Heterogeneous Filaments Research Articles

Novel Conformation-Dependent Tau Antibodies Are Modulated by Adjacent Phosphorylation Sites.

Tau proteins within the adult central nervous system (CNS) are found to be abnormally aggregated into heterogeneous filaments in neurodegenerative diseases, termed tauopathies. These tau inclusions are pathological hallmarks of Alzheimer's disease (AD), Pick's disease (PiD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP). The neuropathological hallmarks of these diseases burden several cell types within the CNS, and have also been shown to be abundantly phosphorylated. The mechanism(s) by which tau aggregates in the CNS is not fully known, but it is hypothesized that hyperphosphorylated tau may precede and further promote filament formation, leading to the production of these pathological inclusions. In the studies herein, we generated and thoroughly characterized two novel conformation-dependent tau monoclonal antibodies that bind to residues Pro218-Glu222, but are sensitive to denaturing conditions and highly modulated by adjacent downstream phosphorylation sites. These epitopes are present in the neuropathological hallmarks of several tauopathies, including AD, PiD, CBD, and PSP. These novel antibodies will further enable investigation of tau-dependent pathological inclusion formation and enhance our understanding of the phosphorylation signatures within tauopathies with the possibility of new biomarker developments.

Rotational Co-extrusion 4D printing of heterogeneous filaments to enable sophisticated shape morphing

Multi-material additive manufacturing has recently flourished due to its capability to fully exploit the potential of 3D printing for constructing sophisticated heterogeneous material structures. However, producing filaments with tunable internal heterogeneous architecture is difficult in current extrusion-based 3D printing processes. Herein, a novel rotational co-extrusion 3D printing method is proposed that can enable the programmable material configuration of extruded filaments, which is achieved by using a rotatable Y-shape nozzle. By coordinating the rotation and movement of the extrusion nozzle, as well as the air pressure, the material conformation of extruded filaments can be controlled. This strategy can achieve a variety of spatial distribution configurations of heterogeneous materials. In addition, different patterns with customized surface textures have been produced. Furthermore, the 4D printed heterogeneous hydrogels with differential stimuli-responsive properties are demonstrated to generate well-regulated shape-morphing as pre-designed. This method provides an intrinsic ability for 3D printing to construct heterogeneous-filament structures, which have potential applications in the fields of reconfigurable structural materials, tunable meta-materials, tissue engineering, soft robots, etc.

Hetero-cellular prototyping by synchronized multi-material bioprinting for rotary cell culture system

Bottom-up tissue engineering requires methodological progress of biofabrication to capture key design facets of anatomical arrangements across micro, meso and macro-scales. The diffusive mass transfer properties necessary to elicit stability and functionality require hetero-typic contact, cell-to-cell signaling and uniform nutrient diffusion. Bioprinting techniques successfully build mathematically defined porous architecture to diminish resistance to mass transfer. Current limitations of bioprinted cell assemblies include poor micro-scale formability of cell-laden soft gels and asymmetrical macro-scale diffusion through 3D volumes. The objective of this work is to engineer a synchronized multi-material bioprinter (SMMB) system which improves the resolution and expands the capability of existing bioprinting systems by packaging multiple cell types in heterotypic arrays prior to deposition. This unit cell approach to arranging multiple cell-laden solutions is integrated with a motion system to print heterogeneous filaments as tissue engineered scaffolds and nanoliter droplets. The set of SMMB process parameters control the geometric arrangement of the combined flow’s internal features and constituent material’s volume fractions. SMMB printed hepatocyte-endothelial laden 200 nl droplets are cultured in a rotary cell culture system (RCCS) to study the effect of microgravity on an in vitro model of the human hepatic lobule. RCCS conditioning for 48 h increased hepatocyte cytoplasm diameter 2 μm, increased metabolic rate, and decreased drug half-life. SMMB hetero-cellular models present a 10-fold increase in metabolic rate, compared to SMMB mono-culture models. Improved bioprinting resolution due to process control of cell-laden matrix packaging as well as nanoliter droplet printing capability identify SMMB as a viable technique to improve in vitro model efficacy.

Thermal vesiculation during volcanic eruptions.

