Linear Filaments Research Articles

DNA-Binding Protein Nanotubes: Learning from Nature's Nanotech Examples

Bacterial pili are nanofibers made of protein subunits. Here we report the controlled assembly of protein nanotubes from an engineered Pseudomonas aeruginosa type IV pilin monomer. The nanotubes are up to 100 μm long with an outer diameter of ∼6 nm and a predicted inner diameter of ∼2 nm. Protein nanotube formation appears to proceed through a hydrophobe-initiated conformational shift in the pilin monomer, which then self-associates to form thin linear filaments that coalesce to form long protein nanotubes. Protein nanotubes are highly attractive for nanotechnology, as protein engineering confers unprecedented control over mechanical and chemical properties. Moreover, like type IV pili, our nanotubes bind DNA, further broadening their appeal in nanotechnological applications.

A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization.

Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as alpha-amino acetylation of the N-terminus of the muscle protein. Nalpha-acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.

The Nature of the Dust Disc Surrounding BD+31°643

Multicolour imaging polarimetry of the main sequence star BD+31°643 shows that the Kalas & Jewitt (1997) disc extends to at least 13000AU. A disc of this radius must be dynamically very young compared to the central star, unlike the situation found in the β Pictoris system. A striking linear filament of 850μm emission detected 50″ south, that parallels the dust disk, is probably not physically associated.

Formin' adherens junctions

Epithelial cells zipper together thanks to linear actin cables—cables that assemble at newly formed adherens junctions to stabilize them, thereby counteracting retractive forces at sites of cell–cell contact. Now, Agnieszka Kobielak, Amalia Pasolli, and Elaine Fuchs (Rockefeller University, New York, NY) have found that formin-1 drives the actin polymerization that creates the cables. Figure Formin-1 (green) localization at cell junctions (left) is lost in an α-catenin knockout (right). Formin-1 entered the story as a binding partner of α-catenin, a component of cadherin adhesion complexes. Cells lacking α-catenin fail to form actin cables at adherens junctions, and the Rockefeller group found that the same was true when the formin-1–α-catenin interaction was disrupted in vivo. In vitro, formin-1 was shown to polymerize actin into linear filaments. Finally, fusion of a β-catenin-binding domain to the actin-polymerization domains of formin-1 restored adhesion ability to cells lacking α-catenin. Actin polymerization is important in two steps of adhesion. First, branched polymerization of actin by Arp2/3 pushes out both filopodia and broad areas of membrane as lamellipodia. Many of the resultant contacts are not productive, and the processes retract. But any surviving contact prompts the formation of an actin cable, which stabilizes the contact. It also pushes on a specific area of membrane so that more adherens junctions form nearby, thus zippering cells together. Just how formin-1 is regulated during this process remains to be determined. ▪ Reference: Kobielak, A., et al., 2003. Nat. Cell Bio. 10.1038/ncb1075.

DYNAMICS AND INTERACTION OF FILAMENTS DURING REENTRY AND FIBRILLATION IN MAMMALIAN VIRTUAL VENTRICULAR TISSUE

Despite its importance, the mechanisms underlying ventricular fibrillation (VF) remain poorly understood. Experimental studies are mainly limited to the study of activity on the heart surface, yet the reentrant mechanisms believed to sustain VF are three-dimensional. In 3D, a reentrant wave rotates around a linear filament, and the properties of these filaments can contribute to the stability of reentry. In this paper we describe how filaments can be detected during reentry in cardiac virtual tissues, and how their length and twist can be extracted from numerical results. As an example, we present some results showing the behavior of reentry in 3D virtual tissues with both idealized slab geometry, and anatomically detailed right ventricular (RV) free wall geometry derived from the Auckland canine ventricles. In the slab geometry reentry was stable even with 120° rotational anisotropy, but with a transmural gradient of action potential duration (APD) to mimic the normal distribution of APD across the ventricular wall, reentry became unstable. In contrast, we found that reentry in the anisotropic RV free wall with uniform APD was unstable, possibly because the rotation of fibers is not uniform as in the slab geometry. With transmural differences in APD the instability was augmented, indicating that transmural differences can act synergistically with other mechanisms to increase instability of reentry. This study shows that numerical models can generate information about the behavior of filaments during reentry in 3D, and highlights the need for a theoretical foundation to explain this observed behavior.

GMRT detection of HI 21 cm associated absorption towards thez = 1.2 red quasar 3C 190

We report the GMRT detection of associated HI 21 cm-line absorption in thez = 1.1946 red quasar 3C 190. Most of the absorption is blue-shifted with respect to the systemic redshift. The absorption, at ∼ 647.7 MHz, is broad and complex, spanning a velocity width of ∼ 600 kms−1. Since the core is self-absorbed at this frequency, the absorption is most likely towards the hotspots. Comparison of the radio and deep optical images reveal linear filaments in the optical which overlap with the brighter radio jet towards the south-west. We therefore suggest that most of the HI 21 cm-line absorption could be occurring in the atomic gas shocked by the south-west jet.

