Characterization of the protein components of Nephila clavipes dragline silk

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable.     

31P-NMR spectroscopy of human cancer cells proliferating in a basement membrane gel

We describe a system in which proliferating human breast cancer cells are monitored by NMR spectroscopy for at least 6 days in basement membrane gel (BMG)1 threads. The cells are perfused under standard sterile cell culture conditions. 31P-NMR spectra obtained continuously for up to 64 h showed an increase in the signals owing to an increasing number of cells. Cell division in the BMG is easily observed by microscope or by the human eye as the gel opacifies. Spectra of cells in the BMG threads at 20% confluency show a more rapid signal increase than at 60% confluency. Cells grown in vivo in nude mice show a spectrum markedly similar to in vitro spectra in BMG threads, whereas the same cells in agarose threads lack peaks owing to Pi, glycerophosphocholine, and glycerophosphoethanolamine. With the high resolution obtained from this system we distinguished intracellular from extracellular Pi in vitro, and found that the intracellular pH is equal to that observed in the same cell line in vivo. This cell-BMG system is in effect a model tumor, but it is composed of a homogeneous cell population that can be observed indefinitely as the cells reproduce. The material needed is inexpensive, the technique is simple and efficient, and no adaptation of the spectrometer is required. This model will be useful for studying intracellular metabolism and the interaction of cells with the basement membrane.     

Differential elution of histones from gel-trapped nuclei

A method has been developed for characterizing at, submicrogram levels the heterogeneity of histones from purified nuclei. The histones are eluted with a smooth concentration gradient from nuclei trapped in polyacrylamide–gel threads and are collected in a micro fraction collector suitable for volumes in the 10–100 μl range. The gradient and fraction collection systems are governed by cam driven syringes. Samples obtained are subjected to electrophoresis in a starch-gel system and the gels are stained with a highly sensitive stain specific for guanidinium groups. Seven major and a similar number of minor components are demonstrated in the histones. The method of differential elution of trapped macromolecules is suitable for use with systems other than nuclei and histones.     

Mutations in Rhizobium phaseoli that lead to arrested development of infection threads.

Two Rhizobium phaseoli mutants, isolated previously by Tn5 mutagenesis, elicited infection threads which ceased development prematurely, usually within root hairs. These infection threads were wide, globular, and otherwise altered in morphology, compared with normal infection threads. Anatomy and division of the root cortical cells during initial stages of nodule morphogenesis appeared normal. However, later nodule differentiation deviated considerably from normal development, and release of bacteria from infection threads was not observed. In tryptone-yeast extract medium the mutants sedimented during growth in shaken cultures and formed rough colonies on agar. Electrophoresis of washed cultures solubilized in dodecyl sulfate revealed that the major carbohydrate band was absent from the mutants. The behavior of this carbohydrate in phenol-water extraction and gel chromatography, its apparent ketodeoxyoctonate content, and its susceptibility to mild acid hydrolysis suggested that it was a lipopolysaccharide. From the results of genetic crosses or reversion analysis, the defect in synthesizing this carbohydrate material and the defect in infection could be attributed to a single mutation in each mutant.     

Byssal proteins of the freshwater zebra mussel,Dreissena polymorpha

The freshwater zebra mussel (Dreissena polymorpha) is a notorious biofouling organism. It adheres to a variety of substrata underwater by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. The byssal proteins are difficult to characterize due to extensive cross-linking of 3,4-dihydroxyphenylalanine (DOPA), which renders the mature structure largely resistant to protein extraction and immunolocalization. By inducing secretion of fresh threads and plaques in which cross-linking is minimized, three novel zebra mussel byssal proteins were identified following extraction and separation by gel electrophoresis. Peptide fragment fingerprinting was used to match tryptic digests of several gel bands against a cDNA library of genes expressed uniquely in the mussel foot, the organ which secretes the byssus. This allowed identification of a more complete sequence of Dpfp2 (D. polymorpha foot protein 2), a known DOPA-containing byssal protein, and a partial sequence of Dpfp5, a novel protein with several typical characteristics of mussel adhesive proteins.     

