that the infectious prion protein, PrPSc, contains a four-rung beta-solenoid structure as a key element of its self-propagating conformation. In particular, amyloid fibrils with their regular and repetitive nature allowed important insights into the structure of PrPSc. X-ray fiber diffraction revealed a height of 19.2 Å per molecule of PrPSc along the fibril axis. Meridional diffraction signals at 4.8, 6.4, and 9.6 Å indicated four rungs of beta structure (4 x 4.8 Å = 19.2 Å), while the lack of a ∼10 Å equatorial reflection implied a beta-solenoid configuration. Unprocessed cryo EM micrographs of PrPSc fibrils displayed 4.8 Å spacings, confirming the cross-beta structure. 3D reconstructions of individual PrPSc fibrils indicated a molecular height of ∼17.7 Å, in good agreement with the X-ray fiber diffraction results. Moreover, single particle averaging of short fibril segments revealed periodicities of ∼2 and ∼4 nm. The first measurement is consistent with the molecular height of PrPSc, while the latter suggests a higher order such as a head-to-head dimerization. The four-rung beta-solenoid structure provides insights into the PrPC to PrPSc conversion mechanism. The folding properties of regular beta-solenoid proteins revealed a nearly universal occurrence of “capping structures” that prevent the propagation of the beta-solenoid conformation. These caps consist of short alpha-helices, loops, or other structures and their removal resulted in aggregation and amyloid formation. The absence of capping structures in PrPSc allows the propagation of its beta-solenoid conformation to neighboring PrPC molecules. The putative head-to-head arrangement of the PrPSc dimers implies a role for matching sequences in the conversion mechanism. The fidelity of the templating process, which is a hallmark of the PrPSc propagation, can thus be attributed to molecular properties of the PrPSc structure.

Burial of the Polymorphic Residue 129 in Amyloid Fibrils of Prion Stop Mutants

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

Rab27a Regulates Exocytosis of Large Dense-Core Granules in Mouse Chromaffin Cells

Rab-GTPases exert important roles in secretory vesicle cycles, with Rab3 and Rab27 participating in exocytotic throughput. While Rab3 and Rab27 share many effectors, some are unique, making it critical to identify specific functional roles. Studies on regulation of neurotransmitter release have focused principally on Rab3 and much less on Rab27. Among Rab-GTPases, Rab27 is unique in binding effectors in either the GTP- or GDP-bound state. We here compare secretory responses from ashen mice, which lack functional Rab27a protein, with strain-matched controls. Secretion was evaluated using measurements of membrane capacitance under whole-cell recording in isolated mouse chromaffin cells. UV flash uncaging of Ca2+ raised [Ca2+]i uniformly and induced catecholamine neurotransmitter release. RT-PCR demonstrated the presence of Rab27 in chromaffin cells, and immunoblotting further characterized the expressed isoform as Rab27a, not Rab27b. The exocytic burst (1s post-UV flash) of membrane capacitance increase (ΔCm) was fitted by a double exponential curve, from which the amount and fusion kinetics of granules from the readily- (RRP) and slowly-releasable pools (SRP) were extracted. Exocytic burst ΔCm was greater, and the size of both the RRP and SRP larger, in wild-type cells (RRP, 347 fF; SRP, 616 fF) than in Rab27a-/- cells (RRP, 157fF; SRP, 438 fF), while the kinetics of release from both pools remained unchanged. Expression of Rab27a in Rab27a-/- cells, using electroporation, rescued the wild-type phenotype. Rab 27a is therefore important for priming into the RRP and SRP. To assess dense-core vesicle docking at the plasma membrane, we analyzed granule distribution from E18 chromaffin cell EM micrographs. These data show that while Rab27a-/- cells possess fewer total granules, morphological docking of granules at the plasma membrane is unaffected. Further work is necessary to identify through which effector Rab27a is exerting its effects.

ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952–14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.

