Electron microscopic observations of an unusual cytoplasmic inclusion in the calcium-starved white mutant (LU887 × LU897) of Physarum polycephalum

Electron micrographs of Physarum polycephalum microplasmodia (LU887 × LU897) reveal cytoplasmic inclusions that appear “striated” at low magnifications; at higher magnifications these exhibit a structure that we have interpreted as microtubule bundles. The light and dark regions in the inclusions are due to the affinity of some microtubules for osmic acid; these appear to have dense regions while other microtubules remain electron lucent. The diameters of the microtubules are about 32–33nm; the subunits forming the tubule walls measure about 8–9nm in diameter. The diameter measurements are slightly larger than the dimensions assigned to vertebrate microtubules (28nm); however, the diameter of the subunits in the microtubule wall measures about 8–9nm which is essentially the same measurement reported for vertebrate tubulin dimers.     

A soft X-ray synchrotron study of the charge state of iron ions in the ferrihydrite core of the ferritin Dps protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Escherichia coli Dps protein belongs to a specific family of bacterial ferritins; it is a nanosized particle that contains an inorganic core (~5 nm in diameter) and a protein shell with a size of 8–9 nm. The protein shell consists of 12 identical subunits with the known crystal structure of a dodecamer. The composition and structure of the core have been less studied. The core formation is associated with the oxidation products of Fe2+ ions in the ferroxidase centers of the protein. Thus, Fe2O3 oxides are the main compounds of the core. However, the mineralization properties of Fe2+ ions under anaerobic conditions in vitro may indicate a more complicated composition of the core in the native Dps protein. This paper presents a technique for the preparation of purified Dps samples for ultrahigh vacuum synchrotron experiments by X-ray absorption near edge structure spectroscopy of the iron absorption edge in the soft X-ray region. The conducted synchrotron experiments have revealed the presence of both trivalent and divalent iron ions in the octahedral and tetrahedral environment of oxygen atoms in the prepared biological samples. This points to a complex ionic composition of the core even in the native Dps protein, which has been isolated from aerobically grown bacteria.     

Flagellar hook structures of Caulobacter and Salmonella and their relationship to filament structure

Native flagellar hooks from a polarly flagellated bacterium, Caulobacter crescentus, and polyhooks from a peritrichously flagellated bacterium, Salmonella typhimurium. have been studied by densitometry of electron micrographs of negatively stained specimens, followed by computerized Fourier analysis and three-dimensional reconstruction. The two structures are remarkably similar. In both cases, the subunits are arranged along a right-handed basic helix of 2.3 nm pitch with successive subunits separated by an azimuthal angle of 64 to 65 °, and there is a pronounced system of continuous 6-start grooves and ridges on the surface of the structures. The subunit of Salmonella (M_r 42,000, versus 70,000 for Caulobacter) is somewhat thinner and yields a smaller overall hook diameter. The “bent finger” subunit shape and orientation in both cases suggests that the hook could bend readily by a sliding motion in the 11-start direction at inner radii, with the 6-start groove preventing collision at outer radii. The basic helical pitch of the Salmonella hook structure, and the number of subunits per basic helical turn (5.56) makes it highly compatible with the Salmonella flagellar filament (2.6 nm pitch. 5.51 subunits per turn); so also does the elongated shape and tilt angle of the hook and flagellin subunits in the respective structures. The two structures may therefore conjoin directly in the intact flagellum, although participation of a minor protein is not ruled out by the data.     

Small-angle X-ray studies of Lumbricus terrestris haemoglobin

Small-angle X-ray scattering of Lumbricus terrestris haemoglobin was measured in dilute solutions in 0.1 M Tris HCl buffer, pH 7.0. The following molecular parameters were determined: radius of gyration 11.2 nm, volume 7700 nm³, maximum diameter 29 nm, molecular weight 3.95 × 10⁶. The experimental scattering curve was compared with the scattering curves and distance distribution functions calculated for various models. The overall shape of the haemoglobin could be approximated by a hollow cylinder with the following dimensions: outer radius 13.5 nm, inner radius 5.4 nm, height 16.0 nm. The best fit was obtained with a model which consists of 12 large subunits arranged in two superimposed hexagonal rings with a number of smaller subunits between the large subunits and in the centre of the molecule.     

