The monomeric, tetrameric, and fibrillar organization of Fib: the dynamic building block of the bacterial linear motor of Spiroplasma melliferum BC3

Spiroplasmas belong to the class Mollicutes, representing the minimal, free-living, and self-replicating forms of life. Spiroplasmas are helical wall-less bacteria and the only ones known to swim by means of a linear motor (rather than the near-universal rotary bacterial motor). The linear motor follows the shortest path along the cell's helical membranal tube. The motor is composed of a flat monolayered ribbon of seven parallel fibrils and is believed to function in controlling cell helicity and motility through dynamic, coordinated, differential length changes in the fibrils. The latter cause local perturbations of helical symmetry, which are essential for net directional displacement in environments with a low Reynolds number. The underlying fibrils' core building block is a circular tetramer of the 59-kDa protein Fib. The fibrils' differential length changes are believed to be driven by molecular switching of Fib, leading consequently to axial ratio and length changes in tetrameric rings. Using cryo electron microscopy, diffractometry, single-particle analysis of isolated ribbons, and sequence analyses of Fib, we determined the overall molecular organization of the Fib monomer, tetramer, fibril, and linear motor of Spiroplasma melliferum BC3 that underlies cell geometry and motility. Fib appears to be a bidomained molecule, of which the N-terminal half is apparently a globular phosphorylase. By a combination of reversible rotation and diagonal shift of Fib monomers, the tetramer adopts either a cross-like nonhanded conformation or a ring-like handed conformation. The sense of Fib rotation may determine the handedness of the linear motor and, eventually, of the cell. A further change in the axial ratio of the ring-like tetramers controls fibril lengths and the consequent helical geometry. Analysis of tetramer quadrants from adjacent fibrils clearly demonstrates local differential fibril lengths.     

The bacterial linear motor of Spiroplasma melliferum BC3: from single molecules to swimming cells

Spiroplasma melliferum BC3 are wall-less bacteria with internal cytoskeletons. Spiroplasma, Mycoplasma and Acholeplasma belong to the Mollicutes, which represent the smallest, simplest and minimal free-living and self-replicating forms of life. The Mollicutes are motile and chemotactic. Spiroplasma cells are, in addition, helical in shape. Based on data merging, obtained by video dark-field light microscopy of live, swimming helical Spiroplasma cells and by cryoelectron microscopy, unravelling the subcellular structure and molecular organization of the cytoskeleton, we propose a functional model in which the cytoskeleton also acts as a bacterial linear motor enabling and controlling both dynamic helicity and swimming. The cytoskeleton is a flat, monolayered ribbon constructed from seven contractile fibrils (generators) capable of changing their length differentially in a co-ordinated manner. The individual, flat, paired fibrils can be viewed as chains of tetramers approximately 100 A in diameter composed of 59 kDa monomers. The cytoskeletal ribbon is attached to the inner surface of the cell membrane (but is not an integral part of it) and follows the shortest helical line on the coiled cellular tube. We show that Spiroplasma cells can be regarded, at least in some states, as near-perfect dynamic helical tubes. Thus, the analysis of experimental data is reduced to a geometrical problem. The proposed model is based on simple structural elements and functional assumptions: rigid circular rings are threaded on a flexible, helical centreline. The rings maintain their circularity and normality to the centreline at all helical states. An array of peripheral, equidistant axial lines forms a regular cylindrical grid (membrane), by crossing the lines bounding the rings. The axial and peripheral spacing correspond to the tetramer diameter and fibril width (100 A) respectively. Based on electron microscopy data, we assign seven of the axial grid lines in the model to function as contractile generators. The generators are clustered along the shortest helical paths on the cellular coil. In the model, the shortest generator coincides with the shortest helical line. The rest, progressively longer, six generators follow to the right or to the left of the shortest generator in order to generate the maximal range of lengths. A rubbery membrane is stretched over (or represented by) the three-dimensional grid to form a continuous tube. Co-ordinated, differential length changes of the generators induce the membranal cylinder to coil and uncoil reversibly. The switch of helical sense requires equalization of the generators' length, forming a straight cylindrical tube with straight generators. The helical parameters of the cell population, obtained by light microscopy, constitute several subpopulations related, most probably, to cell size and age. The range of molecular dimensions in the active cytoskeleton inferred from light microscopy and modelling agrees with data obtained by direct measurements of subunit images on electron micrographs, scanning transmission electron microscopy (STEM) and diffraction analysis of isolated ribbons. Swimming motility and chemotactic responses are carried out by one or a combination of the following: (i). reciprocating helical extension and compression ('breathing'); (ii). propagation of a deformation (kink) along the helical path; (iii). propagation of a reversal of the helical sense along the cell body; and (iv). irregular flexing and twitching, which is functionally equivalent to standard bacterial tumbling. Here, we analyse in detail only the first case (from which all the rest are derived), including switching of the helical sense.     

