Characterization of paired helical filaments by scanning transmission electron microscopy

Paired helical filaments (PHFs) are abnormal twisted filaments composed of hyperphosphorylated tau protein. They are found in Alzheimer's disease and other neurodegenerative disorders designated as tauopathies. They are a major component of intracellular inclusions known as neurofibrillary tangles (NFTs). The objective of this review is to summarize various structural studies of PHFs in which using scanning transmission electron microscopy (STEM) has been particularly informative. STEM provides shape and mass per unit length measurements important for studying ultrastructural aspects of filaments. These include quantitative comparisons between dispersed and aggregated populations of PHFs as well as comparative studies of PHFs in Alzheimer's disease and other neurodegenerative disorders. Other approaches are also discussed if relevant or complementary to studies using STEM, e.g., application of a novel staining reagent, Nanovan. Our understanding of the PHF structure and the development of PHFs into NFTs is presented from a historical perspective. Others goals are to describe the biochemical and ultrastructural complexity of authentic PHFs, to assess similarities between authentic and synthetic PHFs, and to discuss recent advances in PHF modeling.     

Time-resolved cryo-electron microscopy of vitrified muscular components

Biological objects may be arrested in defined stages of their activity by fast freezing and may then be structurally examined. If the time between the start of activity and freezing is controlled, structural rearrangements due to biological function can be determined. Cryo-electron microscopy shows great potential for the study of such time-dependent phenomena. This study examines the actin polymerization process using cryo-electron microscopy of vitrified specimens. Actin filaments are shown to undergo a structural change during polymerization. In the early stages of the polymerization process (t < 2 min), filaments exhibit a pronounced structural variation and frequently show a central low-density area. In the later stages of the polymerization, F-actin-ADP filaments have a more uniform appearance and rarely display a central low-density area. These findings, analysed on the basis of a previously proposed polymerization model, suggest that polymerization intermediates (F-actin-ATP and more probably F-actin-ADP-P_i) and filaments at steady state (F-actin-ADP) have different structures. To investigate the physiological relevance of these results at the cellular level, the potential of cryo-substitution in preserving the structure of muscular fibre was assessed. Optical diffraction patterns of relaxed and contracted frog cutaneous muscle are similar to the corresponding X-ray diffraction patterns. The resolution of the images extends to about 7 nm. These results show that dynamic study of muscle contraction is possible using cryo-substitution.     

Filopodia formation induced by active mDia2/Drf3

Filopodia are rod-shaped cell surface protrusions composed of a parallel bundle of actin filaments. Since filopodia frequently emanate from lamellipodia, it has been proposed that they form exclusively by the convergence and elongation of actin filaments generated in lamellipodia networks. However, filopodia form without Arp2/3-complex, which is essential for lamellipodia formation, indicating that actin filaments in filopodia may be generated by other nucleators. Here we analyzed the effects of ectopic expression of GFP-tagged full length or a constitutively active variant of the human formin mDia2/Drf3. By contrast to the full-length molecule, which did not affect cell behaviour and was entirely cytosolic, active Drf3 lacking the C-terminal regulatory region (Drf3DeltaDAD) induced the formation of filopodia and accumulated at their tips. Low expression of Drf3DeltaDAD induced rod-shaped or tapered filopodia, whereas over-expression resulted in multiple, club-shaped filopodia. The clubs were filled with densely bundled actin filaments, whose number but not packing density decreased further away from the tip. Interestingly, clubs frequently increased in width after protrusion beyond the cell periphery, which correlated with increased amounts of Drf3DeltaDAD at their tips. These data suggest Drf3-induced filopodia form and extend by de novo nucleation of actin filaments instead of convergent elongation. Finally, Drf3DeltaDAD also induced the formation of unusual, lamellipodia-like structures, which contained both lamellipodial markers and the prominent filopodial protein fascin. Microarray analyses revealed highly variable Drf3 expression levels in different commonly used cell lines, reflecting the need for more detailed analyses of the functions of distinct formins in actin cytoskeleton turnover and different cell types.     