A new virus disease in the housefly, Musca domestica (Diptera)

Reovirus particles were isolated from adults in laboratory colonies of the housefly, Musca domestica. These particles were spherical in outline, 57–76 nm in diameter, and were found only in hemocyte cytoplasm, where virions have been disclosed by a new technique. Virions were present in large numbers, and viral inclusion bodies were identified. The virus particles had pentagonal and hexagonal shapes resembling a simple icosahedral structure. The virus was shown to be infectious and pathogenic to adult flies through injection or by feeding them suspensions from flies that had died of the virus. Electron micrographs of midgut sections from infected flies showed that the midgut cells were packed with dark undulating threads which were not present in uninfected flies. However, no virus particles or inclusion bodies could be seen in these cells. On the basis of their association with infected flies, and the similarity to results from other studies on reoviruses and insect viruses, it is suggested that these threads are an alternative replicative form of the reovirus. When the virus suspensions from heavily infected flies were dialyzed against weak alkaline solutions, the threads showed an inner component of coiled material, 12 nm in diameter, inside an envelope with a diameter of 50–83 nm, mean 60.3 ± 7.5, composed of subunits 7–8 nm long and 7–8 nm across.     

Die Feinstruktur der lamellären Einschlußkörper in den Zellkernen des Nektariums von Catalpa bungei

Summary In the nectary of Catalpa bungei the gland cells contain nuclear inclusions consisting of stacks of lamellae 12.7 nm thick. Each lamella is composed of globular particles with a diameter of approximately 12.7 nm. The particles are arranged in a monolayer, revealing a regular square pattern in face view. In adjacent lamellae the globular subunits are almost exactly superimposed; each of them is probably built up of smaller subunits having a diameter of 0.4 nm.

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Herrn Prof. Dr. Horst Drawert nachträglich zum 60. Geburtstag gewidmet     

On the egg investments and fertilization reaction inPomatoceros triqueter L.

Summary After a specific time of glutaraldehyde-acrolein fixation, microtubule walls appear to be composed of single 6.5–7.5 nm diameter osmiophilic subunits. Variations in the duration of glutaraldehyde-acrolein and also glutaraldehyde-osmium fixation reveal a two layered wall containing osmiophilic subunits, 4.0–4.5 nm in diameter, arranged radially, in tandem. The double-layered wall is demonstrated by microdensitometer traces. These observations are discussed in relation to previously proposed models of microtubule substructure.     

The role of the T-pilus in horizontal gene transfer and tumorigenesis

The T-pilus is a flexuous filamentous appendage that is essential for Agrobacterium tumefaciens virulence. T-pilus subunits are derived from a VirB2-processing reaction that generates cyclized polypeptide subunits. The T-pilus filament has a diameter of 10 nm and contains a lumen approximately 2 nm in diameter. Biogenesis of the T-pilus requires all 11 VirB proteins, but not the VirD4 protein, which is used in conjugal plasmid transfer. VirB4 and VirB11 are two ATPases that may form homohexameric rings within the transport apparatus, which is composed of VirB6-10 proteins.     

Crystal Structure of Chlorite Dismutase, a Detoxifying Enzyme Producing Molecular Oxygen

Chlorite dismutase (Cld) is a key enzyme of perchlorate and chlorate respiration. This heme-based protein reduces the toxic compound chlorite into the innocuous chloride anion in a very efficient way while producing molecular oxygen. A sequence comparison between Cld homologues shows a highly conserved family. The crystal structure of Azospira oryzae strain GR-1 Cld is reported to 2.1 Å resolution. The structure reveals a hexameric organization of the Cld, while each monomer exhibits a ferredoxin-like fold. The six subunits are organized in a ring structure with a maximal diameter of 9 nm and an inner diameter of 2 nm. The heme active-site pocket is solvent accessible both from the inside and the outside of the ring. Moreover, a second anion binding site that could accommodate the assumed reaction intermediate ClO‾ for further transformation has been identified near the active site.The environment of the heme cofactor was investigated with electron paramagnetic resonance spectroscopy. Apart from the high-spin ferric signal of the five-coordinate resting-state enzyme, two low-spin signals were found corresponding to six-coordinate species. The current crystal structure confirms and complements a recently proposed catalytic mechanism that proceeds via a ferryl species and a ClO‾ anion. Our structural data exclude cooperativity between the iron centers.     

Isolation and characterization of wheat ribulose-1,5-diphosphate carboxylase

Wheat ribulose-1,5-diphosphate carboxylase purified to homogeneity had a MW of 540 000, sedimentation coefficient (S_{20, W}) of 18.5 S, apparent diffusion constant (D_app) of 3.07 × 10^?7 cm²/sec, Stoke's radius 5.44 nm, and fractional ratio of 1.17. Electron microscopy revealed particles of 10–12 nm diameter. The enzyme was dissociated by sodium dodecyl sulphate into two subunits of MW 53 000 (S_{20, W} = 3.0 S) and 13 500 (S_{20, W} = 1.7 S). The total amino acid residues in the large and small subunits were 481 and 117, respectively. Tryptic peptide maps of the two subunits confirmed the estimated numbers of Arg and Lys residues. Although the amino acid pattern of the large subunit closely resembled that from barley, rather than that for spinach, beet or tobacco, the pattern of the small subunit was markedly different from those of all the other species.     

