Molecular organization and in vivo function of the cytoskeleton of amphibian erythrocytes

One prominent cytoskeletal feature of non-mammalian vertebrate erythrocytes is the marginal band (MB), composed of microtubules. However, there have been several reports of MB-associated F-actin. We have further investigated the function of MB-associated F-actin, using newt erythrocytes having large, thick MBs. Confocal microscopy revealed a distinctive band of F-actin colocalizing point- by-point with MB microtubules. Furthermore, the F-actin band was present in isolated elliptical MBs, but absent in membrane skeletons lacking MBs. F-actin depolymerizing agents did not affect F-actin band integrity in isolated MBs, indicating its non-dynamic state. However, exposure to elastase resulted in F-actin removal and MB circularization. These results provide evidence of a strong association of F-actin with MB microtubules in mature ellipsoidal erythrocytes. To assess the true extent of mechanical stress on the cytoskeleton, erythrocytes were observed by video microscopy during flow in vivo. Moving with long axis parallel to flow direction, cells underwent reversible shape distortion as they collided vigorously with other erythrocytes and vessel walls. In addition, cells twisted into figure-8 shapes, a cytoskeletal property that may provide physiological advantages during flow. Our results, together with those of others, yield a consistent picture in which developing erythrocytes undergo transition from spheroids to immature discoids to mature ellipsoids. The causal step in discoid formation is biogenesis of circular MBs with sufficient flexural rigidity to determine cell shape. F-actin binding to MB microtubules then creates a composite system, enhancing flexural rigidity to produce and maintain ellipsoidal shape during the physical challenges of blood flow in vivo.     

Experimental Study of the Flexural and Compression Performance of an Innovative Pultruded Glass-Fiber-Reinforced Polymer-Wood Composite Profile

The plate of a pultruded fiber-reinforced polymer or fiber-reinforced plastic (FRP) profile produced via a pultrusion process is likely to undergo local buckling and cracking along the fiber direction under an external load. In this study, we constructed a pultruded glass-fiber-reinforced polymer-light wood composite (PGWC) profile to explore its mechanical performance. A rectangular cross-sectional PGWC profile was fabricated with a paulownia wood core, alkali-free glass fiber filaments, and unsaturated phthalate resin. Three-point bending and short column axial compression tests were conducted. Then, the stress calculation for the PGWC profile in the bending and axial compression tests was performed using the Timoshenko beam theory and the composite component analysis method to derive the flexural and axial compression rigidity of the profile during the elastic stress stage. The flexural capacity for this type of PGWC profile is 3.3-fold the sum of the flexural capacities of the wood core and the glass-fiber-reinforced polymer (GFRP) shell. The equivalent flexural rigidity is 1.5-fold the summed flexural rigidity of the wood core and GFRP shell. The maximum axial compressive bearing capacity for this type of PGWC profile can reach 1.79-fold the sum of those of the wood core and GFRP shell, and its elastic flexural rigidity is 1.2-fold the sum of their rigidities. These results indicate that in PGWC profiles, GFRP and wood materials have a positive combined effect. This study produced a pultruded composite material product with excellent mechanical performance for application in structures that require a large bearing capacity.     

Ultrastructure of the Parabasalid Protist Holomastigotoides

ABSTRACT. The ultrastructure of two species of Holomastigotoides is presented. The basic unit of organization of these large cells is the flagellar band. Each flagellar band consists of a row of flagellar basal bodies linked by three fiber systems. The number of flagellar bands is species dependent. The flagellar bands originate at the cell apex and are arranged in parallel spirals of increasing gyre, thus defining the conical shape of the cell. In the cell apex a striated root called a parabasal fiber is juxtaposed with the basal bodies of each flagellar band. Linear extensions of two parabasal fibers function as the spindle poles for the persistent extra-nuclear spindle. The nucleus is in close contact with the spindle poles and spindle microtubules. Parallel sheets of microtubules which constitute axostyles are nucleated along the underside of the parabasal fibers. The axostyles extend away from the cell apex, with many reaching the basal region of the cell. Some of the axostyles follow the spiral pattern of the flagellar bands. Numerous Golgi bodies are spaced regularly along the flagellar bands. Together the parabasal fiber, axostyles and Golgi bodies associated with a flagellar band are termed a parabasal complex.     