Doppelbrechung und Orientierungsrate der Leptonen fließenden Protoplasmas

Summary At defined speeds threads of protoplasm have been drawn out mechanically from a fusion of isolated gymnoplasts using an experimental arrangement described by Gerendás [3]. The polarization device, as well as the method of measuring the birefringence by means of a variable azimuth compensator of fixed retardation, viz. a rotating /30-mica plate, is the same as described formerly [7g-i. Then in dependence on the variable speeds of formation the protoplasmic threads show a different intensity of negative birefringence, and, what is more, it is found a distinct parallelism between birefringence, as an expression of the orientation rate of the leptones, and the speed of spinning. Always when a birefringent gel like protoplasm is put under tension, the intensity of its original birefringence or its orientation rate increases (and in the reverse order). It is to conclude that in order to analyse the leptonic structures in dependence on the effective forces, there is a great need for a quantitative approach to the anisotropy of protoplasmic constituents.     

Developmental regulation of nodule-specific genes in alfalfa root nodules

We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins. Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa. One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa. The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel. Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.     

Capture thread extensibility of orb-weaving spiders: testing punctuated and associative explanations of character evolution

Spider orb-webs contain sticky prey capture threads and non-sticky support threads. Primitive orb-weavers of the Deinopoidea produce dry cribellar threads made of thousands of silk fibrils that surround supporting axial fibres, whereas the viscous threads of modern Araneoidea orb-weavers produce adhesive threads with an aqueous solution that coalesces as droplets around the axial fibres. We have previously shown that the greater diversity of the Araneoidea is phylogenetically significant and attributed this disparity to a number of advantages, considered key innovations, that adhesive thread has over cribellar thread. An important putative advantage of adhesive thread demonstrated by Kohler and Vollrath in their 1995 study is its greater extensibility, a feature that better adapts it to absorb the kinetic energy of a prey strike. However, this conclusion is based on a two-species comparison that does not take advantage of the modern comparative method that requires hypotheses to be tested in a phylogenetic context. Using a transformational analysis to examine threads produced by nine species, our study finds no support for the punctuated explanation that adhesive thread has a greater extensibility than cribellar thread. Instead, it strongly supports the associative null hypothesis that capture thread extensibility is tuned to spider mass and to architectural features of the web, including its capture area, capture spiral spacing, and capture area per radius.     

Tension generation by threads of contractile proteins

Threads of contractile proteins were formed via extrusion and their isometric tensions and isotonic contraction velocities were measured. We obtained reproducible data by using a new and sensitive tensiometer. The force-velocity curves of actomyosin threads were similar to those of muscle, with isometric tensions of the order of 10g/cm2 and maximum contraction velocites of the order of 10(-2) lengths/s. The data could be fitted by Hill's equation. Addition of tropomyosin and troponin to the threads increased isometric tension and maximum contraction velocity. Threads which contained troponin and tropomyosin required Ca++ for contraction and the dependence of their isometric tension on the level of free Ca++ was like that of muscle. The dependence of tension or of contraction velocity upon temperature or upon ionic strength is similar for actomyosin threads and muscle fibers. In contrast, the dependence of most parameters which are characteristic of the actomyosin interaction in solution (or suspension) upon these variables is not similar to the dependence of the muscle fiber parameters. The conclusion we have drawn from these results is that the mechanism of tension generation in the threads is similar to the mechanism that exists in muscle. Because the protein composition of the thread system can be manipulated readily and because the tensions and velocities of the threads can be related directly to the physiological parameters of muscle fibers, the threads provide a powerful method for studying contractile proteins.     

Spatial distribution of proteins in the quagga mussel adhesive apparatus

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious ‘anchor’ (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.     

Metabolic studies of mammalian cells by 31P-NMR using a continuous perfusion technique

Levels of ATP and Pi in metabolically active Chinese hamster lung fibroblasts were monitored noninvasively by 31P-NMR over many hours and under a variety of conditions. The cells were embedded in a matrix of agarose gel in the form of fine threads which were continuously perfused in a standard NMR tube. The small diameter of the thread allows rapid diffusion of metabolites and drugs into the cells. The changes in ATP and Pi levels were followed as a function of time in response to perfusion with a glucose-containing medium, with isotonic saline and with a medium containing 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. This gel-thread perfusion method should enable routine NMR studies of cellular metabolism, and may have other potential biological applications.     

Incorporation of macromolecules in microparticles: preparation and characteristics.