High resolution analysis of the formation of cellulose synthesizing complexes inVaucheria hamata

Summary Ultrastructure and assembly of cellulose terminal synthesizing complexes (terminal complexes, TCs) in the algaVaucheria hamata (Waltz) were investigated by high resolution analytical techniques for freeze-fracture replication.Vaucheria TCs consist of many diagonal rows of subunits located on the inner leaflet of the plasma membrane. Each row contains about 10–18 subunits. The subunits themselves are rectangular, approx. 7×3.5 nm, and each has a single elliptical hole which may be the site of a single glucan chain polymerization. The subunits are connected with extremely small filaments (0.3–0.5 nm). Connections are more extensive in a direction parallel to the subunit rows and less extensive perpendicular to them. Nascent TC subunits are found to be packed within globules (15–20 nm in diameter) which are larger than typical intramembranous particles (IMPS are 10–11 nm in diameter) distributed in the plasma membrane. The subunits in the globule, which may be a zymogenic precursor of the TC, are generally exhibited in the form of doublets. Approximately 6 doublets are connected to a center core with small filaments. The globules are inserted into the plasma membrane together with IMPS by the fusion of cytoplasmic (Golgi derived) vesicles. Two or three globules attach to each other, unfold, and expand to form the first subunit rows of the TC on the inner leaflet of the plasma membrane. More globules attach to the structure and unfold until the nascent TC consists of a few rows of subunits. These rows are arranged almost parallel to each other. Two formation centers of subunits appear at both ends of an elongating TC. New subunits carried by the globules are added at each of these centers to create new rows until the elongating TC structure is completed. On the basis of this study, a model of TC assembly and early initiation of microfibril formation inVaucheria is proposed.Abbreviations IMPS intramembranous particles - MF microfibril - TC terminal complex     

Structure of D-glyceraldehyde-3-phosphate dehydrogenase fromPalinurus versicolor in a tetragonal crystal form

D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell parameters a=15.49 nm, c=8.03 nm and two subunits per asymmetric unit. The crystal structure at 0.34 nm was determined by the molecular replacement method. The final model has crystallographic Rfree and R factors of 0.274 and 0.262, and r.m.s. deviations of 0.002 nm for bond lengths and 2.33?for bond angles. The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure, but also in average temperature factors. This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits. This further supports that holo-GAPDH has a good 222 molecular symmetry.     

Plasticity of Intermediate Filament Subunits

Intermediate filaments (IFs) assembled in vitro from recombinantly expressed proteins have a diameter of 8–12 nm and can reach several micrometers in length. IFs assemble from a soluble pool of subunits, tetramers in the case of vimentin. Upon salt addition, the subunits form first unit length filaments (ULFs) within seconds and then assembly proceeds further by end-to-end fusion of ULFs and short filaments. So far, IF subunits have mainly been observed by electron microscopy of glycerol sprayed and rotary metal shadowed specimens. Due to the shear forces during spraying the IF subunits appear generally as straight thin rods. In this study, we used atomic force microscopy (AFM), cryo-electron microscopy (cryo-EM) combined with molecular modeling to investigate the conformation of the subunits of vimentin, desmin and keratin K5/K14 IFs in various conditions. Due to their anisotropic shape the subunits are difficult to image at high resolution by cryo-EM. In order to enhance contrast we used a cryo-negative staining approach. The subunits were clearly identified as thin, slightly curved rods. However the staining agent also forced the subunits to aggregate into two-dimensional networks of dot-like structures. To test this conformational change further, we imaged dried unfixed subunits on mica by AFM revealing a mixture of extended and dot-like conformations. The use of divalent ions such as calcium and magnesium, as well as glutaraldehyde exposure favored compact conformations over elongated ones. These experimental results as well as coarse-grained molecular dynamics simulations of a vimentin tetramer highlight the plasticity of IF subunits.     