Development and characterization of flexible heating fabric based on conductive filaments

Active heating garments will be a promising product for improving life quality of rheumatisants and old people in the future. This paper presented an improved fabrication method of flexible heating fabrics (FHFs) which were developed by weaving intermittently silver filaments (SFs) or coated silver yarns (CSYs) into plain fabrics, this method can control more easily density of conductive filaments and rated power of FHFs in terms of requirements. Resistance thermostability and utmost load current of several kinds of conductive materials were utilized to determine an appropriate conductive material of FHFs. Subsequently, performances of FHF with SFs were characterized by a temperature controlling and power measuring device that was developed in our laboratory. Experimental results show SF can load over 0.3 A current, but the CSY and coated silver knitted fabric (CSKF) can load less than 0.1 A current. However, resistance variation ratios (RVRs) of SF are less than etc. 1.66%, but the RVRs of CSY and CSKF exceed 10% and 20%, when environment temperature increases from room temperature to 80 and 100 °C. Furthermore, strong positive linear correlations are between rated power and utmost ascending temperature of FHFs and between power consumption and presetting equilibrium temperatures of FHFs. In general, measurement data and analysis results can provide help for the design and prediction of physical properties of FHFs.     

Paired helical filaments (PHFs) are a family of single filament structures with a common helical turn period: negatively stained PHF imaged by TEM and measured before and after sonication, deglycosylation, and dephosphorylation

Isolated paired helical filaments (PHFs) were visualized on glutaraldehyde vapor-treated thin approximately 10-nm thick indirect carbon films using high-resolution transmission electron microscopy (TEM) and the negative stain, phosphotungstate acid (PTA) at near neutral pH of 6.8. PHF preparations were prepared with and without 1 minute of sonication. These same PHF were also deglycosylated with endoglycosidase F/N-glycosidase F for 1 hour or the PHF were dephosphorylated with PP-2A for 1 hour. The negatively stained PHF filaments were quantitatively studied by measuring their wide regions (W) their thin regions (T) and their helical turn period (L) and these separate parameters were averaged for each filament. In the unsonicated PHF preparation there were PHF, cylindrical filaments with periodic thin regions (CF-PT), cylindrical filaments (CF), as well as 2.0-nm tau polymer-like filaments. The CF-PT were characterized by W, T, and L measurements and the CF were characterized by diameter measurements. The paired helical filament model proposed by Kidd (1963, Nature 197:192-193) of two approximately 10 nm filaments twisting around each other every approximately 80 nm with a thin region of 10 nm and a wide region of 25 nm does not correspond to the PHF structures found. None of the PHF we observed were composed of a pair of filaments and all of the PHF appear to be a single filament. The wide regions ranged from 12.5-27 nm and the thin regions ranged from 4.5-12.3 nm. The helical turn periods ranged from 76-85 nm and were generally about 80 nm. Only the helical turn period of approximately 80 nm was a common property of the whole family of PHF structures. The CF-PT appear to be a PHF precursor filament. Deglycosylation of the PHF and CF-PT reduced their sizes by 0.5-0.6 nm and 0.7-1.0 nm, respectively, and the right-hand helicity of the PHF was lost after deglycosylation. Dephosphorylation with PP-2A reduced the PHF wide regions by 6.0 nm and the thin regions by 2.6 nm.     

Organization and functional roles of the cytoskeleton in oligodendrocytes

Mature oligodendrocytes are characterized by their numerous cytoplasmic extensions and flat membranous sheets. These sheets contain an extensive cytoskeletal network of microtubules (MTs) that maintain the cellular morphology, are specifically important for cellular sorting, and provide the rails for organelle trafficking. Mitochondria are localized in the primary and secondary processes and follow the tracks of the MTs in the cytoplasmic extensions. Oligodendrocytes express microtubule associated proteins (MAPs), specifically MAP2 and tau, which might be involved in the regulation and stabilization of the dynamic MT network in the myelin-containing cellular processes. Tau and MAP2 heterogeneity increases during oligodendroglia maturation, and in mature oligodendrocytes tau mRNA with four MT binding domains are more prominent than in progenitor cells. Filamentous cell inclusions are a unifying mechanism underlying a variety of late-onset neurodegenerative disorders and have mainly been viewed as neuron-specific. Recent evidence indicated that glial changes occur in CNS degenerative diseases and seem to be a more common feature than previously thought. Glial fibrillary tangles (GFTs) in oligodendrocytes were observed in familial multiple system tauopathy, and glial cytoplasmic inclusions (GCIs) and oligodendroglia degeneration are the histological hallmark of multiple system atrophy (MSA). GCIs are associated with MTs and contain stress proteins and MAPs. Thus, neurons and glial cells share common cytoskeletal pathologies. During health and disease, MAPs might be important regulators of the structural stability and plasticity of the oligodendroglia cytoskeleton.     