The Origin of X-Ray Emission from a Galactic Center Molecular Cloud: Low-Energy Cosmic-Ray Electrons

The Galactic center region near l ≈ 02 hosts a mixture of nonthermal linear filaments and thermal radio continuum features associated with the radio arc. Chandra observations of this region reveal an X-ray filament and diffuse emission with an extent of roughly 60'' × 2'' and 5' × 3', respectively. The X-ray filament lies at the edge of the nonthermal radio filaments and the dense molecular shell G0.13-0.13 that has an unusually high kinetic temperature ≥70 K. These observations demonstrate that the G0.13-0.13 molecular cloud and the nonthermal radio filaments of the arc are interacting. The diffuse X-ray emission is correlated with the molecular shell and is fitted either by two-temperature (1 and 10 keV) thermal emission or by power-law and 1 keV thermal gas. Fluorescent 6.4 keV line emission is also detected throughout the molecular shell. This cloud coincides within the error circle of a steady unidentified EGRET source, 3EG J1746-2851. We argue that low-energy cosmic-ray electrons produce the power-law continuum by bremsstrahlung and 6.4 keV line emission from the filament and the diffuse cloud with the implication on the origin of the Galactic ridge X-ray emission. The strong 6.4 keV Fe line emission seen from other Galactic center clouds could be produced in a similar fashion rather than via fluorescent emission induced by a transient hard X-ray source in the Galactic center. In addition, heating by ionization induced by low-energy cosmic-ray electrons are also responsible for the high temperature of G0.13-0.13. The gamma-ray source is a result of bremsstrahlung by the high-energy tail of the electron energy distribution.

Oligomerization of polyalanine expanded PABPN1 facilitates nuclear protein aggregation that is associated with cell death.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)(6) repeat to (GCG)(8-13) in the PABPN1 gene, which results in the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of the protein. Mutated PABPN1 (mPABPN1) is able to induce nuclear protein aggregation and form filamentous nuclear inclusions, which are the pathological hallmarks of OPMD. PABPN1, when bound to poly(A) RNA, forms both linear filaments and discrete-sized, compact oligomeric particles in vitro. In the absence of poly(A) RNA, PABPN1 can form oligomers. Here we report that: (i) oligomerization of PABPN1 is mediated by two potential oligomerization domains (ODs); (ii) inactivating oligomerization of mPABPN1 by deletions of 6-8 amino acids in either of the ODs prevents nuclear protein aggregation; (iii) expression of mPABPN1 in COS-7 cells is associated with cell death; and (iv) preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. These findings suggest that oligomerization of PABPN1 plays a crucial role in the formation of OPMD nuclear protein aggregation, while the expanded polyalanine stretch is necessary but not sufficient to induce OPMD protein aggregation, and that the nuclear protein aggregation might be toxic and cause cell death. These observations also imply that inactivation of oligomerization of mPABPN1 might be a useful therapeutic strategy for OPMD.

Effect of Hydrostatic Pressure on Aggregation and Viscoelastic Properties of Tilapia (Orechromis niloticus) Myosin

ABSTRACT: Hydrostatic pressures from 500 to 2000 atmospheres (atm) were applied at 0°C to determine the aggregation and viscoelastic properties of tilapia (Orechromis niloticus) myosin. Native myosins were present as long, linear, and single filaments. After a 500‐atm treatment, these filaments unfolded and their volume decreased. Upon 1000‐atm and 1500‐atm treatments, myosins aggregated and formed inseparable network structures. Further, they transformed from viscous sol to elastic gels with a pressure of 500 to 1000 atm. At 2000 atm, the myosin formed irregular aggregates. This study reveals that at 500 atm, myosins unfolded; at 1000 atm, they aggregated, and beyond 1500 atm, they formed both a precipitate and gel.

A Straight and Narrow Ionized Filament

We report the discovery of a extremely narrow, extremely linear, ionized filament. The filament is 2.5 degrees long and has an H$\alpha$ surface brightness of 0.5 rayleighs. The filament is approximately ``Y'' shaped. The widest separation of the two diagonal segments is 5 arcminutes. We discuss four possible origins for this feature: (1) an extremely low density, nearby jet, (2) an unusually linear filament associated with some large-scale nearby nebula, perhaps even the Local Bubble, (3) an ionized trail left by mechanical input from a star or compact object moving through the ISM, or (4) an ionized trail left by photoionization (``Fossil \stromgren Trail'') from a star or compact object. We favor this last hypothesis, and derive some of the basic properties for an ionized trail. Regardless of whether this latter hypothesis applies to this specific filament, the basic properties of such a trail, its length, width, and brightness, are interesting, predictable, and should be observable behind some white dwarfs. We suggest future tests for ascertaining the origin of this filament, and discuss how this structure might be useful to constrain the thermal and velocity structure of the nearby interstellar medium.