Shaping and moving a spiroplasma

The Mollicutes (Spiroplasma, Mycoplasma and Acholeplasma) are the most minimal cells known to exist, being the smallest and simplest free-living and self-replicating forms of life. Phylogenetically, the Mollicutes are related to gram-positive bacteria and have evolved, by regressive evolution and genome reduction, from Clostridia. The smallest genome in this group (Mycoplasma genitalium - 5.77 x 10(5) bp) is only twice that of a large virus (e.g., Entomopox viruses). The largest Mollicute genome (Spiroplasma LB12 - 2.2 x 10(6) bp) is only about half that of, e.g., Escherichia coli. Structurally, the Mollicutes lack cell walls and flagella, but have internal cytoskeletons and are motile and chemotactic. Only a cholesterol-containing unit membrane envelops the cells. No analogs to the bacterial chemotactic and motility (che, mot, fla) genes, genes for a two-component signal transduction system, genes associated with gliding, or genomic homologs for the eukaryotic cytoskeleton and motor proteins were found in the Mollicutes. The Spiroplasmas are unique amongst the Mollicutes in having a well-defined basic helical cell geometry. In this respect, the Spiroplasma cell can, essentially, be viewed as a helical dynamic membranal tube (diameter approximately 0.2 microm; equivalent to that of one eukaryotic flagellar axoneme or to a bacterial flagellar bundle). A flat cytoskeletal ribbon of parallel fibrils is attached to the inside of the cellular tube. Both tube and cytoskeleton are mutually coiled into a dynamic helix driven by differential length changes of the fibrils, which function as linear motors. The cytoskeletal ribbon follows the shortest (inner) helical line on the inner surface of the cellular tube. Being helical allows for further analytical reduction and consequent structural quantification of Spiroplasma. Of particular importance is the ability to correlate light and electron microscopy data and to calculate the fibril lengths (and corresponding molecular dimensions) in the helical and nonhelical dynamic states. The structural unit of the contractile cytoskeleton is a approximately 50-Angstrom-wide filament comprised of pairs of the 59-kD fib gene product. The monomers are arranged in pairs with opposite polarities allowing for a approximately 100-Angstrom-long axial repeat. The functional unit of the contractile cytoskeletal ribbon is a fibril comprised of an aligned pair of filaments. Neighboring repeats form a tetrameric ring with a lateral repeat of approximately 100 A. The axial length of the rings may shorten by approximately 40%, driving the changes in the fibril lengths and, consequently, helical dynamics. Local length changes result in helical symmetry breaking and nonreciprocating cell movements allowing for net directional displacement. Flexing allows for changes in swimming direction.     