Effect of intrinsic and extrinsic factors on the simulated D‐band length of type I collagen

A signature feature of collagen is its axial periodicity visible in TEM as alternating dark and light bands. In mature, type I collagen, this repeating unit, D, is 67 nm long. This periodicity reflects an underlying packing of constituent triple‐helix polypeptide monomers wherein the dark bands represent gaps between axially adjacent monomers. This organization is visible distinctly in the microfibrillar model of collagen obtained from fiber diffraction. However, to date, no atomistic simulations of this diffraction model under zero‐stress conditions have reported a preservation of this structural feature. Such a demonstration is important as it provides the baseline to infer response functions of physiological stimuli. In contrast, simulations predict a considerable shrinkage of the D‐band (11–19%). Here we evaluate systemically the effect of several factors on D‐band shrinkage. Using force fields employed in previous studies we find that irrespective of the temperature/pressure coupling algorithms, assumed salt concentration or hydration level, and whether or not the monomers are cross‐linked, the D‐band shrinks considerably. This shrinkage is associated with the bending and widening of individual monomers, but employing a force field whose backbone dihedral energy landscape matches more closely with our computed CCSD(T) values produces a small D‐band shrinkage of < 3%. Since this force field also performs better against other experimental data, it appears that the large shrinkage observed in earlier simulations is a force‐field artifact. The residual shrinkage could be due to the absence of certain atomic‐level details, such as glycosylation sites, for which we do not yet have suitable data. Proteins 2015; 83:1800–1812. © 2015 Wiley Periodicals, Inc.     

Rigidity of microtubules is increased by stabilizing agents

Microtubules are rigid polymers that contribute to the static mechanical properties of cells. Because microtubules are dynamic structures whose polymerization is regulated during changes in cell shape, we have asked whether the mechanical properties of microtubules might also be modulated. We measured the flexural rigidity, or bending stiffness, of individual microtubules under a number of different conditions that affect the stability of microtubules against depolymerization. The flexural rigidity of microtubules polymerized with the slowly hydrolyzable nucleotide analogue guanylyl-(alpha, beta)- methylene-diphosphonate was 62 +/- 9 x 10(-24) Nm2 (weighted mean +/- SEM); that of microtubules stabilized with tau protein was 34 +/- 3 x 10(-24) Nm2; and that of microtubules stabilized with the antimitotic drug taxol was 32 +/- 2 x 10(-24) Nm2. For comparison, microtubules that were capped to prevent depolymerization, but were not otherwise stabilized, had a flexural rigidity of 26 +/- 2 x 10(-24) Nm2. Decreasing the temperature from 37 degrees C to approximately 25 degrees C, a condition that makes microtubules less stable, decreased the stiffness of taxol-stabilized microtubules by one-third. We thus find that the more stable a microtubule, the higher its flexural rigidity. This raises the possibility that microtubule rigidity may be regulated in vivo. In addition, the high rigidity of an unstabilized, GDP-containing microtubule suggests that a large amount of energy could be stored as mechanical strain energy in the protein lattice for subsequent force generation during microtubule depolymerization.     

Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging

The addition of malondialdehyde to erythrocytes in vitro causes a decrease in bands 1 and 2 of spectrin and an increase in high molecular weight protein polymers. Additionally, this agent causes the formation of fluorscent chromolipids characteristic of those produced during the peroxidation of endogenous membrane phospholipids. These same alterations in proteins and lipids are observed in the membranes of older cells fractionated from freshly drawn blood and in the membranes of reticulocytes induced by treatment of animals with phenylhydrazine, but not in reticulocytes induced by bleeding. The former reticulocytes have a much shorter half-life in the circulation than do either normal erythrocytes or reticulocytes produced consequent to bleeding. These experiments and the apparent paradox of "young" reticulocytes with short half-lives suggest that the in vivo polymerization of membrane proteins consequent to radical-induced peroxidation of membrane lipids may contribute to the altered rheological behavior and hence to the splenic sequestration of cells. They also suggest that increases in intrinsic membrane rigidity due to lipid peroxidation, malondialdehyde, and protein polymerization may be a common feature of both aging in normal erythrocytes and in the accelerated aging that accompanies the administration of radical-generating, hemolytic agents. However, it is cautioned that other polymerization reactions involving disulfides, calcium, or direct radical attack on protein monomers may also be important determinants of the visco-elastic properties of erythrocyte membranes.     

The rigidity of bacterial flagellar filaments and its relation to filament polymorphism.

We determined and correlated the rigidity of Salmonella typhimurium, Escherichia coli, and Rhizobium lupini flagellar filaments representing various structural and polymorphic states (plain, complex, straight, superhelical, and right- and left-handed). Persistence length, from which the filament's rigidity and other parameters (Young's modulus, bending force constant, buckling persistence length, flexural deformation, and flexural time) were derived, was determined from electron micrographs of isolated, negatively stained filaments. Outer diameters and radii of strong intersubunit connectivity were determined from three-dimensional image reconstructions and radial mass density profiles from scanning transmission electron microscopy. All filaments appear to be highly rigid with no evident correlation with their helical sense or superhelicity. The complex filament of R. lupini is rigid to the extent that it becomes brittle. The overall flexibility of the flagellum seems to stem mainly from the hook and not from the filament. Polymorphism is probably related to the propelling properties and hydrodynamic shape of the filament rather than to its rigidity.     