Tau binds ATP and induces its aggregation

Tau is a microtubule‐associated protein mainly found in neurons. The protein is associated with process of microtubule assembly, which plays an important role in intracellular transport and cell structure of the neuron. Tauopathies are a group of neurodegenerative diseases specifically associated with tau abnormalities. While a well‐defined mechanism remains unknown, most facts point to tau as a prominent culprit in neurodegeneration. In most cases of Tauopathies, aggregates of hyperphosphorylated tau have been found. Two proposals are present when discussing tau toxicity, one being the aggregation of tau proteins and the other points toward a conformational change within the protein. Previous work we carried out showed tau hyperphosphorylation promotes tau to behave abnormally resulting in microtubule assembly disruption as well as a breakdown in tau self‐assembly. We found that tau's N‐terminal region has a putative site for ATP/GTP binding. In this paper we demonstrate that tau is able to bind ATP and not GTP, that this binding induces tau self‐assembly into filaments. At 1 mM ATP the filaments are 4–7 nm in width, whereas at 10 mM ATP the filaments appeared to establish lateral interaction, bundling and twisting, forming filaments that resembled the Paired Helical Filaments (PHF) isolated from Alzheimer disease brain. ATP‐induced self‐assembly is not energy dependent because the nonhydrolysable analogue of the ATP induces the same assembly. Microsc. Res. Tech. 77:133–137, 2014. © 2013 Wiley Periodicals, Inc.     

Form and distribution of actin and myosin in non-muscle cells: a study using cultured chick embryo fibroblasts.

Attempting to throw light on the mechanical basis of movement of non-muscle (cf. muscle) cells, the present work aims to determine the form and distribution of actin and myosin in chick embryo fibroblasts. These cells were cultured on formvar, fixed in glutaraldehyde then osmium tetroxide vapours, dehydrated, critical-point dried and examined, in toto, in the electron microscope (EM). Stereoscopic pairs of micrographs were studied to define more exactly the form and distribution of cytoplasmic filaments topographically associated with deformations of the cell surface and with organelle movements through the cytoplasm. Permeating the cytoplasm, interconnecting long and short filaments closely surrounded all organelles, linked with microtubules and polyribosomes and joined to the plasma membrane. These filaments, which varied greatly in width (2-13 nm) were closely associated with large numbers of 'comma-shaped' globoid bodies of approximately 15 nm diameter. Attempting to establish the identity, form and distribution of cytoplasmic myosin, cultured cells were extracted with a cold (4 degrees C) glycerol/pyrophosphate solution for 24 h before being fixed and critical-point dried. EM examination of these cells revealed a residual three-dimensional network of branching and anastomosing 4-13 nm diameter smooth filaments, devoid of fine (2 nm) filaments and globoid bodies. Examination of fixed, critical-point dried, skeletal muscle heavy meromyosin showed globoid structures similar in form and size to the globoid bodies found in cultures fibroblasts. Similarly fixed and critical-point dried paracrystals of actin, polymerized in the presence of Mg2+, appeared as branching interconnecting filaments which, in form and dimensions, resembled the network filaments observed in pyrophosphate-extracted cells. It is concluded that the pyrophosphate-extractable globoid bodies found in cultured fibroblasts represent monomers of myosin, that the broader filaments to which these attach represent actin in Mg2+ paracrystalline form and that the various subcellular movements are brought about by interactions between the two, analogous to those occurring in muscle cells.     

Alzheimer paired helical filaments (PHFs) studied by high-resolution TEM: what can vertical Pt-C replication tell us about the organization of the pronase-digested PHF core?

Untreated paired helical filaments (PHFs) and pronase-digested PHF-core filaments were stereoscopically imaged with a freeze-drying vertical platinum-carbon replication preparation method for TEM. The untreated PHF have an average wide region (W) = 22.8 +/- 2.4 nm, a narrow region (T) = 10.6 +/- 1.7 nm, and a helical turn period (L) = 78.6 +/- 13.4. The surfaces of the untreated PHF's fuzzy coat appears disorganized. The widths of the pronase-treated PHF-core filaments were significantly reduced (W(d) = 14.8 +/- 1.2 nm, T(d) = 5.7 +/- 1.0 nm, and L(d) = 75.4 +/- 17 nm). The surfaces of the untreated PHF contained approximately 1.1 nm strands, the same size as tau monomer ( approximately 1.0 nm). The pronase-digested PHF cores mostly contained approximately 1.6 +/- 0.3 nm strands although strand diameters ranged from 0.6-2.5 nm. The strands sometimes appear to be wrapped around the filament axis; less often, they appear to be roughly parallel to the PHF axis, and otherwise appear to be randomly oriented. Images of pronase-digested PHF core images are discussed in relation to the core's biochemical composition, its proposed beta structure, and structural subunit models. Images of the untreated and the pronase-digested PHF support a helical ribbon morphology.     

