Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments

The mammalian neurofilament triplet proteins (210, 160 and 68 × 10³M_r proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 10³M_r protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 10³M_r protein to 68 × 10₃M_r protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 10₃M_r protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments.     

Characteristics of Hormogonia Formation by Symbiotic Nostoc spp. in Response to the Presence of Anthoceros punctatus or Its Extracellular Products

Nostocacean cyanobacteria typically produce gliding filaments termed hormogonia at a low frequency as part of their life cycle. We report here that all Nostoc spp. competent in establishing a symbiotic association with the hornwort Anthoceros punctatus formed hormogonial filaments at a high frequency in the presence of A. punctatus. The hormogonia-inducing activity was produced by A. punctatus under nitrogen-limited culture conditions. The hormogonia of the symbiotically competent Nostoc spp. were characterized as motile (gliding) filaments lacking heterocysts and with distinctly smaller cells than those of vegetative filaments; the small cells resulted from a continuation of cell division uncoupled from biomass increase. An essentially complete conversion of vegetative filaments to hormogonia occurred within 12 h of exposure of Nostoc sp. strain 7801 to A. punctatus growth-conditioned medium. Hormogonia formation was accompanied by loss of nitrogen fixation (acetylene reduction) and by decreases in photosynthetic CO₂ fixation and in vivo NH₄⁺ assimilation of 30% and approximately 40%, respectively. The rates of acetylene reduction and CO₂ fixation returned to approximately the control rates within 72 to 96 h after hormogonia induction, as the cultures of Nostoc sp. strain 7801 differentiated heterocysts and reverted to the vegetative growth state. The relationship between hormogonia formation and symbiotic competence is discussed.     

Formation of a flagella-like but straight polymer of Salmonella flagellin

Salmonella flagellin (monomer) polymerizes into flagellar filaments with the addition of (NH₄)₂SO₄ (Ada et al., 1963; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high concentration of NaCl (about 1.5 m), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flagellins derived from strains SJ670, SJ25, SJ30 and SJ814. When the straight filament, suspended in 0.15 m-NaCl, was heated, it depolymerized to the monomer, which could in turn be polymerized into flagellar filaments by the addition of short fragments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsuccessful. In 0.15 m-NaCl, straight filaments prepared from the four kinds of flagellins had markedly different heat stabilities, which were much lower than that of any kind of flagella. When monomeric flagellin dissolved in 3.5 m-NaCl was seeded with short fragments of straight filaments, the monomer polymerized onto the ends of the short fragments, which consequently grew into long straight filaments. In this type of experiment, monomers and seeds derived from the four strains were able to interact in any combination, suggesting that straight filaments consisting of the four kinds of flagellins have the same substructures. Whether the concentration of added NaCl was 0.15 m or 3.5 m, fragments of flagella (or straight filaments) were unable to act as seeds for the formation of straight filaments (or flagellar filaments). From this and other experimental results, it was concluded that in the two filamentous structures, flagellin molecules may be packed in different ways.     

Treadmilling of actin at physiological salt concentrations: An analysis of the critical concentrations of actin filaments

Treadmilling of actin was investigated at physiological salt concentrations (100 mm-KCl, 0.5 to 2.0 mm-MgCl₂, 200 μm-ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid or 50 μm-CaCl₂ at 37 °C. The concentration at which monomers bind to the lengthening end of filaments with the same rate as subunits are released (low critical concentration c₁ was determined by mixing unmodified actin filaments with various concentrations of monomeric actin labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Above a monomeric actin concentration of about 0.12 μm, incorporation of actin molecules into filaments was detected, whereas below this concentration no incorporation was found (c₁ = 0.12 μm). Combination of various concentrations of labeled monomers with labeled filaments permitted determination of the net critical concentration ($\text{c}{}_1$) at which filaments lengthen at one end with the same rate as they shorten at the other end ($\text{c}{}_1\text{= 0.16}\text{μm}$). A lower limit of the high critical concentration at the shortening end (c_h) was estimated by measuring the release of subunits from labeled filaments in the presence of various concentrations of unlabeled monomers (c_h >0.5 μm). The differences in the three critical concentrations demonstrate that under physiological conditions actin filaments lengthen at one end by, on the average, one subunit during the time that four association reactions take place at the two ends (efficiency parameters $\text{s =}\text{1}\text{4}$). The small difference between the low and the net critical concentration suggests that the rates of both association and dissociation are considerably greater at the lengthening end than at the shortening end of actin filaments.     