Terrestrial volcanic eruptions are the consequence of magmas ascending to the surface of the Earth. This ascent is driven by buoyancy forces, which are enhanced by bubble nucleation and growth (vesiculation) that reduce the density of magma. The development of vesicularity also greatly reduces the 'strength' of magma, a material parameter controlling fragmentation and thus the explosive potential of the liquid rock. The development of vesicularity in magmas has until now been viewed (both thermodynamically and kinetically) in terms of the pressure dependence of the solubility of water in the magma, and its role in driving gas saturation, exsolution and expansion during decompression. In contrast, the possible effects of the well documented negative temperature dependence of solubility of water in magma has largely been ignored. Recently, petrological constraints have demonstrated that considerable heating of magma may indeed be a common result of the latent heat of crystallization as well as viscous and frictional heating in areas of strain localization. Here we present field and experimental observations of magma vesiculation and fragmentation resulting from heating (rather than decompression). Textural analysis of volcanic ash from Santiaguito volcano in Guatemala reveals the presence of chemically heterogeneous filaments hosting micrometre-scale vesicles. The textures mirror those developed by disequilibrium melting induced via rapid heating during fault friction experiments, demonstrating that friction can generate sufficient heat to induce melting and vesiculation of hydrated silicic magma. Consideration of the experimentally determined temperature and pressure dependence of water solubility in magma reveals that, for many ascent paths, exsolution may be more efficiently achieved by heating than by decompression. We conclude that the thermal path experienced by magma during ascent strongly controls degassing, vesiculation, magma strength and the effusive-explosive transition in volcanic eruptions.

Mechanical Heterogeneity Favors Fragmentation of Strained Actin Filaments

We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency and location along filaments. The emergent behavior of mechanically heterogeneous filaments, particularly under confinement, emphasizes that severing in cells is likely to be influenced by multiple physical and chemical factors.

Force Spectroscopy of Complex Biopolymers with Heterogeneous Elasticity

Cellular biopolymers can exhibit significant compositional heterogeneities as a result of the non-uniform binding of associated proteins, the formation of microstructural defects during filament assembly, or the imperfect bundling of filaments into composite structures of variable diameter. These can lead to significant variations in the local mechanical properties of biopolymers along their length. Existing spectral analysis methods assume filament homogeneity and therefore report only a single average stiffness for the entire filament. However, understanding how local effects modulate biopolymer mechanics in a spatially resolved manner is essential to understanding how binding and bundling proteins regulate biopolymer stiffness and function in cellular contexts. Here, we present a new method to determine the spatially varying material properties of complex biopolymers from the observation of passive thermal fluctuations of the filament conformation. We develop new statistical mechanics-based approaches for heterogeneous filaments that estimate local bending elasticities as a function of the filament arc-length. We validate this methodology using simulated polymers with known stiffness distributions, and find excellent agreement between derived and expected values. We then determine the bending elasticity of microtubule filaments of variable composition generated by repeated rounds of tubulin polymerization using either GTP or GMPCPP, a nonhydrolyzable GTP analog. Again, we find excellent agreement between mechanical and compositional heterogeneities.

Force spectroscopy of complex biopolymers with heterogeneous elasticity

Cellular biopolymers can exhibit significant compositional heterogeneities as a result of the non-uniform binding of associated proteins, the formation of microstructural defects during filament assembly, or the imperfect bundling of filaments into composite structures of variable diameter. These can lead to significant variations in the local mechanical properties of biopolymers along their length. Existing spectral analysis methods assume filament homogeneity and therefore report only a single average stiffness for the entire filament. However, understanding how local effects modulate biopolymer mechanics in a spatially resolved manner is essential to understanding how binding and bundling proteins regulate biopolymer stiffness and function in cellular contexts. Here, we present a new method to determine the spatially varying material properties of individual complex biopolymers from the observation of passive thermal fluctuations of the filament conformation. We develop new statistical mechanics-based approaches for heterogeneous filaments that estimate local bending elasticities as a function of the filament arc-length. We validate this methodology using simulated polymers with known stiffness distributions, and find excellent agreement between derived and expected values. We then determine the bending elasticity of microtubule filaments of variable composition generated by repeated rounds of tubulin polymerization using either GTP or GMPCPP, a nonhydrolyzable GTP analog. Again, we find excellent agreement between mechanical and compositional heterogeneities.