Macromolecules can easily be incorporated in microparticles of polyacrylamide by copolymerization with acrylamide in a water-in-oil emulsion. The microparticles preferably arount 1-3 mum in diameter, will have a macroporous structure formed by the polymeric network. The amount of incorporation of the macromolecules will depend on the structure of the network, which, in turn, will depend on the total amount of monomer (T) and the relative amount of cross-linking agent (C) in the monomeric solution. Two mechanisms are responsible for the incorporation; all macromolecules are, independently of the size, fixed in the threads of polyacrylamide and large ones are entrapped within the network formed by the threads. The amount entrapped will depend on the size of the macromolecule and the mean pore radius of the gel. In microparticles with a total concentration of monomers of 8% and a cross-linking of 25% (T-C=8-25) the biological properties of incorporated macromolecules are retained, due to the macroporous structure, as found in binding studies with albumin. The density of the particles will also depend on C and T and, to some extent, on the protein concentration. Due to the fixation in the polyacrylamide threads, some of the incorporated macromolecules will be exposed on the surface, allowing them to react with, for instance, cells, which cannot penetrate the particles. The optimal conditions for the incorporation of macromolecules in the microparticles are investigated.     

Prolonged antibacterial action of synthetic suture materials containing gentamicin]

A new sutural material in the form of polyacrylamide threads containing gentamicin in an amount of 10 per cent by the thread weight was studied. After storage for a year under refrigerator conditions the threads preserved their antibacterial activity when studied in vitro. Since gentamicin was included within the threads by means of the ionic links its release from the threads to the environment either in vitro or in vivo was gradual and uniform within at least 10 days. The quantity of the released antibiotic depended on its content per unit of thread length. The marked antibacterial effect within the prolonged period is a basis for the use of the threads in surgical practice for the prevention of infections in the early postoperative period.     

Adhesive efficiency of spider prey capture threads

Cribellar capture threads are comprised of thousands of fine silk fibrils that are produced by the spigots of a spider's cribellum spinning plate and are supported by larger interior axial fibers. This study examined factors that constrain the stickiness of cribellar threads spun by members of the orb-weaving family Uloboridae in the Deinopoidea clade and compared the material efficiency of these threads with that of viscous capture threads produced by members of their sister clade, the Araneoidea. An independent contrast analysis confirmed the direct relationship between cribellar spigot number and cribellar thread stickiness. A model based on this relationship showed that cribellar thread stickiness is achieved at a rapidly decreasing material efficiency, as measured in terms of stickiness per spigot. Another limitation of cribellar thread was documented when the threads of two uloborid species were measured with contact plates of four widths. Unlike that of viscous threads, the stickiness of cribellar threads did not increase as plate width increased, indicating that only narrow bands along the edges of thread contact contributed to their stickiness. As thread volume increased, the gross material efficiency of cribellar threads decreased much more rapidly than that of viscous threads. However, cribellar threads achieved their stickiness at a much greater gross material efficiency than did viscous threads, making it more challenging to explain the transition from deinopoid to araneoid orb-webs.     

Adhesive compatibility of cribellar and viscous prey capture threads and its implication for the evolution of orb-weaving spiders

Evolution of orb-weaving spiders that comprise the Orbiculariae clade involved a transition in the composition of prey capture thread that has been challenging to explain. The primitive cribellar threads spun by members of the Deinopoidea subclade resemble the capture threads of their non-orb-web-weaving ancestors and are formed of thousands of fine, dry, protein cribellar fibrils. In contrast, the derived viscous capture threads spun by members of the Araneoidea subclade have regularly spaced, aqueous adhesive droplets. When second instar deinopoid spiderlings emerge from egg sacs they are unable to spin cribellar threads, and, therefore, do not construct orb-webs; whereas second instar araneoids spin capture threads and construct orb-webs. If, as we hypothesize, viscous material evolved to enable second instar spiderlings to construct orb-webs, early araneoids may have spun composite cribellar-viscous capture threads. To examine the functional feasibility of such intermediate capture threads, we compared the adhesion of cribellar threads, viscous threads, and combined cribellar-viscous threads. The stickiness of these combined threads was greater than that of native cribellar or viscous threads alone. The viscous material of Araneus marmoreus threads exhibited a substantial increase in stickiness when combined with cribellar fibrils and that of Argiope aurantia threads a small increase in stickiness when combined with cribellar fibrils. Thus, if early araneoids retained their ability to spin cribellar threads after having evolved glands that produced viscous material, their composite threads could have formed a functional adhesive system that achieved its stickiness at no loss of material economy.     

The silk road of <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: is it a single or a double lane?