Amino acid substitutions of coiled-coil protein Tpr abrogate anchorage to the nuclear pore complex but not parallel, in-register homodimerization.

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil--forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent alpha-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of approximately 7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed.

Topological testing of the mechanism of homology search promoted by RecA protein.

To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA-single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA-ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA-ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA-ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson-Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA-ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.

Jet‐ and Wind‐driven Ionized Outflows in the Superbubble and Star‐forming Disk of NGC 3079

Hubble Space Telescope WFPC2 images are presented that span the inner ~19 kpc diameter of the edge-on galaxy NGC 3079; they are combined with optical, emission-line imaging spectrophotometry and VLA images of radio polarization vectors and rotation measures. Ionized gas filaments within 9 kpc diameter project up to 3 kpc above the disk, with the brightest forming the ≈1 kpc diameter superbubble. They are often resolved into strands ≈03 (25 pc) wide, which emerge from the nuclear CO ring as five distinct streams with large velocities and velocity dispersions (FWHM ≈ 450 km s-1). The brightest stream emits ≈10% of the superbubble Hα flux and extends for 250 pc along the axis of the VLBI radio jet to one corner of the base of the superbubble. The other four streams are not connected to the jet, instead curving up to the vertical ≈0.6 kpc above the galaxy disk, then dispersing as a spray of droplets each with ≈103 M☉ of ionized gas (the volume filling factor f > 3 × 10-3). Shredded clumps of disk gas form a similar structure in hydrodynamical models of a galaxy-scale wind. The pattern of magnetic fields and the gaseous kinematics also suggest that a wind of mechanical luminosity Lw ≈ 1043 ergs s-1 has stagnated in the galaxy disk at a radius of ~800 pc, has flared to larger radii with increasing height as the balancing interstellar medium pressure reduces above the disk, and has entrained dense clouds into a mushroom vortex above the disk. Hα emissivity of the filaments limits densities to ne > 4.3f-1/2 cm-3, hence kinetic energy and momentum to (0.4-5) × 1055 ergs and (1.6-6) × 1047 dyne s, respectively; the ranges result from uncertain space velocities. A prominent star-forming complex elsewhere in the galaxy shows a striking spray of linear filaments that extend for hundreds of parsecs to end in unresolved bullets.

The effect of linear density of polyamide filaments on the comfort and performance of ladies hosiery

This study used 49 female consumers, to investigate the effect of variation in the linear density of polyamide filaments used in the construction of ladies opaque tights, on a range of comfort and performance factors. Two sets of tights were used. Each set consisted of three types of tights manufactured from micro, semi-micro and conventional filaments respectively. The first set incorporated Lycra in alternate courses while the second set incorporated Lycra in every course. The hosiery was assessed by means of a number of ranking and rating scales after having been worn and washed five times. The tights made from micro and semi-micro filaments were generally much preferred to those made from conventionally thicker filaments. The differences between the perceptions of tights made from the micro and semi-micro filaments were small, and there appears little to be gained by using the lowest linear density micro filaments. Consumers preferences with regard to overall comfort agreed closely with preferences emerging from initial handle assessments. The hypothesis is advanced that for clothing worn next to the skin handle assessments could obviate the need for more expensive, time consuming and lengthy wearer trials.

Development and bioreactor cultivation of a novel semidifferentiated tissue suspension derived from the marine plant Acrosiphonia coalita

A semidifferentiated tissue culture consisting of linear filaments in liquid suspension was established from Acrosiphonia coalita, a cold-water green macroalga known to express pharmacologically active oxylipins deriving from lipoxygenase metabolism of linolenic acid. The tissue was vegatively propagated by blending the filaments down to 1 to 5 mm in length prior to subculture. The filamentous A. coalita tissue suspension was successfully cultivated in an illuminated, 3-L stirred-tank bioreactor at 12 degrees C, 0.46-vvm aeration rate, 250-rpm mixing speed, and incident illumination intensity of 77 microE m(-2)s(-1). The mean specific growth rate over the exponential phase was 0.185 day(-1) and a final cell density of 1083 mg dry cell weight (DCW) L(-1) was achieved within 15 days of cultivation from an initial cell density of 200 mg DCW L(-1). The addition of 3500 ppm CO(2) to the aeration gas provided a maximum CO(2) transfer rate of six times the maximum CO(2) consumption rate and stabilized the pH to 8.0 during the light phase of growth, but did not improve biomass productivity.