Cellulose-synthesizing terminal complexes and microfibril structure in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)*

The brown alga Sphacelaria rigidula Kützing synthesizes cellulose microfibrils as determined by CBH I-gold labeling. The cellulose microfibrils are thin, ribbon-like structures with a uniform thickness of about 2.6 nm and a variable width in the range of 2.6-30 nm. Some striations appear along the longitudinal axis of the microfibrils. The developed cell wall in Sphacelaria is composed of three to four layers, and cellulose micro-fibrils are deposited in the third layer from the outside of the wall. A freeze fracture investigation of this alga revealed cellulose-synthesizing terminal complexes (TCs), which are associated with the tip of microfibril impressions in the plasmatic fracture face of the plasma membrane. The TCs consist of subunits arranged in a single linear row. The average diameter of the sub-units is about 6 nm, and the intervals between the neighboring subunits, about 9 nm, are relatively constant. The number of subunits constituting the TC varies between 10 and 100, so that the length of the whole TC varies widely. A model that has been proposed for the assembly of thin, ribbon-like microfibrils was applied to microfibril assembly in Sphacelaria.     

The formation and development of cellulose-synthesizing linear terminal complexes (TCs) in the plasma membrane of the marine red algaErythrocladia subintegra Rosenv.

Summary The formation and development of linear terminal complexes (TCs), the putative cellulose synthesizing units of the red algaErythrocladia subintegra Rosenv., were investigated by a freeze etching technique using both rotary and unidirectional shadowing. The ribbon-like cellulose fibrils ofE. subintegra are 27.6 ± 0.8 nm wide and only 1–1.5 nm thick. They are synthesized by TCs which are composed of repeating transverse rows formed of four particles, the TC subunits. About 50.4 ± 1.7 subunits constitute a TC. They are apparently more strongly interconnected in transverse than in longitudinal directions. Some TC subunits can be resolved as doublets by Fourier analysis. Large globular particles (globules) seem to function as precursor units in the assembly and maturation of the TCs. They are composed of a central hole (the core) with small subunits forming a peripheral ridge and seem to represent zymogenic precursors. TC assembly is initiated after two or three gobules come into close contact with each other, swell and unfold to a nucleation unit resembling the first 2–3 transverse rows of a TC. Longitudinal elongation of the TC occurs by the unfolding of globules attached to both ends of the TC nucleation unit until the TC is completed. The typical intramembranous particles observed inErythrocladia (unidirectional shadowing) are 9.15 ± 0.13 nm in diameter, whereas those of a TC have an average diameter of 8.77 ± 0.11 nm. During cell wall synthesis membranes of vesicles originating from the Golgi apparatus and which seem to fuse with the plasma membrane contain large globules, 15–22 nm in diameter, as well as tetrads with a particle diameter of about 8 nm. The latter are assumed to be involved in the synthesis of the amorphous extracellular matrix cell wall polysaccharides. The following working model for cellulose fibril assembly inE. subintegra is suggested: (1) the ribbon-like cellulose fibril is synthesized by a single linear TC; (2) the number of glucan chains per microfibril correlates with the number of TC subunits; (3) a single subunit synthesizes 3 glucan chains which appear to stack along the 0.6 nm lattice plane; (4) lateral aggregation of the 3-mer stacks leads to the crystalline microfibril.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Fibrous and tubular inclusions in four xylariaceous fungi

Summary Intranuclear and cytoplasmic fibrous inclusions and cytoplasmic tubular inclusions have been studied using electron microscope techniques. The fibrous inclusions are composed of closely packed, rod-like structures. Each rod has an outside diameter of ± 24 nm, a hollow centre and lateral projections along its length. The tubular inclusions occur as densely packed bundles or loose arrays of 10–13 nm diameter tubules which are composed of subunits arranged in a double helical structure. The distribution, origin and possible function of these and similar inclusions is discussed.     

Gonadotropin and subunit conformation.