Efficient Light Harvesting by Photosystem II Requires an Optimized Protein Packing Density in Grana Thylakoids

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Serial EM analysis of a copepod larval nervous system: Naupliar eye, optic circuitry, and prospects for full CNS reconstruction

The medial eye and optic center of the first nauplius of Dactylopusia (= Dactylopodia) tisboides, a harpacticoid copepod, were reconstructed from serial EM micrographs. Axons from the eye project to a set of matching cartridges defined by glial cells processes, and input is then processed in sequence through two synaptic fields. A single class of local relay neurons provides the main pathway between these, subject to modulatory input from a class of densely stained neurons with distinctive dense terminals. The importance of other outside sources of synaptic input to the second synaptic field indicates that the latter is a major site for integrating the optic input with signals originating elsewhere in the CNS. This accords with physiological data on the shadow response in barnacles, whose visual system is also derived from a naupliar eye. With a body length of ca. 80 microns, copepod larvae like that of Dactylopusia are arguably among the smallest functional metazoans with a complex nervous system. Hence they are promising subjects for full reconstruction of neural circuitry at the EM level that could, in principle, reveal where key decision-making functions are localized.

Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1(K38A)) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-beta peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1(K38A). Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.

SIGNATURE: A single-particle selection system for molecular electron microscopy

SIGNATURE is a particle selection system for molecular electron microscopy. It applies a hierarchical screening procedure to identify molecular particles in EM micrographs. The user interface of the program provides versatile functions to facilitate image data visualization, particle annotation and particle quality inspection. The system design emphasizes both functionality and usability. This software has been released to the EM community and has been successfully applied to macromolecular structural analyses.

A mechanism of airway injury in an epithelial model of mucociliary clearance.

We studied the action of sodium metabisulphite on mucociliary transport in a frog palate epithelial injury model, hypothesizing that it may be useful for the study of mechanisms of airway injury. Sodium metabisulphite (MB) releases SO2 on contact with water. SO2 is a pollutant in automobile fumes and may play a role in the exacerbation of airway disease symptoms. We first investigated its effect on mucociliary clearance. MB 10-1 M, increased mucociliary clearance time (MCT) by 254.5 ± 57.3% of control values, (p < 0.001, n = 7). MB 10-4 and 10-2 M did not interfere with mucus clearance time compared to control values. In MB-treated frog palates, MCT did not return to control values after one hour (control, 97.3 ± 6.3% vs. MB, 140.9 ± 46.3%, p < 0.001, n = 7). Scanning EM images of epithelial tissue were morphometrically analyzed and showed a 25 ± 12% loss of ciliated cells in MB palates compared to controls with an intact ciliary blanket. Intact cells or groups of ciliated cells were found in scanning EM micrographs of mucus from MB-treated palates. This was associated with increased matrix metalloproteinase (MMP-9) activity in epithelial tissue and mucus. We suggest that the loss of ciliated cells as a result of MMP-9 activation prevented full recovery of MCT after MB 10-1 M. The mechanism of action may be on epithelial cell-cell or cell-matrix attachments leading to cell loss and a disruption of MCT. Further studies are warranted to determine whether this is an inflammatory mediated response or the result of a direct action on epithelial cells and what role this mechanism may play in the progression to chronic airway diseases with impaired mucociliary clearance.

Transient formation of nano-crystalline structures during fibrillation of an Abeta-like peptide.

During the first few minutes of fibrillation of a 14-residue peptide homologous to the hydrophobic C-terminal part of the Abeta-peptide, EM micrographs reveal small crystalline areas (100 to 150 nm, repeating unit 47 A) scattered in more amorphous material. On a longer time scale, these crystalline areas disappear and are replaced by tangled clusters resembling protofilaments (hours), and eventually by more regular amyloid fibrils of 60 A to 120 A diameter (days). The transient population of the crystalline areas indicates the presence of ordered substructures in the early fibrillation process, the diameter of which matches the length of the 14-mer peptide in an extended beta-strand conformation.