The cytoskeleton of spiroplasma: a complex linear motor

Spiroplasma are wall-less, helical bacteria from the class Mollicutes. The Mollicutes (Mycoplasma, Acholeplasma, Spiroplasma) evolved by regressive evolution to generate one of the simplest and minimal free-living and self-replicating forms of life. The spiroplasmas are the more advanced members in the class and are the closest to their clostridial ancestors. Spiroplasmas were discovered and identified as such only in 1972 and the finding of a unique and well-defined internal cytoskeleton, believed to be uncommon in bacteria, followed in 1973. Structural analysis suggests that the core of the spiroplasmal cytoskeleton is a flat, monolayered ribbon comprised of the 59-kDa fib gene product. The ribbon follows the shortest helical line of the polar cell from end to end. The structural building blocks of the cytoskeletal ribbon are fibrils assembling into a structure with approximately 10-nm axial and lateral repeats. Differential length changes of the fibrils may generate a wide dynamic spectrum of helical and non-helical geometries allowing for directional motility in low Reynolds number environments. The presence of other cytoskeletal elements (FtsZ, FtsA, EF-TU, MreB) has been demonstrated only recently in Spiroplasma cells. The cellular and molecular structure and dynamics of spiroplasmas and their cytoskeletal elements are reviewed.     

Motility modes of Spiroplasma melliferum BC3: a helical,wall-less bacterium driven by a linear motor

Spiroplasma are members of the Mollicutes (Mycoplasma, Acholeplasma and Spiroplasma) - the simplest, minimal, free-living and self-replicating forms of life. The mollicutes are unique among bacteria in completely lacking cell walls and flagella and in having an internal, contractile cytoskeleton, which also functions as a linear motor. Spiroplasma are helical, chemotactic and viscotactic active swimmers. The Spiroplasmal cytoskeleton is a flat ribbon composed of seven pairs of fibrils. The ribbon is attached to the inner side of the cell membrane along its innermost (shortest) helical line. The cell's geometry and dynamic helical parameters, and consequently motility, can be controlled by changing differentially and in a co-ordinated manner, the length of the fibrils. We identified several consistent modes of cell movements and motility originating, most likely, as a result of co-operative or local molecular switching of fibrils: (i). regular extension and contraction within the limits of helical symmetry (this mode also includes straightening, beyond what is allowed by helical symmetry, and reversible change of helical sense); (ii). spontaneous and random change of helical sense originating at random sites along the cell (these changes propagate along the cell in either direction and hand switching is completed within approximately 0.08 second); (iii). forming a deformation on one of the helical turns and propagating it along the cell (these helical deformations may travel along the cell at a speed of up to approximately 40 microm s-1); (iv). random bending, flexing and twitching (equivalent to tumbling). In standard medium (viscosity = 1.147 centipoise) the cells run at approximately 1.5 microm s-1, have a Reynolds number of approximately 3.5 x 10-6 and consume approximately 30 ATP molecules s-1. Running velocity, duration, persistence and efficiency increase with viscosity upon adding ficoll, dextran and methylcellulose to standard media. Relative force measurements using optical tweezers confirm these findings.     

A bacterial linear motor: cellular and molecular organization of the contractile cytoskeleton of the helical bacterium Spiroplasma melliferum BC3

The Mollicutes (Mycoplasma, Acholeplasma, and Spiroplasma) are the smallest, simplest and most primitive free-living and self-replicating known cells. These bacteria have evolved from Clostridia by regressive evolution and genome reduction to the range of 5.8 x 10(5)-2.2 x 10(6) basepairs (bp). Structurally, the Mollicutes completely lack cell walls and are enveloped by only a cholesterol containing cell membrane. The Mollicutes contain what can be defined as a bacterial cytoskeleton. The Spiroplasmas are unique in having a well-defined, dynamic, helical cell geometry and a flat, monolayered, membrane-bound cytoskeleton, which follows, intracellularly, the shortest helical line on the cellular coil. By applying cryo-electron-microscopy to whole cells, isolated cytoskeletons and cytoskeletal fibrils and subunits, as well as by selective extraction of cellular components, we determined, at a resolution of approximately 25 A, the cellular and molecular organization of the cytoskeleton. The cytoskeleton is assembled from a 59 kDa protein. The 59 kDa protein, has an equivalent sphere diameter of approximately 50 A. Given the approximately 100 A axial and lateral spacings in the cytoskeletal ribbons and the near-circular shape of the subunit, we suggest that the subunit is a tetramer of 59 kDa monomers; the tetramers assemble further into flat fibrils, seven of which form a flat, monolayered, well-ordered ribbon. The cytoskeleton may function as a linear motor by differential and coordinated length-changes of the fibrils driven by conformational changes of the tetrameric subunits, the shape of which changes from near circular to elliptical. The cytoskeleton controls both the dynamic helical shape and the consequent motility of the cell. A stable cluster of proteins co-purifies with the cytoskeleton. These apparent membrane and membrane-associated proteins may function as anchor proteins.     