Circular dichroic spectra have been obtained and resolved for the gonadotropins, ovine pituitary luteinizing hormone and human chorionic (urinary) gonadotropin, their subunits and glycopeptides. Much of the gonadotropin ellipticity above 250 nm can be attributed to the disulfide chromophore, although there are discernible contributions from tyrosyl and phenylalanyl residues as well. Of the two dissimilar subunits, the β-subunit makes the greatest contribution to the near-ultraviolet circular dichroic spectrum of the gonadotropins. From the position of the 0-0 tyrosyl band, i.e., 286–287 nm, one can ascertain that at least some of the tyrosyl residues of the gonadotropins are located in a hydrophobic environment. A positive circular dichroic extremum at 232.5 nm, present in luteinizing hormone but not in chorionic gonadotropin, can be ascribed to the α-subunit and probably results from tyrosines 21 and/or 30 in luteinizing hormone.An analysis of the circular dichroic difference spectrum above 230 nm, generated by subtracting the sum of the molecular ellipticities of the respective subunits from the molecular ellipticities of each gonadotropin, indicates that the local environment of disulfides and of tyrosyl residues is altered when gonadotropins dissociate. Circular dichroic difference spectra between the two α-subunits and between the two β-subunits indicated major contributions from- tyrosyl residues, presumably arising from tyrosyl substitutions.Between 200 and 230 nm, both gonadotropins exhibit negative circular dichroic extrema. The extremum occurs at 210 nm for luteinizing hormone and at 207.5 nm for chorionic gonadotropin. Each extremum can be described by two negative resolved bands, one at 215 nm and the other between 207 and 208.5 nm. The 215-nm resolved band is assigned to the peptide chromophore in a β-pleated sheet conformation and there is no evidence of α-helicity. The lower-wavelength resolved band is believed to have a significant contribution from the N-acetyl groups of glucosamine, galactosamine, and sialic acid, particularly since the glycopeptide fractions, prepared from each gonadotropin by digestion of the S-carboxymethyl derivatives with Pronase, exhibited a negative circular dichroic extremum at about 207 nm.The extent of β-structure in both gonadotropins is estimated to be about 28% whereas the separated subunits contain less β-structure, e.g., about 21 and 13% for the α- and β-subunits, respectively. The sum of the subunit β-structure, corrected for the respective molecular weight of each subunit, is about 17%. This is substantially less than that of the native hormone, thus indicating that significant conformational changes occur during gonadotropin dissociation to the biologically inactive subunits. Also, part of the gonadotropin β-structure may arise from intermolecular hydrogen bonding involving a pleated sheet arrangement between the subunits.     

Fine structure in cellulose microfibrils: NMR evidence from onion and quince

It has been controversial for many years whether in the cellulose of higher plants, the microfibrils are aggregates of ‘elementary fibrils’, which have been suggested to be about 3.5 nm in diameter. Solid-state NMR spectroscopy was used to examine two celluloses whose fibril diameters had been established by electron microscopy: onion (8–10 nm, but containing 40% of xyloglucan as well as cellulose) and quince (2 nm cellulose core). Both of these forms of cellulose contained crystalline units of similar size, as estimated from the ratio of surface to interior chains, and the time required for proton magnetisation to diffuse from the surface to the interior. It is suggested that the onion microfibrils must therefore be constructed from a number of cellulose subunits 2 nm in diameter, smaller than the ‘elementary fibrils’ envisaged previously. The size of these subunits would permit a hexagonal arrangement resembling the cellulose synthase complex.     

Microtubules with different diameter, protofilament number and protofilament spacing in Ornithogalum umbellatum ovary epidermis cells

Microtubules present in the epidermis of Ornithogalum umbellatum ovary in the area of lipotubuloids (i.e. aggregates of lipid bodies surrounded by microtubules) are 25-51 nm in diameter. They consist mainly of 10 and 11, sometimes 9 and 12 protofilaments. An average diameter of microtubule consisting of 9 subunits is about 32 nm, of 10-35 nm, of 11-38 nm and of 12-43 nm, however, individual microtubules in each category significantly vary in size. These differences result from varying distance between protofilaments in microtubule walls and diameters of protofilaments: in thin microtubules they are densely packed and smaller while in thicker ones they are loosely arranged and bigger. A hypothesis has been put forward that changes in microtubule diameter depend on structural changes associated with their functional status and are executed by modifications of protofilament arrangement density and their diameters in microtubule wall. The above hypothesis seems to be in agreement with the opinion formed on the basis of in vitro image of microtubules, that lateral contact between tubulin subunits in neighboring protofilaments indicates some flexibility and changeability during microtubule function.     

Morphogenesis and ultrastructure of Geotrichum candidum septa

The ultrastructure and mode of formation of septa of Geotrichum candidum were investigated by light and electron microscopy. The invaginations of the lateral membrane and wall appear to initiate at multiple points around the circumference of the cell; the immature septum subsequently assumes a cart-wheel shape, with branched spokes radiating from the center of the septum. Each face of the septum is covered with a membrane possessing hitherto undescribed structural differentiation; the membrane substructures are comprised of two central subunits encircled by 12 identical subunits. The diameter of the entire 12 plus 2 structure is 24 to 25 nm, and the diameter of each individual subunit is approximately 4 nm. The maturation of the septum appears to occur by further deposition of material along the branched skeletal regions. Numerous small openings (micropores), formed as a result of incomplete deposition, ultimately give rise to plasmodesmata. During arthrospore formation, the plasmodesmal canals and associated micropores are occluded by electron-dense materials, rendering each segment of the hyphae completely independent of the rest of the hyphae.