EM single particle analysis of the ATP-dependent BchI complex of magnesium chelatase: an AAA + hexamer

BchI, belonging to the AAA +-protein family, forms the enzyme magnesium chelatase together with BchD and BchH. This enzyme catalyses the insertion of Mg 2+ into protoporphyrin IX upon ATP hydrolysis. Previous studies have indicated that BchI forms ATP-dependent complexes and it is a member of the AAA +-protein family (ATPases associated with various cellular activities) and it was suggested based on structural homology that the BchI formed hexameric complexes. AAA +-proteins are Mg 2+-dependent ATPases that normally form oligomeric ring complexes in the presence of ATP. Single particle analysis of fully formed ring complexes of BchI observed by negative staining EM indicate that the BchI has strong 6- and 2-fold rotational symmetries and a weaker 4-fold rotational symmetry which are reminiscent of DNA helicase. A 2D average of the fully formed BchI–ATP ring complex is presented here from images of the complex obtained from negative staining EM. Other complexes are also observed in the EM micrographs and the class averages of these are indicative of the fragility and dynamic nature of the BchI complex which has been reported and they are suggestive of partially circular complexes with six or less protomers per particle. The resolution of the average circular complex is estimated at ∼30 Å and it is similar in shape and size to an atomic resolution hexameric model of BchI rendered at 30 Å.

A binary segmentation approach for boxing ribosome particles in cryo EM micrographs

Three-dimensional reconstruction of ribosome particles from electron micrographs requires selection of many single-particle images. Roughly 100,000 particles are required to achieve approximately 10 Å resolution. Manual selection of particles, by visual observation of the micrographs on a computer screen, is recognized as a bottleneck in automated single-particle reconstruction. This paper describes an efficient approach for automated boxing of ribosome particles in micrographs. Use of a fast, anisotropic non-linear reaction-diffusion method to pre-process micrographs and rank-leveling to enhance the contrast between particles and the background, followed by binary and morphological segmentation constitute the core of this technique. Modifying the shape of the particles to facilitate segmentation of individual particles within clusters and boxing the isolated particles is successfully attempted. Tests on a limited number of micrographs have shown that over 80% success is achieved in automatic particle picking.

The influence of the molar ratio of cholesteryl hemisuccinate/dipalmitoylphosphatidylcholine on ‘liposome’ formation after lipid film hydration

The morphology of the structures formed after hydration of lipid films of cholesteryl hemisuccinate/dipalmitoylphosphatidylcholine (CHEMS/DPPC) was investigated in low ionic strength solutions. The importance of addition of a charge inducing agent/geometrical structure such as CHEMS for the formation of stable vesicle dispersions upon hydration was demonstrated. The encapsulated volume measured for CHEMS/DPPC ratios below 1:50 was low. For a ratio of CHEMS/DPPC of 1:30 EM micrographs showed mainly small unilamellar vesicles, with particle sizes between 0.07 and 0.3 μm, together with a small number of much larger vesicles. For ratios of CHEMS/DPPC above 0.1 only unilamellar vesicles and no bilayer stacks were found. The results confirm the hypothesis by Hauser ( Biochim. Biophys. Acta 772 (1984) 37–50), that the structures formed upon hydration of charged phospholipid films are unilamellar vesicles, while for neutral phospholipid films upon hydration bilayer stacks and multilamellar vesicles are formed. The effect of CHEMS on the liposome bilayer structure can be mainly ascribed to its charge inducing properties and presumably to a minor extent to its molecular geometry, or to a combination of both.

Morphometric analysis on myocardial injury related to the use of high volume potassium cardioplegic solution during ischemic arrest

We correlated the effects of high volumes of K+ cardioplegic solution on myocardial structure and function in 16 dogs following open-heart surgery. Eight animals received high volume potassium cardioplegic solution (25 cc/kg body weight, every 30 min) during 90 min of ischemic arrest (HVK-C group). The others received sufficient cardioplegic solution to maintain complete electrical arrest as defined by voltage monitoring criteria (VM group). Cardiac index (CI), left ventricular stroke work index (LVSWI), and myocardial contractility (dp/dt) were determined before arrest and after 90 min of ischemia and 45 min of reperfusion. Biopsies were taken for EM ultrastructure and ATP estimation. Morphometric analysis of EM micrographs found increased volume of damaged mitochondria (DMR) (p less than 0.025), damaged myofibrils (DMF) (p less than 0.001), intermyofibrilar edema (p less than 0.005), T-tubule and sarcoplasmic reticulum (p less than 0.05) in the HVK-C group. Left ventricular (LV) function was more depressed in animals receiving HVK-C. CI decreased by 1.8 +/- 0.4 l/min/square meter (p less than 0.01), LVSWS fell by 3.3 +/- 0.8 gm-m/beat/Kg (p less than 0.01), dp/dt decreased by 684 +/- 135 (p less than 0.0025). ATP decreased by 26% in HVK-C and by 12% in VM group (0.1 less than p less than 0.05). Structural damage (scores of injured volume of mitochondria and myofibrils) correlated with post-ischemic depression of LV function (Cardiac output and myocardial contractility), r = -0.72 and -0.66 (p less than 0.001 and 0.004).