Electric birefringence of bacterial flagellar protein filaments: evidence for field-induced interactions.

The time course for the build-up and decay of birefringence induced by a rectangular voltage pulse was measured on solutions of flagellar filaments from Salmonella equi-abortus (strain SJ25). These filaments are tubular polymers of protein (degree of polymerization ≈ 10³) constituted by non-covalent linkage of flagellin monomers of molecular weight 4 × 10⁴. The effect on electro-optical properties of solutions of filaments due to variations in temperature, concentration and mean length of protein filaments, and the duration and intensity of the applied electric field is described. Analysis of the field intensity dependence of the birefringence and comparison of the build-up and decay processes indicate that orientation in the field is due primarily to the existence of a permanent dipole moment in the filaments. At 18 °C the following values were obtained for a solution of filaments with mean length and standard deviation of 0.39 and 0.30 μm: specific Kerr constant (K_sp) = 6.14 × 10⁻³ electrostatic units; optical anisotropy factor (g₁ — g₂) = 5.66 × 10⁻³; dipole moment (μ) = 1.01 × 10⁵ Debye units; and mean relaxation time ($\text{}\text{̄}$gt) = 9.20 ms. At temperatures below 20 °C there is a marked increase in the optical anisotropy factor of the filaments which may be due to a change in their flexibility. The large values of K_sp obtained indicate the highly responsive nature of these filaments to an electric field. The birefringence decay curves were decomposed by computer into a specified number of exponential terms from which both the mean length and the size distribution of these polydisperse filaments were calculated. The results obtained were in substantial agreement with the values of these parameters observed by electron microscopy. A cumulative field effect dependent on field intensity and filament concentration was observed. Repeated pulsing of electric field, above threshold values of field intensity and filament concentration, produced decreases in the birefringence near 60% of its initial value. The effect was reversible with a time constant greater than two minutes. No appreciable change in the relaxation time for decay of birefringence was observed on multiple pulsing of these solutions. These results are interpreted consistently to arise from the sidewise aggregation of filaments induced by electrical impulses of sufficient intensity and duration. These properties appear relevant to bacterial motility: variations in electric potential along the membrane of the bacterium might serve first to orient these organelles and then to induce their coalescence to “bundles” of filaments. The latter structures are commonly observed in vivo. In this way the activity of flagella might be co-ordinated.     

The location and periodicity of a troponin-T-like protein in the myofibril of the horseshoe crab Limulus polyphemus

Localization studies show that a 59,000 M_r, protein from Limulus muscle, thought to be a troponin-T-like protein, is associated with thin filaments. Immunoelectron microscopy indicates that its periodicity on thin filaments is 380 to 390 Å, a distribution characteristic of proteins comprising the troponin complex.     

Nucleotide dependent differences between the alpha-skeletal and alpha-cardiac actin isoforms

The thermodynamic properties of the actin filaments prepared from cardiomyocytes were investigated with differential scanning calorimetry. This method could distinguish between the α-cardiac and α-skeletal components of the actin filaments polymerised from ADP-actin monomers by their different melting temperatures (T_m). Similar separation was not possible with filaments polymerised from ATP-actin monomers. Further analyses revealed that the activation energy (E_act) was greater for filaments of α-skeletal actin than for α-cardiac actin monomers when the filaments were polymerised from ADP-actin monomers. These results showed that the α-cardiac actin filaments were thermodynamically less stable than the filaments of α-skeletal actin and their difference was nucleotide dependent. Based on these results and considering previous observations it was concluded that the existence of two actin isoforms and their nucleotide dependent conformational differences are part of the tuning regulatory mechanism by which the cardiac muscle cells can maintain their biological function under pathological conditions.     

Localization of magnetized electrons in current filaments as a fundamental cause of Coulomb explosion

Mechanisms for generating current filaments in a dense plasma under the action of focused laser pulses and in a Z-pinch configuration are discussed. The main properties of current filaments with a zero and nonzero electron vorticity Ω _e =B?(c/e)?×p _e that originate at magnetic fields in the range 4πn _e m _e c²?B²?4πn _i m _i c² are investigated under the conditions of Coulomb explosion at currents below the ion Alfvén current. A study is made of the equilibrium configurations of nonquasineutral current filaments in a purely longitudinal (B_z) and a purely azimuthal (B_θ) magnetic field and also in a more general case of a helical magnetic field, having two components, under conditions such that the charge separation occurs on a spatial scale on the order of the magnetic Debye radius r_B ? |B|/(4πen_e. It is shown that strong electric fields generated in the current filaments are comparable in magnitude to the atomic field and are capable of accelerating ions to energies of several tens of megaelectronvolts. The ion dynamics in strong electric fields of the filaments is calculated numerically and is shown to lead to the formation of collisionless shock waves on time scales on the order of several inverse ion plasma frequencies ω _pi ^?1 . The possible formation of current filaments on different spatiotemporal scales is considered.     

Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350 nm vs. 1750 nm) transport by myoVa motors. Five thousand simulated liposomes transported within each network adopted one of three states: transport, tug-of-war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug-of-wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, N_c. N_c is a geometric property of the actin network that depends only on the position and polarity of the actin filaments, transport distance, and the liposome diameter, as evidenced by a fivefold increase in liposome diameter resulting in a fivefold decrease in N_c. Thus in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell’s physiological demands.     

Nonquasineutral relativistic current filaments and their X-ray emission

Nonquasineutral electron current filaments with the azimuthal magnetic field are considered that arise due to the generation of electron vorticity in the initial (dissipative) stage of evolution of a current-carrying plasma, when the Hall number is small (σB/en _e c ? 1) because of the low values of the plasma conductivity and magnetic field strength. Equilibrium filamentary structures with both zero and nonzero net currents are considered. Structures with a zero net current type form on time scales of t < t _sk = (r ₀ω _pe /c)² t _st, where t _sk is the skin time, t _st is the typical time of electron-ion collisions, and r ₀ is the radius of the filament. It is shown that, in nonquasineutral filaments in which the current is carried by electrons drifting in the crossed electric (E _r ) and magnetic (B _θ) fields, ultrarelativistic electron beams on the typical charge-separation scale r _B = B/(4πen _e ) (the so-called magnetic Debye radius) can be generated. It is found that, for comparable electron currents, the characteristic electron energy in filaments with a nonzero net current is significantly lower than that in zero-net-current filaments that form on typical time scales of t < t _sk. This is because, in the latter type of filaments, the oppositely directed electron currents repel one another; as a result, both the density and velocity of electrons increase near the filament axis, where the velocities of relativistic electrons are maximum. Filaments with a zero net current can emit X rays with photon energies ? ω up to 10 MeV. The electron velocity distributions in filaments, the X-ray emission spectra, and the total X-ray yield per unit filament length are calculated as functions of the current and the electron number density in the filament. Analytical estimates of the characteristic lifetime of a radiating filament and the typical size of the radiating region as functions of the plasma density are obtained. The results of calculations are compared with the available experimental data.     

Effect of temperature on the in vitro assembly of bacterial flagella

The temperature dependence for the rate of reconstitution or polymerization (k₊) at neutral pH of the protein, flagellin, to flagella was measured using Ostwald-type viscometers. Similarly, the kinetics for the reverse process, the thermally-induced depolymerization of flagella filaments to the flagellin monomer (k_?) was measured. The temperature at which k_? equals zero was used to define the thermal dissociation temperature or melting point of flagella filaments. The remarkable similarity of melting points obtained (36.8 ± 0.2 deg. C) for flagella isolated from three Salmonella strains (SJ670, SJ25 and SJ30 bearing H-antigen types i, 1.2 and e, n, x, respectively) suggests that the structural stability of these different protein filaments is also similar.On increasing the temperature between 12 and 28°C, k₊ increased smoothly and had a Q₁₀ of 1.8. Above 28.0, k₊ decreased rapidly and fell to zero at a temperature near 37°C, its precise value varying with the bacterial strain. This result supports the prior hypothesis (Gerber &; Noguchi, 1967) that on heating, a reversible co-operative transconformation occurs between different states of the protein; in one state, flagellin (M) can polymerize to flagella, whereas its conformational isomer(s) may do so with difficulty or not at all.For strains SJ25 and SJ30 the rates of polymerization and depolymerization both fall to zero near 37°C. Therefore, mixtures of monomer and flagella fragments (short polymers or “seeds”), in all ratios, appear to be in equilibrium at temperatures near this critical temperature, and neither polymerization of flagellin to flagella nor melting of polymers is apparent.Measurements made on flagella from strain SJ670 showed that k₊ and k_? approached zero at 45 and 37°C, respectively. Within this temperature range the conc entration of monomer in equilibrium with filaments was determined. By a null -point type experiment, solutions of monomer and seed were mixed to find the ratio that showed neither increases (polymerization) nor decreases (depolyme rization) in viscosity with time. An unexpected finding was that the temperature defines a critical monomer concentration, which exists in equilibrium with any concentration of filaments (and not the ratio of monomer-to-filament concentrations). Thus, the polymerization of fiagellin to flagella corresponds to a phase change akin to either crystallization or condensation.Application, of the Clapeyron-Clausius equation to the results obtained yields a heat of condensation of 70 kcal/mol of monomeric protein. The enthalpy change associated with M ? M_i is estimated as 110 kcal/mol of protein. Since the heat content of these various forms of flagella protein lies in the order M_i > F > M, by difference we estimate the enthalpy change for the conversion of monomers to polymers to be 40 kcal/mol of monomer.     