Signal peptide of FadA adhesin from Fusobacterium nucleatum plays a novel structural role by modulating the filament’s length and width

FadA, a novel adhesin of periodontal pathogen Fusobacterium nucleatum is composed of two forms, pre-FadA and mature FadA (mFadA), constituting the functional FadA complex (FadAc). By electron microscopy, we observed that mFadA formed uniformly long and thin filaments, while FadAc formed heterogeneous filaments of varying lengths and widths, as well as “knots”. Mutants in signal peptide or in the non-alpha-helical loop retaining heterogeneous structures had binding activity while those forming aggregates or long filaments lost activity. These observations suggest short filaments and knots may be the active forms of FadA. This is the first demonstration that a signal peptide is required for the assembly of a bacterial adhesin. Structured summary of protein interactions mFadA and pre-FadA interaction by transmission electron microscopy ( View interaction)

Three- and Four-repeat Tau Coassemble into Heterogeneous Filaments: AN IMPLICATION FOR ALZHEIMER DISEASE

Tau filaments are the pathological hallmark of numerous neurodegenerative diseases including Alzheimer disease, Pick disease, and progressive supranuclear palsy. In the adult human brain, six isoforms are expressed that differ by the presence or absence of the second of four semiconserved repeats. As a consequence, half of the tau isoforms have three repeats (3R tau), whereas the other half of the isoforms have four repeats (4R tau). Tauopathies can be characterized based on the isoform composition of their filaments. Alzheimer disease filamentous inclusions contain all isoforms. Pick disease filaments contain 3R tau. Progressive supranuclear palsy filaments contain 4R tau. Here, we used site-directed spin labeling of recombinant tau in conjunction with electron paramagnetic resonance spectroscopy to obtain structural insights into these filaments. We find that filaments of 4R tau and 3R tau share a highly ordered core structure in the third repeat with parallel, in-register arrangement of β-strands. This structure is conserved regardless of whether full-length isoforms (htau40 and htau23) or truncated constructs (K18 and K19) are used. When mixed, 3R tau and 4R tau coassemble into heterogeneous filaments. These filaments share the highly ordered core in the third repeat; however, they differ in their overall composition. Our findings indicate that at least three distinct types of filaments exist: homogeneous 3R tau, homogeneous 4R tau, and heterogeneous 3R/4R tau. These results suggest that individual filaments found in Alzheimer disease are structurally distinct from those in the 3R and 4R tauopathies.

Determinants of Myosin II Cortical Localization during Cytokinesis

Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.

Effect of fluctuation in step size on actin-myosin sliding motion

It is possible that the step size, or power stroke, of a skeletal muscle myosin is not constant; rather, it fluctuates for each force generation. The estimated widths of the fluctuation are as large as the estimated values of the step size. Although such non-negligible fluctuation is presumed to affect the sliding motion, these effects remain unclear. We examined a system driven by a single myosin molecule sliding along an actin filament to reveal its basic effects. First, we calculated the sliding velocity of the system for each fluctuation width and found that the mean velocity increased with the fluctuation width. We also found that the estimated fluctuation widths satisfied the conditions for maximizing the sliding velocity. Next, we examined the sliding motion along a heterogeneous filament, on which binding sites for myosins were distributed randomly. We found that the loss in sliding velocity that was attributable to heterogeneity of the filament became small when fluctuation in the step size existed. This finding implied that the fluctuation stabilized velocity sliding along possible heterogeneous filaments. These benefits of fluctuation in step size might be used in biological systems, such as a muscle system, and are applicable to fabricated micromachines.

Actin- and protein-4.1-containing filaments link nuclear pore complexes to subnuclear organelles in Xenopus oocyte nuclei.

We imaged the interiors of relatively intact Xenopus oocyte nuclei by field emission scanning electron microscopy (feSEM) and visualized a network of filaments that attach to nuclear pore complexes and extend throughout the nucleus. Within the nucleus, these 'pore-linked filaments' (PLFs) were embedded into spherical structures 100 nm to approximately 5 microm in diameter. A subset of spheres was identified as Cajal bodies by immuno-gold labeling; the rest were inferred to be nucleoli and snurposomes both of which are abundant in Xenopus oocyte nuclei. Most PLFs were independent of chromatin. The thickness of a typical PLF was 40 nm (range, approximately 12-100 nm), including the 4 nm chromium coat. PLFs located inside the nucleus merged, bundled and forked, suggesting architectural adaptability. The PLF network collapsed upon treatment with latrunculin A, which depolymerizes actin filaments. Jasplakinolide, which stabilizes actin filaments, produced PLFs with more open substructure including individual filaments with evenly-spaced rows of radially projecting short filaments. Immuno-gold labeling of untreated oocyte nuclei showed that actin and protein 4.1 each localized on PLFs. Protein 4.1-gold epitopes were spaced at approximately 120 nm intervals along filaments, and were often paired ( approximately 70 nm apart) at filament junctions. We suggest that protein 4.1 and actin contribute to the structure of a network of heterogeneous filaments that link nuclear pore complexes to subnuclear organelles, and discuss possible functions for PLFs in nuclear assembly and intranuclear traffic.