Degreening in Canola ( Brassica napus cv. Westar) Embryos under Optimum Conditions

An HPLC method for rapid identification and quantification of picogram quantities of chlorophylls, chlorophyllides, pheophytins and pheophorbides in canola seeds is described. With this method, pigment changes during seed maturation in canola (Brassica napus cv. Westar) were analyzed. During seed maturation, the embryos undergo controlled degradative processes resulting in loss of chlorophyll. Both chlorophyll a and b degradation is biphasic. The breakdown products, chlorophyllide a and pheophytin a reach a peak during the period of rapid chlorophyll a degradation. Thereafter, loss of these pigments and chlorophyll a are linearly correlated. The relationship between chlorophyll b and its degradation products is not clear. However, this may reflect the low levels of these pigments in the seed rather than a lack of causal relationship. Despite the fact that green canola oil contains a high proportion of pheophytin, neither pheophytin nor pheophorbide accounted for a large proportion of the pigments in the seed. Electrophoretic profiles and EM micrographs indicate the presence of normal chloroplasts with pigments bound in chlorophyll-protein complexes. Furthermore, there is a gradual breakdown of the chlorophyllprotein complexes during degreening. The mature nongreen seed does not contain any components of the photosystems.

Ultrastrctural identification of viral infections in paraffin sections stained by immunohistochemistry and in situ hybridization

Confirmation of viral infections in surgical biopsies is sometimes limited to routine paraffin sections because a specimen may not have been set aside for culture or culture results are negative. Since viral infections are frequently focal and localized to only a few cells, definitive histopathological diagnosis can be difficult to achieve even with immunohistoGhemistry (IHC), in situ hybridization (ISH) or electron microscopy. In this paper, we would like to demonstrate a siirple and direct approach to confirming viral infections in paraffin sections , using two immuno-labelling techniques coupled with electron microscopy.Paraffin sections of surgical specimens are deparaffinized in xylene and rehydrated through graded ethyl alcohol. Herpes simplex virus (Figure 1) and Toxoplasma gondii are localized immunohistochemically by the peroxidase antiperoxidase technique and Pneumocystis carinii (Figure 2) by 3 step ipdirect method, using 3,3'-diaminobenzidine tetrahydrochloride as the chrmogen, CMV ENA probe (ENZO Biochemicals) is used for in situ hybridization, following kit instructions (Figure 3 &amp; 4). After the final chromogen reaction, wet sections are examined with a light microscope. Slides in vrfiich focal positive staining is difficult to interpret as specific are fixed in osmium tetroxide, dehydrated with graded acetone, and infiltrated with Poly/Bed 812. After polymerization, the epoxy blocks containing area of interest can be easily separated from the glass slides by heating on a 100 C hotplate for approximately 20 seconds . The area of interest should be positioned in the center of the block face for ultrathin sectioning. With careful adjustments of knife and microtcme, the area of interest can be precisely sectioned without loss of tissue. Ultrathin sections are collected on uncoated mesh grids for higher contrast and then directly examined by TEM. Peroxidase-labelled microorganisms or viral DHA can be easily localized and identified without heavy metal staining. Cellular details can be further increased by staining with uranyl acetate and lead citrate. Tissue remaining in block face after sectioning is available for IM comparison with the EM micrographs.

Scanning electron microscopic studies of pore formation in mica schist insulating brick

AbstractSEM micrographs showing porous structures and bonding of pores through broken mica flakes are presented. Pores of varying dimensions (10–400 μm) are formed. Interlayer spaces in mica are shown to be the preferential direction for heat and subsequently crack propagation during heat treatment for producing cellular insulating bricks at 900°C. The cracks end up with the formation of large pores in the presence of foaming agent and sodium silicate.