THE CONTRACTILE PROCESS IN THE CILIATE, STENTOR COERULEUS : I. The Role of Microtubules and Filaments

The structural basis for the function of microtubules and filaments in cell body contractility in the ciliate Stentor coeruleus was investigated. Cells in the extended state were obtained for ultrastructural analysis by treatment before fixation with a solution containing 10 mM EGTA, 50–80 mM Tris, 3 mM MgSO₄, 7.5 mM NH₄Cl, 10 mM phosphate buffer (pH 7.1). The response of Stentor to changes in the divalent cation concentrations in this solution suggests that Ca⁺² and Mg⁺² are physiologically important in the regulation of ciliate contractility. The generation of motive force for changes in cell length in Stentor resides in two distinct longitudinal cortical fiber systems, the km fibers and myonemes. Cyclic changes in cell length are associated with (a) the relative sliding of parallel, overlapping microtubule ribbons in the km fibers, and (b) a distinct alteration in the structure of the contractile filaments constituting the myonemes. The microtubule and filament systems are distinguished functionally as antagonistic contractile elements. The development of motive force for cell extension is accomplished by active microtubule-to-microtubule sliding generated by specific intertubule bridges. Evidence is presented which suggests that active shortening of contractile filaments, reflected in a reversible structural transformation of dense 4-nm filaments to tubular 10–12-nm filaments, provides the basis for rapid cell contraction.     

Experimental verification of the effect of different preionization systems on the implosion dynamics of an argon double gas puff

Experiments are reported on the implosion of argon double gas puffs in the GIT-12 current generator (T _fr=0.25 μs, I _m =2.3 MA). The gas-puff medium was preionized by different methods. The experimental data provide evidence for a strong effect of the initial conditions for the formation of the current-carrying shell on the implosion process. Emphasis is given to a discussion of the following issues: the enhanced scatter in both the emission power and X-ray yield in the Ar K-lines, the existence of a large number of current filaments, the uncertainty in the process by which the generator current is redistributed among a progressively smaller number of current filaments, and the redistribution of the generator current between the inner and outer gas-puff shells. __________ Translated from Fizika Plazmy, Vol. 27, No. 7, 2001, pp. 584–591. Original Russian Text Copyright ? 2001 by Rousskikh, Baksht, Labetsky, Shishlov, Fedyunin.     

AUTORADIOGRAPHIC LOCALIZATION OF 13N AFTER FIXATION OF 13N-LABELED NITROGEN GAS BY A HETEROCYST-FORMING BLUE-GREEN ALGA

¹³N, generated by proton bombardment of ¹³C powder, is rapidly and easily converted to ¹³N-N₂, 0.01 atm pressure, ca. 10 mCi/ml, by automated Dumas combustion. ¹³N fixed (as ¹³N-N₂) by algal filaments was localized by an autoradiographic technique which permits track autoradiography with isotopes having short half-lives. Our findings show directly that a minimum of about 25% of the N₂ fixation by intact, aerobically grown filaments of Anabaena cylindrica is carried out by the heterocysts. If all of the N₂ fixation takes place in the heterocysts, then the movement of nitrogen along the filaments can be characterized by a constant τ < ca. 5 s (cell^-2).     

Attractive Interactions among Intermediate Filaments Determine Network Mechanics In Vitro