Peptide conformation and supramolecular organization in amylin fibrils: constraints from solid-state NMR

The 37-residue amylin peptide, also known as islet amyloid polypeptide, forms fibrils that are the main peptide or protein component of amyloid that develops in the pancreas of type 2 diabetes patients. Amylin also readily forms amyloid fibrils in vitro that are highly polymorphic under typical experimental conditions. We describe a protocol for the preparation of synthetic amylin fibrils that exhibit a single predominant morphology, which we call a striated ribbon, in electron microscopy and atomic force microscopy images. Solid-state nuclear magnetic resonance (NMR) measurements on a series of isotopically labeled samples indicate a single molecular structure within the striated ribbons. We use scanning transmission electron microscopy and several types of one- and two-dimensional solid-state NMR techniques to obtain constraints on the peptide conformation and supramolecular structure in these amylin fibrils and to derive molecular structural models that are consistent with the experimental data. The basic structural unit in amylin striated ribbons, which we call the protofilament, contains four layers of parallel beta-sheets, formed by two symmetric layers of amylin molecules. The molecular structure of amylin protofilaments in striated ribbons closely resembles the protofilament in amyloid fibrils with a similar morphology formed by the 40-residue beta-amyloid peptide that is associated with Alzheimer's disease.     

Purified trout egg vitelline envelope proteins VEbeta and VEgamma polymerize into homomeric fibrils from dimers in vitro

The rainbow trout egg vitelline envelope (VE) is composed of three proteins, called VEalpha ( approximately 58-60kDa Mr), VEbeta ( approximately 52kDa Mr), and VEgamma ( approximately 47kDa Mr). Each of these proteins is related to mouse egg zona pellucida (ZP) glycoproteins, called ZP1, ZP2, and ZP3, and possesses a ZP domain that has been implicated in the polymerization of the proteins into long, interconnected fibrils or filaments. Here, trout egg VEbeta and VEgamma were purified to homogeneity and analyzed under various experimental conditions (SDS-PAGE, Blue Native-(BN-)PAGE, size-exclusion chromatography, and transmission electron microscopy) to determine whether individual VE proteins would polymerize into fibrils in vitro. Such analyses revealed that in the presence of 6M urea each VE protein is present primarily as monomers and as small oligomers (dimers, tetramers, etc.). However, either a reduction in urea concentration or a complete removal of urea results in the polymerization of VEbeta and VEgamma dimers into very large oligomers. Mixtures of VEbeta and VEgamma also give rise to large oligomers. Under these conditions, VE proteins are visualized by transmission electron microscopy as aggregates of long fibrils, with each fibril composed of contiguous beads located periodically along the fibril. The relationship between the behavior of fish egg VE proteins and mouse ZP glycoproteins, as well as other ZP domain-containing proteins, is discussed.     

The circularization of amyloid fibrils formed by apolipoprotein C-II

Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a "cross-beta" x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common beta-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons approximately 2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm +/- 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross-beta superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.     

