Localization of α-galactomannan on the surface of Schizosaccharomyces pombe cells by scanning electron microscopy

Galactomannan was localized by scanning and transmission electron microscopy on the cells and cell walls of Schizosaccharomyces pombe. The markers were prepared from colloidal gold granules labelled with an -galactopyranosyl-binding lectin isolated from the seeds of Bandeiraea simplicifolia. Part or all of this -galactomannan was present in the outer layer of the cell wall and was uniformly distributed even on the fission scars.Non-Standard Abbreviations Au lectin-labelled colloid - SEM scanning electron microscopy - TEM transmission electron microscopy     

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.     

Purification of protein–DNA complexes by native gel electrophoresis for electron microscopy study

Electrophoretic separation under native conditions may be used for purification of protein molecules and their complexes with DNA and other ligands. Here, we employed this approach to separate protein-DNA complexes with a molecular weight of approximately 200 kDa: mono- and dinucleosomes. The purified mononucleosomes were subjected to single particle electron microscopy study using negative stain contrasting, and the two-dimensional projections of the nucleosomes at 25 Å resolution were obtained. A comparison of the nucleosome projections before and after separation in the native PAGE revealed different orientation of particles on the carbon film.     

Localization of E1–E2 conformational transitions of sarcoplasmic reticulum Ca-ATPase by tryptic cleavage and hydrophobic labeling

Summary Tryptic peptides of Ca-ATPase in E_t and E₂ conformational states (Andersen, J. P., Jørgensen, P. L.,J. Membrane Biol. 88:187–198 (1985)) have been isolated by size exclusion high performance liquid chromatography in sodium dodecyl sulfate. This permitted unambiguous localization of a conformational sensitive tryptic split at Arg 198 by N-terminal amino acid sequence analysis. Other splits at Arg 505 and at Arg 819-Lys 825 were insensitive to E₁–E₂ transitions. Tryptic cleavage of Ca-ATPase after phosphorylation by inorganic phosphate showed that this enzyme form has a conformation similar to that of the vanadate-bound E₂ state, both in membranous and in soluble monomeric Ca-ATPase.Hydrophobic labeling of Ca-ATPase in sarcoplasmic reticulum vesicles with the photoactivable reagent trifluoromethyl-[¹²⁵I]iodophenyl-diazirine indicated that E₂ and E₂V states are more exposed to the membrane phase than E₁ and E₁P (Ca²⁺-occluded) states. The preferetial hydrophobic labeling in E₂ forms was found to be localized in the A₁ tryptic fragment.     

Oligomerization–function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor–ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag–probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~ 10%), whereas ~ 70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20 s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90 s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~ 1 nM) was lower than that for half-maximum autophosphorylation (~ 8 nM), which suggested the presence of an inactive dimer binding a single EGF molecule.     

Localization and distribution of laminin in the basal lamina of certain mechanoreceptors—an immunogold study

The applied immunogold cytochemical technique in investigating the cytologic distribution of the laminin (LAM) molecule in the capsulated Pacinian and Herbst mechanoreceptors shows the presence of LAM around most elements of the receptor structures. The LAM immunoreactivity (LAM-IR) is best expressed in the vicinity of the perineural capsule cells of both receptor types, where it is primarily concentrated around the perinuclear regions as well as the cytoplasmic lamellae. Such a localization overlaps with the already known ultrastructural localization of a basal lamina (BL) around these cells. Laminin immunoreactivity is less well expressed around the modified Schwann cells. Even in these cells, however, there is an apparent immunoreaction around the cytoplasmic lamellae regardless of the lamellar location. In both receptor types, there is no LAM-IR in the cells of the subcapsular space. Of particular significance we consider the localization of gold particles (respectively the presence of a BL) between the innermost lamellae of the modified Schwann cells and the non-myelinated part of the receptor nerve fiber and their endings, as well as around the axoplasmic protrusions of the nerve endings. We discuss the role of the BL and LAM in the investigated rapidly adapting mechanoreceptors and their trophic influence upon the sensory regions. We also assume the arresting and selective effect of these membranes in building up the ion channels of the axolemma which probably has a certain importance in mechanotransduction.     

Structure of the dimeric RC–LH1–PufX complex from Rhodobaca bogoriensis investigated by electron microscopy

Electron microscopy and single-particle averaging were performed on isolated reaction centre (RC)—antenna complexes (RC–LH1–PufX complexes) of Rhodobaca bogoriensis strain LBB1, with the aim of establishing the LH1 antenna conformation, and, in particular, the structural role of the PufX protein. Projection maps of dimeric complexes were obtained at 13 Å resolution and show the positions of the 2 × 14 LH1 α- and β-subunits. This new dimeric complex displays two open, C-shaped LH1 aggregates of 13 αβ polypeptides partially surrounding the RCs plus two LH1 units forming the dimer interface in the centre. Between the interface and the two half rings are two openings on each side. Next to the openings, there are four additional densities present per dimer, considered to be occupied by four copies of PufX. The position of the RC in our model was verified by comparison with RC–LH1–PufX complexes in membranes. Our model differs from previously proposed configurations for Rhodobacter species in which the LH1 ribbon is continuous in the shape of an S, and the stoichiometry is of one PufX per RC.     