Mechanical and structural properties of K8/K18 and vimentin intermediate filament (IF) networks have been investigated using bulk mechanical rheometry and optical microrheology including diffusing wave spectroscopy and multiple particle tracking. A high elastic modulus G ₀ at low protein concentration c, a weak concentration dependency of G ₀ (G ₀∼c ^0.5±0.1) and pronounced strain stiffening are found for these systems even without external crossbridgers. Strong attractive interactions among filaments are required to maintain these characteristic mechanical features, which have also been reported for various other IF networks. Filament assembly, the persistence length of the filaments and the network mesh size remain essentially unaffected when a nonionic surfactant is added, but strain stiffening is completely suppressed, G ₀ drops by orders of magnitude and exhibits a scaling G ₀∼c ^1.9±0.2 in agreement with microrheological measurements and as expected for entangled networks of semi-flexible polymers. Tailless K8Δ/K18ΔT and various other tailless filament networks do not exhibit strain stiffening, but still show high G ₀ values. Therefore, two binding sites are proposed to exist in IF networks. A weaker one mediated by hydrophobic amino acid clusters in the central rod prevents stretched filaments between adjacent cross-links from thermal equilibration and thus provides the high G ₀ values. Another strong one facilitating strain stiffening is located in the tail domain with its high fraction of hydrophobic amino acid sequences. Strain stiffening is less pronounced for vimentin than for K8/K18 due to electrostatic repulsion forces partly compensating the strong attraction at filament contact points.     

Scanning electron microscopy of the gills of a freshwater catfish,Rita Rita

Variations in the gross morphology and surface architecture of the gill filaments and secondary lamellae of a freshwater catfish (Rita rita) have been investigated using scanning electron microscopy. Heterogeneity of the gill has been correlated with the distribution of lamellar water-flow at different regions of a gill filament. Higher lamellar water flow (cc/pore/cmH₂O/sec) was estimated for the middle region of the filaments. The filaments are covered with epithelial cells whose surface is provided with well-developed microridges. The lamellae are generally covered with microvillous epithelial cells. The variations in surface architecture of the gill filaments and secondary lamellae have been correlated with their probable functions.     

Light and dark reactions of the uptake hydrogenase in anabaena 7120

Reactions of the uptake hydrogenase from Anabaena 7120 (A.T.C.C. 27893, Nostoc muscorum) were examined in whole filaments, isolated heterocysts, and membrane particles. Whole filaments or isolated heterocysts that contained nitrogenase consumed H₂ in the presence of C₂H₂ or N₂ in a light-dependent reaction. If nitrogenase was inactivated by O₂ shock, filaments catalyzed H₂ uptake to an unidentified endogenous acceptor in the light. Addition of NO₃⁻ or NO₂⁻ enhanced these rates. Isolated heterocysts consumed H₂ in the dark in the presence of electron acceptors with positive midpoint potentials, and these reactions were not enhanced by light. With acceptors of negative midpoint potential, significant light enhancement of H₂ uptake occurred. Maximum rates of light-dependent uptake were approximately 25% of the maximum dark rates observed. Membrane particles prepared from isolated heterocysts showed similar specificity for electron acceptors. These particles catalyzed a cyanide-sensitive oxyhydrogen reaction that was inactivated by O₂ at O₂ concentrations above 2%. Light-dependent H₂ uptake to low potential acceptors by these particles was inhibited by dibromothymoquinone but was insensitive to cyanide. In the presence of O₂, light-dependent H₂ uptake occurred simultaneously with the oxyhydrogen reaction. The pH optima for both types of H₂ uptake were near 7.0. These results further clarify the role of uptake hydrogenase in donating electrons to both the photosynthetic and respiratory electron transport chains of Anabaena.     

Light microscope study of mixed helices in reconstituted Salmonella flagella

In this morphological study of bacterial flagella, single flagellar filaments in solutions were photographed by dark-field light microscopy to determine parameters to describe their intact shapes. First, I measured pitches of helices I, II and III assumed by copolymers of flagellins from Salmonella strains SJ25 and SJ814 (Asakura & Iino, 1972), and combined the data with electron microscopic data of the contour length per period of each filament to calculate the pitch angles of the three helices. (The pitch angle is the angle between a tangent to the filament and the helical axis.)Secondly, filaments which consisted of two blocks assuming different helices were prepared by two-step copolymerization of SJ25 and SJ814 flagellins and the configurations of these mixed-type filaments were examined. In filaments of any mixed type, the axes of the constituent blocks were oriented at the same angle, called the “block angle” Ψ. This angle was found to be approximated by Ψ = 180 ° ? ¦θ₁ ? θ₂¦, where θ₁ and θ₂ are the pitch angles of the mixed helices. On the basis of this finding, the morphology of mixed-type filaments is discussed.     

Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a_w). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a_w for long periods, but minimum humectant concentrations of 8% NaCl (a_w, 0.95), 96% sucrose (a_w, 0.94), and 32% glycerol (a_w, 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a_w, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At a_w values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a_w conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a_w highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a_w (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a_w storage. If Salmonella strains form filaments in food products that have low a_w values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.