Self-assembly in aqueous solution of a modified amyloid beta peptide fragment

The self-assembly in films dried from aqueous solutions of a modified amyloid beta peptide fragment is studied. We focus on sequence Abeta(16-20), KLVFF, extended by two alanines at the N-terminus to give AAKLVFF. Self-assembly into twisted ribbon fibrils is observed, as confirmed by transmission electron microscopy (TEM). Dynamic light scattering reveals the semi-flexible nature of the AAKLVFF fibrils, while polarized optical microscopy shows that the peptide fibrils crystallize after an aqueous solution of AAKLVFF is matured over 5 days. The secondary structure of the fibrils is studied by FT-IR, circular dichroism and X-ray diffraction (XRD), which provide evidence for beta-sheet structure in the fibril. From high resolution TEM it is concluded that the average width of an AAKLVFF fibril is (63+/-18) nm, indicating that these fibrils comprise beta-sheets with multiple repeats of the unit cell, determined by XRD to have b and c dimensions 1.9 and 4.4 nm with an a axis 0.96 nm, corresponding to twice the peptide backbone spacing in the antiparallel beta-sheet.     

A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple ‘letter-G-like’ β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops.     

Normal and reversed supramolecular chirality of insulin fibrils probed by vibrational circular dichroism at the protofilament level of fibril structure

Fibrils are β-sheet-rich aggregates that are generally composed of several protofibrils and may adopt variable morphologies, such as twisted ribbons or flat-like sheets. This polymorphism is observed for many different amyloid associated proteins and polypeptides. In a previous study we proposed the existence of another level of amyloid polymorphism, namely, that associated with fibril supramolecular chirality. Two chiral polymorphs of insulin, which can be controllably grown by means of small pH variations, exhibit opposite signs of vibrational circular dichroism (VCD) spectra. Herein, using atomic force microscopy (AFM) and scanning electron microscopy (SEM), we demonstrate that indeed VCD supramolecular chirality is correlated not only by the apparent fibril handedness but also by the sense of supramolecular chirality from a deeper level of chiral organization at the protofilament level of fibril structure. Our microscopic examination indicates that normal VCD fibrils have a left-handed twist, whereas reversed VCD fibrils are flat-like aggregates with no obvious helical twist as imaged by atomic force microscopy or scanning electron microscopy. A scheme is proposed consistent with observed data that features a dynamic equilibrium controlled by pH at the protofilament level between left- and right-twist fibril structures with distinctly different aggregation pathways for left- and right-twisted protofilaments.     

Growth of Collagen Fibril Seeds from Embryonic Tendon: Fractured Fibril Ends Nucleate New Tip Growth

Collagen fibrils are the principal tensile element of vertebrate tissues where they occur in the extracellular matrix as spatially organised arrays. A major challenge is to understand how the mechanisms of nucleation, growth and remodelling yield fibrils of tissue-specific diameter and length. Here we have developed a seeding system whereby collagen fibrils were isolated from avian embryonic tendon and added to purified collagen solution, in order to characterise fibril surface nucleation and growth mechanisms. Fragmentation of tendon in liquid nitrogen followed by Dounce homogenisation generated fibril length fragments. Most (> 94%) of the fractured ends of fibrils, which show an abrupt square profile, were found to act as nucleation sites for further growth by molecular accretion. The mechanism of this nucleation and growth process was investigated by transmission electron microscopy, atomic force microscopy and scanning transmission electron microscopy mass mapping. Typically, a single growth spur occurred on the N-terminal end of seed fibrils whilst twin spurs frequently formed on the C-terminal end before merging into a single tip projection. The surface nucleation and growth process generated a smoothly tapered tip that achieved maximum diameter when the axial extension reached ∼ 13 μm. Lateral growth also occurred along the entire length of all seed fibrils that contained tip projections. The data support a model of collagen fibril growth in which the broken ends of fibrils are nucleation sites for propagation in opposite axial directions. The observed fibril growth behaviour has direct relevance to tendon matrix remodelling and repair processes that might involve rupture of collagen fibrils.     