Structural studies of glycerinated skeletal muscle. I. A-band length and cross-bridge period in ATP-contracted fibers.

An electron microscope study is reported of structural changes during ATP-induced contraction of glycerinated rabbit psoas. In the absence of ATP, A-band length is constant at sarcomere lengths above 1.9 micron, with average length of 1.54 mu. In ATP-treated fibers, A-band length is also constant at sarcomere lengths above 2.0 microns, but the apparent length of A-band decreases to approximately 1.3 micron, as sarcomere length decreases from 1.9 micron to 1.5 mu. The occurrence of short A-bands cannot be attributed to crumpling of thick filaments against Z-lines, since I-bands remain patent; nor to the presence of heterogeneous filaments, since resting muscle does not show comparable heterogeneity, nor to compressive artifacts, which are minor when knife edge is oriented parallel with fiber axis during microtomy . The decrease of A-band length appears related, at least in part, to disarray of terminal cross-bridges as the thick filaments encroach upon the N-line, a structure which becomes evident within the I-band during contraction of glycerinated fibers. In preliminary studies, optical transforms of A-bands from individual sarcomeres reveal a characteristic myosin layer-line pattern as low as 1.5 micron sarcomere length. A cross-bridge repeat of 143 A is obtained for sarcomeres above 1.6 micron length; however, an appreciable proportion of sarcomeres in the range from 1.5 micron to 1.9 mu length generate meridional reflections less than 143 A, and as low as 130 A.

Unidirectional growth of Salmonella flagella in vitro

In previous papers (Asakura, Eguchi & Iino, 1964, 1966), in vitro reconstitution of flagellar filaments from monomeric flagellins, using strains of Salmonella, was shown to be similar to crystallization. At physiological ionic strength and pH, reconstitution consists only of growth, that is, the polymerization of a monomer on to the ends of added fragments of flagella or “seeds”. The present study was undertaken to investigate whether growth takes place at two ends of each fragment or only at one of the two ends. For this purpose, we used two kinds of flagella, having i- and 1,2-antigens, respectively, together with anti-flagella sera, anti-i and anti-1,2. Monomers and seeds derived from i- and 1,2-flagella were cross mixed to produce heterogeneous filaments or “block copolymers”, which, after treatment with either one of the two antisera, were observed in an electron microscope with negative staining. When a product of copolymerization was treated with anti-i, for example, a part of each filment containing i-flagellin was uniformly labelled with the antibody and could be distinguished from another unlabelled part composed only of 1,2-flagellin. In a large number of the filaments observed, the great majority were of the form of i-1,2; filaments of the form of i-1,2-i or 1,2-i-1,2, were never found. On the other hand, it was shown that the addition of a small amount of seed to a mixture of i- and 1,2-monomers results in the formation of long flagellar filments, each of which is uniformly labelled with anti-i and anti-1,2, respectively. In view of thes observations, it was concluded that during reconstitution flagellar filaments grow in a unidirectional manner. Abram, Koffler & Vatter (Abst. 45, 2nd Int. Cong. Biophys., Vienna, 1966) have reported that when negatively stained fragments of flagella (Bacillus) were observed in an electron microscope, only their distal ends appeared to be frayed. It will be shown in this paper that this end corresponds to the end where in vitro growth takes place.

Experimental production of filaments and networks in cytoplasm of Cichorieae

1. 1. The cytoplasmic participate of epidermal cells of Lactuca saliva L. and of a number of plants belonging to the tribe Cichorieae consists of mitochondria, plastids and spherosomes. Their identification is effected on the hases of differential staining with Janus green B, different properties exhibited under the phase contrast microscope, and by experimental virescence of colorless plastids. 2. 2. When living epidermal cells of Cichorieae are mounted on a slide under anaerobic conditions for more than 3 hours, long filamentous structures appear in the cytoplasm. They branch, anastomose, form loops, networks, and change shape constantly. 3. 3. Spherical elements of the cytoplasm do not participate in the formation of branching filaments. 4. 4. Filaments and networks may also be produced in cells mounted under anaerobic conditions in liquid petrolatum and in olive oil. 5. 5. The filaments and networks are not dissolved in 30 per cent acetic acid. 6. 6. The heterogeneous filaments formed under anaerobic conditions are considered to be composed of modified plastids joined end-to-end and enveloped by a membrane which could be either an extension of the plastid membrane, or an interface membrane between the plastids and the cytoplasm.