Manipulation of individual viruses: friction and mechanical properties.

We present our results on the manipulation of individual viruses using an advanced interface for atomic force microscopes (AFMs). We show that the viruses can be dissected, rotated, and translated with great facility. We interpret the behavior of tobacco mosaic virus with a mechanical model that makes explicit the competition between sample-substrate lateral friction and the flexural rigidity of the manipulated object. The manipulation behavior of tobacco mosaic virus on graphite is shown to be consistent with values of lateral friction observed on similar interfaces and the flexural rigidity expected for macromolecular assemblies. The ability to manipulate individual samples broadens the scope of possible studies by providing a means for positioning samples at specific binding sites or predefined measuring devices. The mechanical model provides a framework for interpreting quantitative measurements of virus binding and mechanical properties and for understanding the constraints on the successful, nondestructive AFM manipulation of delicate samples.     

The cytoskeleton of isolated murine primitive erythrocytes

Summary Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among the cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitve erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.     

Interaction of the aldolase and the membrane of human erythrocytes.

Up to 80% of cellular aldolase (EC 4.1.2.13) was retained in the membrane fraction isolated following hemolysis of human erythrocytes under appropriate conditions. Binding was reversed by increasing the pH and ionic strength. Millimolar levels of the substrate, fructose 1,6-bisphosphate, selectively eluted aldolase from the membrane, while related metabolites did not. Using the membrane as a high affinity adsorbant, electrophoretically pure aldolase of high specific activity was prepared in high yield. The reassociation of pure aldolase and membranes was characterized. The sole site of human erythrocyte aldolase binding was shown to be the cytoplasmic surface domain of band 3, the predominant membrane-spanning polypeptide. One aldolase molecule was bound per band 3 polypeptide. Upon binding to either whole membranes, solubilized band 3, or proteolytic fragments from the cytoplasmic surface pole of band 3, aldolase underwent a profound loss of catalytic activity, reversed by raising the substrate concentration.     

A Comprehensive Study of the Polypropylene Fiber Reinforced Fly Ash Based Geopolymer

As a cementitious material, geopolymers show a high quasi-brittle behavior and a relatively low fracture energy. To overcome such a weakness, incorporation of fibers to a brittle matrix is a well-known technique to enhance the flexural properties. This study comprehensively evaluates the short and long term impacts of different volume percentages of polypropylene fiber (PPF) reinforcement on fly ash based geopolymer composites. Different characteristics of the composite were compared at fresh state by flow measurement and hardened state by variation of shrinkage over time to assess the response of composites under flexural and compressive load conditions. The fiber-matrix interface, fiber surface and toughening mechanisms were assessed using field emission scan electron microscopy (FESEM) and atomic force microscopy (AFM). The results show that incorporation of PPF up to 3 wt % into the geopolymer paste reduces the shrinkage and enhances the energy absorption of the composites. While, it might reduce the ultimate flexural and compressive strength of the material depending on fiber content.     

Isolation of DNA from single microsurgically excised bands of polytene chromosomes of Chironomus

A method is described for excising by a glass knife single bands of isolated polytene chromosomes of the salivary glands of Chironomus tentans larvae. DNA strands were isolated from cut-out bands and their contour lengths were determined on electron micrographs. The mean contour length of DNA strands isolated from the double band I-8A was about twice that of the single band I-11B, namely 63 versus 34 m. The described method may be applicable for molecular studies on single bands (e.g., by DNA cloning).     

Role of ionic strength on the relationship of biopolymer conformation,DLVO contributions,and steric interactions to bioadhesion of Pseudomonas putida KT2442