Scanning tunnelling microscopic images of amino acids

We present images of amino acids adsorbed on highly orientated pyrolytic graphite (HOPG) obtained with the scanning tunnelling microscope (STM) in air. Individual molecules can be observed although the majority of adsorbates appear to form clusters. In the case of leucine, methionine, and tryptophan, two molecules often associate together to form a dimer. Single or dimer glycine molecules were not seen, but a cluster of a number of them was observed. The various adsorbed states may be related to the different interactions between the amino acids and the graphite surface. The mechanism of image formation of the amino acids is probably related to charge transfer mechanisms.     

Orthogonal models of structure functions

Approximation of structure functions (SFs) using orthogonal polynomials in most cases, leads to an incorrect determination of the spectral power density for the obtained orthogonal model. A method for constructing orthogonal models of SFs is proposed based on preliminary shifting of the analyzed SF and using orthogonal functions as the bases. In this method, the expansion coefficients are determined using a numerical-analytical approach, which allows one to determine orthogonal models of SFs with a small approximation error and calculate spectral power density with greater accuracy. The implementation of the algorithms for constructing orthogonal models of SFs in the Laguerre and Legendre bases is considered.     

A real space investigation of the dimer defect structure of Si(001)-(2times8)

The atomic arrangement of the Si(001)-(2 times 8) surface has been directly imaged in real space by scanning tunnelling microscopy. The superstructure is impurity induced and stablilized by as little as 1% Ni. Clean Si(001) surfaces do not form the (2times8) phase. The two-dimensional Fourier transforms of the STM topographs correspond well to the (2times8) LEED pattern, but the real space images are in fact considerably more complex than previously concluded on the basis of recent diffraction technique measurements. The STM images clearly demonstrate that the (2times8) surface consists of a complex missing dimer structure, namely one and multiple dimer vacancies. Such a dimer defect appears related to the Ni impurity and intimately related to the ordering of the dimer defects probably by third layer dimerization to produce quasi-ordered ‘channels’.     

Method for forming two-dimensional paracrystals of biological filaments on lipid monolayers

A method is described for electron microscopic observation of two-dimensional paracrystals on unsupported lipid monolayers. The method uses a hydrophobic holey C-coated grid placed on a monolayer made positively charged by the inclusion of stearylamine (SA) and has been used to align scallop thin filaments and reconstituted actin/tropomyosin filaments to form paracrystals. The use of unsupported monolayers allows the paracrystals to be viewed in either negative stain or with cryoelectron microscopy. Those paracrystals in frozen hydrated specimens have better order than those with negative stain. It was found that varying the lipid composition between the less fluid distearolyphosphotidylcholine/SA and the more fluid egg yolk phosphotidylcholine/SA alters the size and order of the paracrystals, the more fluid system having smaller, more ordered paracrystalline domains. The advantage of the technique for studying actin/thin filaments is the ability to form large two-dimensional paracrystals under physiological conditions of [Mg²⁺] and pH.     

STM studies of adsorbed ZDDP on graphite

We have studied the adsorption of zinc O,O-diisopropylphosphoro-dithioate (ZDDP) adsorption on graphite at room temperature by STM. Two different structures corresponding respectively to monomer and dimer were observed on this surface although only the dimer was found to exist in the crystalline state. Molecularly resolved images showed that the molecular packing is significantly different from that in the bulk crystalline state. The monomers stand on the substrate to form a regular structure with a unit cell of 4.6 × 6.5 Ų ( = 110°). The dimers, imaged as elongated protrusions, lie on the substrate to form a regular structure with a unit cell of 6.5 × 9.0 Ų ( = 125°). The coexistence of both monomer and dimer on graphite reflects competition between the molecule-molecule and molecule-substrate interaction for which the former favors the dimeric form and the latter favors the monomeric form.     

铸态Cu-5.7%Cr合金在等径角挤压中铬纤维的原位形成

利用等径角挤压(ECAP)技术对铸态Cu-5.7%Cr合金进行了变形,对变形过程中原位铬纤维的形成进行了研究;根据纤维的形成过程,分析了Segal和Mcnelly剪切变形模型的适用性.结果表明:在挤压变形过程中,树枝状的铬相发生了旋转和延伸,从而形成细长的原位纤维,随着变形量的增大,纤维方向趋于与x轴平行;考虑摩擦的影响,提出了用修正的Mcnelly剪切变形模型来计算形变原位纤维的倾斜角,计算值与试验结果吻合较好.     