Kinetics of fibril formation of bovine kappa-casein indicate a conformational rearrangement as a critical step in the process

S-carboxymethylated (SCM) κ-casein forms in vitro fibrils that display several characteristics of amyloid fibrils, although the protein is unrelated to amyloid diseases. In order to get insight into the processes that prevent the formation of amyloid fibrils made of κ-caseins in milk, we have characterized in detail the reaction and the roles of its possible effectors: glycosylation and other caseins. Given that native κ-casein occurs as a heterogeneous mixture of carbohydrate-free and carbohydrate-containing chains, kinetics of fibril formation were performed on purified glycosylated and unglycosylated SCM κ-caseins using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and Fourier transform infrared spectroscopy for morphological and structural analyses. Both unglycosylated and glycosylated SCM κ-caseins have the ability to fibrillate. Kinetic data indicate that the fibril formation rate increases with SCM κ-casein concentration but reaches a plateau at high concentrations, for both the unglycosylated and glycosylated forms. Therefore, a conformational rearrangement is the rate-limiting step in fibril growth of SCM κ-casein. Transmission electron microscopy images indicate the presence of 10- to 12-nm spherical particles prior to the appearance of amyloid structure. Fourier transform infrared spectroscopy spectra reveal a conformational change within these micellar aggregates during the fibrillation. Fibrils are helical ribbons with a pitch of about 120-130 nm and a width of 10-12 nm. Taken together, these findings suggest a model of aggregation during which the SCM κ-casein monomer is in rapid equilibrium with a micellar aggregate that subsequently undergoes a conformational rearrangement into a more organized species. These micelles assemble and this leads to the growing of amyloid fibrils. Addition of α_s1-and β-caseins decreases the growth rate of fibrils. Their main effect was on the elongation rate, which became close to that of the limiting conformation change, leading to the appearance of a lag phase at the beginning of the kinetics.     

Collagen fibril formation in a wound healing model

Control of tissue composition and organization will be a key feature in the development of successful products through tissue engineering. However, the mechanism of collagen fibril formation, growth, and organization is not yet fully understood. In this study we have examined collagen fibril formation in a wound healing model in which the newly formed fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial implant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant collagen formation at 14 and 28 days. Individual collagen fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact fibrils. Examination by transmission electron microscopy showed that approximately 80% of the intact fibrils showed a single polarity reversal, with both ends of each fibril comprising collagen amino-terminal domains; the remaining fibrils had no polarity reversal. All fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled fibrils contained both type I and III collagens. These data indicate that this wound healing model provides a system in which collagen fibril formation can be readily followed.     

Echinoderm collagen fibrils grow by surface-nucleation-and-propagation from both centers and ends

Collagen fibrils from sea cucumber (class Holothuroidea) dermis were previously found to grow by coordinated monomer addition at both centers and ends. This analysis of sea urchin (class Echinoidea) collagen fibrils was undertaken to compare the growth characteristics of fibrils from two classes of echinoderms, and to determine whether a single growth model could account for the main features of fibrils from these two taxa. Native collagen fibrils (37-431 micrometer long) from the spine ligaments of the sea urchin Eucidaris tribuloides were studied by scanning transmission electron microscopy and image analysis. The analyses revealed the mass per unit length, and hence the number of molecules in cross-section, along the entire length of each fibril. The fibrils were symmetrically spindle shaped. The maximum mass per unit length occurred in the center of each fibril, where the fibril contains anti-parallel molecules in equal numbers. The two pointed tips of each fibril showed similar linear axial mass distributions, indicating that the two tips retain shape and size similarity throughout growth. The linear axial mass distributions showed that the tips were paraboloidal, similar to those of vertebrate and sea cucumber fibrils. The computed maximum diameters of the fibrils increased linearly with fibril length. The overall shapes of the fibrils showed that they retain geometric similarity throughout growth. Computer modeling showed that the simplest self-assembly mechanism that can account for the features of these fibrils, and of the sea cucumber fibrils that have been described, is one in which the fibril tips produce independent axial growth, while lateral growth takes place through a surface nucleation and propagation mechanism. This mechanism produces coordinated growth in length and diameter as well as geometric similarity, characteristic features of echinoderm collagen fibrils.     