Biopolymers produced extracellularly by Pseudomonas putida KT2442 were examined via atomic force microscopy (AFM) and single molecule force spectroscopy. Surface biopolymers were probed in solutions with added salt concentrations ranging from that of pure water to 1 M KCl. By studying the physicochemical properties of the polymers over this range of salt concentrations, we observed a transition in the steric and electrostatic properties and in the conformation of the biopolymers that were each directly related to bioadhesion. In low salt solutions, the electrophoretic mobility of the bacterium was negative, and large theoretical energy barriers to adhesion were predicted from soft-particle DLVO theory calculations. The brush layer in low salt solution was extended due to electrostatic repulsion, and therefore, steric repulsion was also high (polymers extended 440 nm from surface in pure water). The extended polymer brush layer was "soft", characterized by the slope of the compliance region of the AFM approach curves (-0.014 nN/nm). These properties resulted in low adhesion between biopolymers and the silicon nitride AFM tip. As the salt concentration increased to > or =0.01 M, a transition was observed toward a more rigid and compressed polymer brush layer, and the adhesion forces increased. In 1 M KCl, the polymer brush extended 120 nm from the surface and the rigidity of the outer cell surface was greater (slope of the compliance region = -0.114 nN/nm). A compressed and more rigid polymer layer, as well as a less negative electrophoretic mobility for the bacterium, resulted in higher adhesion forces between the biopolymers and the AFM tip. Scaling theories for polyelectrolyte brushes were also used to explain the behavior of the biopolymer brush layer as a function of salt concentration.     

Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes. The changes are quantitatively described using the pixel height histogram and window size grain analysis.     

Fatty acylation of a 55 kDa membrane protein of human erythrocytes.

The major palmitoylated human erythrocyte membrane protein has an M(r) of 55,000. It is distinct from the glucose transporter and is not derived from band 3 or ankyrin. It resists salt extraction suggesting a high affinity for the membrane. Pulse chase experiments demonstrate that palmitoylation is a dynamic process, and it may therefore have regulatory significance in membrane protein-protein or protein-lipid interaction. Slower dynamics of palmitoylation in erythrocytes from patients suffering from chronic myelogenous leukemia, which are less stable than normal erythrocytes, strengthen this view.     

Delivery to Macrophages of Interleukin 3 Loaded in Mouse Erythrocytes

Mouse carrier erythrocytes containing ¹²⁵I-interleukin 3 have been prepared and treated with band 3 crosslinking reagents. The incorporation of interleukin 3 by hypotonic treatment into mouse erythrocytes reached levels of about 15% of the interleukin 3 added to the medium being predominantly present in the cytosolic fraction (73%). Uptake fell to about 7.4% when using the same conditions but omitting hypotonic shock. The interaction of band 3 crosslinked interleukin 3 loaded erythrocytes with macrophages was also studied. A high level of incorporation of interleukin 3 into macrophages was observed either from band 3 crosslinked, interleukin 3-loaded erythrocytes or from interleukin 3 loaded erythrocytes. The observations encourage the view that the system may be able to deliver and target cytokines and other growth factors to macrophages.     

Characterization of beta-connectin (titin 2) from striated muscle by dynamic light scattering.

Connectin (titin) is a large filamentous protein (single peptide) with a molecular mass of approximately 3 MDa, contour length approximately 900 nm, and diameter approximately 4 nm, and resides in striated muscle. Connectin links the thick filaments to the Z-lines in a sarcomere and produces a passive elastic force when muscle fiber is stretched. The aim of this study is to elucidate some aspects of physical properties of isolated beta-connectin (titin 2), a proteolytic fragment of connectin, by means of dynamic light-scattering (DLS) spectroscopy. The analysis of DLS spectra for beta-connectin gave the translational diffusion coefficient of 3.60 x 10(-8) cm2/s at 10 degrees C (or the hydrodynamic radius of 44.1 nm), molecular mass little smaller than 3.0 MDa (for a literature value of sedimentation coefficient), the root-mean-square end-to-end distance of 163 nm (or the radius of gyration of 66.6 nm), and the Kuhn segment number of 30 and segment length of 30 nm (or the persistence length of 15 nm). These results permitted to estimate the flexural rigidity of 6.0 x 10(-20) dyn x cm2 for filament bending, and the elastic constant of 7 dyn/cm for extension of one persistence length. Based on a simple model, implications of the present results in muscle physiology are discussed.     

Studies on the biochemistry and fine structure of silicia shell formation in diatoms VII. Sequential cell wall development in the pennateNavicula pelliculosa

Summary The development of the wall of synchronized culture ofN. pelliculosa is described. The first step, modification of the 3-2 configuration of the girdle bands of the wall during interphase, occurs immediately before mitotic division by the addition of a third girdl band to the hypotheca. Following cytokenesis, the new valve is initiated when a primary central band is formed within a silica deposition vesicle. This band extends the length of the cell and contains a central nodule. Secondary arms extend from the central nodule, join with extensions of the primary central band, and constitute the raphe rib. Mounds or knolls are formed on the central nodule and disappear as the valve matures. Transapical ribs appear on both the primary central band and secondary arms, and cross extensions join to form the sieve plate areas. The wall appears to be released by a joining of the inner silicalemma and the plasmalemma. An organic coat covers the newly released wall. Two girdle bands are formed and released sequentially.