水-乙二醇液压液极压抗磨添加剂二聚酸钾的研究

本对合成的一种应用于水-乙二醇液压液的极压抗磨添加剂二聚酸钾进行了润滑性能测试和抗磨作用机理研究．四球实验表明,二聚酸钾的水溶性能和抗磨性能优于水溶性改性ZDDP和Lubrizol5642,以二聚酸钾作为极压抗磨添加剂的水-乙二醇液压液KGY的润滑性能与N46抗磨液压油相当。钢球磨斑表面元素EDS分析表明,在极压条件下,二聚酸钾与摩擦表面发生某种摩擦化学反应,形成了一层有机类抗磨膜。     

A technique for exposure of the glycoproteic matrix (zona pellucida and mucus) for scanning electron microscopy.

The fine structure of the zona pellucida (ZP) covering the oocyte and of the mucus covering the surface of the intestinal villi was investigated by using a new method employing ruthenium red (RR), saponin, and osmium-thiocarbohydrazide impregnation. The glycoproteic matrices both appeared constituted by thin filaments (ranging from 22 to 50 nm in thickness) anastomosed to form a very fine network. RR prevented the dissolution and/or alteration of glycoproteins and polyanionic carbohydrates induced by acqueous fixatives. Saponin was a detergent of the soluble proteins. Osmium-thiocarbohydrazide preserved the glycoproteic matrix filaments from the mechanical stress induced by dehydration and critical point drying and reduced filaments packing and shrinkage. The technical improvement was demonstrated by the following results: 1) a regular arrangement of the filaments network; 2) a thickness of mucus filaments smaller than that obtained with other methods of preparation; 3) a homogeneous thickness of ZP filaments. This method allowed a very detailed study of the fine structural organization of the ZP and intestinal mucus. Therefore, this technique can be useful for a better evaluation of the morphodynamic of these and other glycoproteic matrices.     

Actin cytoskeleton in plants: from transport networks to signaling networks.

The plant actin cytoskeleton is characterized by a high diversity in regard to gene families, isoforms, and degree of polymerization. In addition to the most abundant F-actin assemblies like filaments and their bundles, G-actin obviously assembles in the form of actin oligomers composed of a few actin molecules which can be extensively cross-linked into complex dynamic meshworks. The role of the actomyosin complex as a force generating system - based on principles operating as in muscle cells - is clearly established for long-range mass transport in large algal cells and specialized cell types of higher plants. Extended F-actin networks, mainly composed of F-actin bundles, are the structural basis for this cytoplasmic streaming of high velocities On the other hand, evidence is accumulating that delicate meshworks built of short F-actin oligomers are critical for events occurring at the plasma membrane, e.g., actin interventions into activities of ion channels and hormone carriers, signaling pathways based on phospholipids, and exo- and endocytotic processes. These unique F-actin arrays, constructed by polymerization-depolymerization processes propelled via synergistic actions of actin-binding proteins such as profilin and actin depolymerizing factor (ADF)/cofilin are supposed to be engaged in diverse aspects of plant morphogenesis. Finally, rapid rearrangements of F-actin meshworks interconnecting endocellular membranes turn out to be especially important for perception-signaling purposes of plant cells, e.g., in association with guard cell movements, mechano- and gravity-sensing, plant host-pathogen interactions, and wound-healing.     

Evaluating triggers and enhancers of tau fibrillization

Alzheimer's disease is characterized in part by the aggregation of tau protein into filamentous inclusions. Because tau filaments form in brain regions associated with memory retention, and because their appearance correlates well with the degree of dementia, they have emerged as robust markers of disease progression. Yet the discovery that mutations in tau protein can lead directly to filament and tangle formation in humans, and that filament formation is linked to neurodegeneration in model biological systems, suggests that tau aggregation may also contribute directly to degeneration in affected neurons. In this context, the mechanism of tau filament formation and its modulation by mutation and posttranslational modification is of fundamental importance. Here, recent progress on the molecular mechanisms underlying tau aggregation deduced from in vivo and in vitro experimentation is reviewed and a model rationalizing the effect of posttranslational and other structural modifications on assembly kinetics and thermodynamics is presented. We hypothesize that tau aggregation can be described as a heterogeneous nucleation reaction, where exogenous effectors, tau gene mutations, or other modifications that stabilize assembly-competent conformations of tau act to trigger the fibrillization reaction. In contrast, those that modulate postnuclear equilibria can enhance fibrillization by increasing the free energy difference between polymers and unincorporated monomers, resulting in stabilization of filaments at low bulk protein concentrations.