The nuclear poly(A) binding protein, PABP2, forms an oligomeric particle covering the length of the poly(A) tail

The mammalian nuclear poly(A) binding protein, PABP2, controls the length of the newly synthesized poly(A) tail on messenger RNAs. To gain a better understanding of the mechanism of length control, we have investigated the structure of the PABP2·poly(A) complex. Electron microscopy and scanning force microscopy studies reveal that PABP2, when bound to poly(A), forms both linear filaments and discrete-sized, compact, oligomeric particles. The maximum diameter of the filament is 7 nm; the maximum diameter of the particle is 21(±2) nm. Maximum particle size is realized when the PABP2·poly(A) complex is formed with poly(A) molecules 200–300 nt long, which corresponds to the average length of the newly synthesized poly(A) tail in vitro and in vivo. The equilibrium between filaments and particles is highly sensitive to ionic strength; filaments are favored at low ionic strength, while particles predominate at moderate to high ionic strength. Nitrocellulose filter binding and gel mobility shift assays indicate that the PABP2·poly(A) particle formed on A 300 is not significantly more stable than complexes formed with smaller species of poly(A). These results are discussed in the context of the proposed functions for PABP2.

Nev] Imaging of the Cygnus Loop

We present continuum-subtracted images of a 20' × 20' region of the eastern Cygnus Loop (NGC 6995) in the 3425 A forbidden line of Ne V. The images reveal bright linear filaments which are associated with, but not positionally coincident with, bright features seen in Hα, [O III], and other narrowband images. In some areas, the [Ne V] filaments define the edge of X-ray-emitting regions. The filaments exhibit a peak surface brightness of 1.2 × 10-4 photons cm-2 s-1 arcsec-2 at the top of the atmosphere in images with typical detection limits of ~10-5 photons cm-2 s-1 arcsec-2. We present arguments that these structures are produced by radiative shock waves and discuss implications for the shock velocities and the three-dimensional structure of this section of the Cygnus Loop. We place limits on the importance of thermal conduction-driven evaporation as a contribution to the mass of X-ray-emitting gas. Lack of evidence of [Ne V] emission resulting from thermal evaporation may have significance for supernova remnants and interstellar medium models that rely heavily on the importance of thermal evaporation.

Coupled synthesis and self-assembly of nanoparticles to give structures with controlled organization

Colloidal inorganic nanoparticles have size-dependent optical, optoelectronic and material properties that are expected to lead to superstructures with a range of practical applications1,2. Discrete nanoparticles with controlled chemical composition and size distribution are readily synthesized using reverse micelles and microemulsions as confined reaction media3,4,5, but their assembly into well-defined superstructures amenable to practical use remains a difficult and demanding task. This usually requires the initial synthesis of spherical nanoparticles, followed by further processing such as solvent evaporation6,7,8, molecular cross-linking9,10,11,12,13,14 or template-patterning15,16,17,18. Here we report that the interfacial activity of reverse micelles and microemulsions can be exploited to couple nanoparticle synthesis and self-assembly over a range of length scales to produce materials with complex organization arising from the interdigitation of surfactant molecules attached to specific nanoparticle crystal faces. We demonstrate this principle by producing three different barium chromate nanostructures—linear chains, rectangular superlattices and long filaments—as a function of reactant molar ratio, which in turn is controlled by fusing reverse micelles and microemulsion droplets containing fixed concentrations of barium and chromate ions, respectively. If suitable soluble precursors and amphiphiles with headgroups complementary to the crystal surface of the nanoparticle target are available, it should be possible to extend our approach to the facile production of one-dimensional ‘wires’ and higher-order colloidal architectures made of metals and semiconductors.

Diffusion-Induced Vortex Filament Instability in 3-Dimensional Excitable Media

We studied the stability of linear vortex filaments in 3-dimensional (3D) excitable media, using both analytical and numerical methods. We found an intrinsic 3D instability of vortex filaments that is diffusion-induced, and is due to the slower diffusion of the inhibitor. This instability can result either in a single helical filament or in chaotic scroll breakup, depending on the specific kinetic model. When the 2-dimensional dynamics were in the chaotic regime, filament instability occurred via on-off intermittency, a failure of chaos synchronization in the third dimension.

Expression of rat histone H1d in Escherichia coli and its purification.

Histone H1 is involved in the folding of linear polynucleosomal filament into a 30-nm fiber. In an effort to understand the role of different domains of histone H1 in chromatin folding, we have now expressed rat histone H1d in Escherichia coli using pTrc99A expression vector by providing a 6-His tag at the C-terminus to facilitate its purification. The expressed protein histone H1d was purified from the soluble extract of E. coli by employing Ni2+ NTA-agarose and heparin-agarose chromatography. The recombinant histone H1d was shown to be authentic by its N-terminal amino acid analysis, its secondary structural characteristics, and its ability to (a) condense DNA and (b) bind specifically to synthetic four-way junction DNA.