Establishment of a first-order kinetic model of light chain-associated amyloid fibril extension in vitro

Light chain-associated (AL) amyloidosis is a common and fatal systemic amyloidosis. AL amyloid fibrils (fAL) are composed of intact or fragmental monoclonal light chains (AL proteins). To elucidate the molecular mechanisms of fAL formation from AL proteins, we purified fAL and AL proteins from the amyloid-deposited organs of five AL amyloidosis patients. By electron microscopy and fluorometric thioflavin T method, we observed optimal fibril extension at pH 2.0-3.5 for the fibrils obtained from four patients, while at pH 7.5-8.0 for those obtained from one patient. Fragmental AL proteins were more efficient in the extension reaction than intact AL proteins. The fibrils obtained from all five patients showed clear fibril extension electron microscopically at pH 7.5. The extension of the fibrils obtained from all five patients could be explained by a first-order kinetic model, i.e., fibril extension proceeds via the consecutive association of AL proteins onto the ends of existing fibrils. Fibril extension was accelerated by dermatan sulfate proteoglycan, and inhibited by apolipoprotein E, alpha1-microglobulin, fibronectin, and an antioxidant nordihydroguaiaretic acid. These findings contribute to our understanding of the molecular mechanism underlying the pathogenesis of AL amyloidosis, and will be useful for developing a therapeutic strategy against the disease.     

Zonulae occludentes in junctional complex-enriched fractions from mouse liver: preliminary morphological and biochemical characterization

A bile canaliculus-derived preparation containing junctional complexes has been obtained from mouse livers using subcellular fractionation techniques. The junctional complexes include structurally intact zonulae occludentes (ZOs). Extraction of this preparation with the anionic detergent sodium deoxycholate (DOC) left junctional ribbons, the detergent-insoluble zonular remnants of the junctional complexes. When visualized in negative stain electron microscopy, each of these ribbons contained a branching and anastomosing network of fibrils which appears similar to that of ZOs in freeze-fractured whole liver. Comparative measurements of freeze-fracture and negative stain fibril diameters and network densities support this relationship. SDS polyacrylamide gel analysis shows the DOC-insoluble junctional ribbons to be characterized by major polypeptides at 37,000 and at 48,000, with minor bands at 34,000, 41,000, 71,000, 86,000, 92,000, and 102,000. The ZO-containing membrane fractions have been isolated in the presence of EGTA in concentrations and under conditions shown by others to disrupt normal ZO morphology and physiology in whole living epithelia. The network of fibrils visualized in these fractions by negative staining is structurally resistant to treatment with DOC, but is either solubilized or disrupted by N-lauroylsarcosine.     

Helical arrays of Escherichia coli small ribosomal subunits produced in vitro.

In vitro conditions have been determined for obtaining ordered helical ribbons of small ribosomal subunits from Escherichia coli. These ribbons, suitable for study by three-dimensional reconstruction, are the first ordered arrays of ribosomes or ribosomal subunits to be produced in vitro.Although small ribosomal subunits remain in solution for extended periods (up to 6 months) during this procedure, their structural integrity, as assessed by acrylamide/agarose gel electrophoresis, by sucrose gradients, and by electron microscopy, is not significantly altered.Electron micrographs of ribbons of small subunits diffract to 60 Å resolution. Optical diffraction patterns suggest that adjacent subunits within helical ribbons are related by a 2-fold screw parallel to the long axis of the ribbon and the helical repeat distance measured from electron micrographs is 220 Å.