“Chromosome exo-skeleton” in plant cells visualized by scanning electron microscopy

Summary Cytoskeletons surrounding the chromosomes of the root tip cells ofPisum sativum and the generative cells ofAllamanda schottii were visualized using Triton X-100 extraction and scanning electron microscopy. The cytoskeleton surrounding the chromosome consisted of a reticulate network of fibres. This is the first report showing the existence of a chromosome exo-skeleton in plant cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid)     

To be or not to be? Examples of incorrect identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells

Transmission electron microscopy is one of the most sensitive methods to detect autophagic compartments in mammalian cells. The number of scientists that are experts in this technique has declined during recent years, probably because the method was developed over forty years ago and is not in fashion today. This has lead to a situation where, too many times, authors and reviewers alike are not able to interpret electron microscopy pictures correctly. In this commentary I present some common mistakes in identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells.     

“Thiocyanate gold”: small (2–3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy

Summary Reduction of HAuCl₄ by NaSCN or KSCN produces colloidal gold particles of 2.6 nm in diameter and homogeneous in size (coefficient of variation 15%). The Au_SCN sol forms protein-gold complexes. The amount of protein required to form an Au_SCN-protein complex is best determined in the electron microscope, where serial dilutions of protein with gold sol are inspected for the presence of aggregates.By immuno-electron microscopy SCN-gold complexed to protein A is active and visible as is shown by revealing -amylase in rat pancreatic acinar cells.     

Effect of soil waterlogging on below‐ground biomass allometric relations in Norway spruce

Abstract

An increasing importance is assigned to the estimation and verification of carbon stocks in forests. Forestry practice has several long‐established and reliable methods for the assessment of above‐ground biomass; however, we still miss accurate predictors of below‐ground biomass. A major windthrow event exposing the coarse root systems of Norway spruce trees allowed us to assess the effects of contrasting soil stone and water content on below‐ground allocation. Increasing stone content decreases the root/shoot ratio, while soil waterlogging leads to an increase in this ratio. We constructed allometric relationships for below‐ground biomass prediction and were able to show that only soil waterlogging significantly impacts model parameters. We showed that diameter at breast height is a reliable predictor of below‐ground biomass and, once site‐specific parameters have been developed, it is possible to accurately estimate below‐ground biomass in Norway spruce.     

Improved transmission electron microscopy (TEM) of cultured cells through a “floating sheet” method

Cultured cells provide isolated systems for both biochemical and morphological studies. Previous methods of processing cell culture specimens for electron microscopy (EM) have been limited to sectioning either a monolayer or centrifuged cell suspensions which are not morphologically intact. In our improved method, N-butylglycidyl ether is added to cell cultures (2–5 min with agitation) following in situ fixation (3.0% glutaraldehyde in 0.1 M Pipes, pH 7.2, for 20 min, osmium tetraoxide 4% for 20 min). A thin pliable “sheet” of cells floats free from the plastic culture device and can be manipulated (centrifuged or folded) to obtain a vast number of morphologically intact cells for examination. We have examined several cell types (vascular smooth muscle, lung, liver, and endothelial cells) grown in two types of plastic culture flasks (Nunc and Falcon). This new method provides excellent EM morphology, maximizes the number of cells examined, and allows determination of cell orientation since a remnant of the dissolved flask remains loosely bound to the bottom of the cells.     

Divergent growth of Norway spruce on Babia Góra Mountain in the western Carpathians

Growth divergence – i.e. the expression of divergent growth trends of neighboring trees – has certain implications for dendrochronological research, for instance in the context of climate reconstructions but also in terms of estimating net ecosystem productivity. Thus, understanding the underlying mechanisms is essential to extend our fundamental dendroecological knowledge. In this context, the Picea genus plays an important role since several of its species were reported to exhibit growth divergence. Here, we investigate a well sampled Norway spruce (Picea abies (L.) Karst) data set for growth divergence comprising ring-width and Blue Intensity measurements from seven sites on Babia Góra Mountain, at the border between Poland and Slovakia. By means of Principal Component Gradient Analysis, inter-series correlations, and climate growth relationships, we are able to show that I) Norway spruce on Babia Góra expressed growth divergence since the 1970s, II) the definition of groups increased the strength of population signals and the stability of climate-growth relationships, and III) Blue Intensity appeared as a more robust proxy for environmental conditions. We discuss soil heterogeneity, genetics, and air pollution as possible underlying mechanisms, thereby indicating further research avenues to obtain a better understanding of growth divergence.     

Location of two Mn2+ affinity sites in photosystem II detected by pulsed electron–electron double resonance

In this study, we identified two Mn²⁺ sites in apo-Photosystem II (PSII) using the pulsed electron–electron double resonance (PELDOR). A Mn²⁺ ion was bound to apo-PSII on the deactivation of the oxygen-evolving complex. The electron–electron magnetic dipole interaction of the Mn²⁺ to Y_D^· was estimated to be 2.4 MHz. The site was assigned at the position between His332 and Glu189 in the D1 polypeptide, which is close to the Mn1 site in mature PS II. Using recent structures observed under electron microscopes (EM), the location of the Mn²⁺ site on photoactivation was reevaluated. The position between Asp170 and Glu189 in the D1 polypeptide is a good candidate for the initial high-affinity site for photoactivation. Based on a comparison with the PELDOR results, the two EM structures were evaluated.