Tetranychus urticae (Acari: Tetranychidae) is a phytophagous mite that forms huge colonies. All active members of a colony (immatures and matures, females and males) spin silken threads. These mites construct a common web that protects the colony from external aggression. The silk coverage is well-known to provide advantages to the colony but very little is known about the characteristics of the threads themselves. Here is the first quantification of the diameter of silken threads spun by two different stages (adult females and larvae) and its relationship with body size of the spinning individuals. Moreover, we observed how silk was deposited on the substrate through their two pedipalps. Threads were observed by means of transmission electron and fluorescence microscopy. Silken threads spun by larvae (0.055 ± 0.018 μm) were significantly thinner than threads spun by adult females (0.111 ± 0.038 μm). In the first step of the silk depositing behaviour, the mite attached the thread to the substrate by putting its pedipalps in contact with the surface (adhesion, double silken threads). When walking, silken threads became detached from the substrate and spitted up (silken threads were free). Finally, silken threads adhered to the surface. The presence of single and double threads makes thread diameter highly variable.     

Persistent stickiness of viscous capture threads produced by araneoid orb-weaving spiders

The most commonly encountered spider orb-webs rely on sticky, viscous capture threads to retain prey. These threads are composed of supporting fibers covered by a complex aqueous solution that forms a series of droplets, each with a glycoprotein granule that confers adhesion. This adhesive system normally functions for less than a day before being replaced. Despite their ephemeral nature, we found that the stickiness of viscous threads persists for much longer. When measured over the course of 7 days, small decreases in the adhesion of Larinioides cornutus threads were not statistically significant. Threads of Araneus marmoreus, Argiope trifasciata, and Metepeira labyrinthea were aged for 8-10 months and remeasured under environmental conditions similar to those under which initial measurements were made. When returned to humidity similar to that under which measurements were initially made, neither the droplet volumes nor the stickiness of aged threads differed significantly from those of newly spun threads. These observations indicate that when viscous threads are protected from contamination, the compounds responsible for their hydrophilic and adhesive properties do not degrade easily.     

Persistence of DNA threads in human anaphase cells suggests late completion of sister chromatid decatenation

PICH (Plk1-interacting checkpoint helicase) was recently identified as an essential component of the spindle assembly checkpoint and shown to localize to kinetochores, inner centromeres, and thin threads connecting separating chromosomes even during anaphase. In this paper, we have used immuno-fiber fluorescence in situ hybridization and chromatin-immunoprecipitation to demonstrate that PICH associates with centromeric chromatin during anaphase. Furthermore, by careful analysis of PICH-positive anaphase threads through FISH as well as bromo-deoxyurdine and CREST labeling, we strengthen the evidence that these threads comprise mainly alphoid centromere deoxyribonucleic acid. Finally, by timing the addition of ICRF-193 (a specific inhibitor of topoisomerase-II alpha) to cells synchronized in anaphase, we demonstrate that topoisomerase activity is required specifically to resolve PICH-positive threads during anaphase (as opposed to being required to prevent the formation of such threads during earlier cell cycle stages). These data indicate that PICH associates with centromeres during anaphase and that most PICH-positive threads evolve from inner centromeres as these stretch in response to tension. Moreover, they show that topoisomerase activity is required during anaphase for the resolution of PICH-positive threads, implying that the complete separation of sister chromatids occurs later than previously assumed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Wang and Schwarzbraun both contributed equally to this study.     

Elastic properties and beta-sheet structure of connectin threads

Connectin is a very long and flexible protein of striated muscle, linking myosin filaments to z discs in a sarcomere. Isolated native connectin in solution frequently forms elastic threads upon concentration of the solution, by side-by-side association of molecules. An X-ray diffraction study was performed to examine the presence of beta-sheet structure in artificially prepared threads. The elastic properties of such threads were measured at various temperatures. Negative temperature dependence of the elastic coefficient suggests that the elasticity of connectin threads is due to deformation of the three-dimensional structure and not to rubber-like behavior.     

Mapping chemical gradients within and along a fibrous structural tissue, mussel byssal threads

The byssal thread of a mussel is an extraorganismic connective tissue that exhibits a striking end-to-end gradient in mechanical properties and thus provides a unique opportunity for studying how gradients are made. Mfp-1 (Mytilus foot protein-1) is a conspicuous component of the protective outer cuticle of byssal threads given its high 3,4-dihydroxyphenylalanine (Dopa) content at 10-15 mol %. Amino acid analysis of mfp-1 extracted from successive foot sections of Mytilus galloprovincialis reveals a post-translationally mediated gradient with highest Dopa levels present in mfp-1 from the accessory gland near the tip of the foot decreasing gradually toward the base. The Dopa content of successive segments of byssal threads decreases from the distal to the proximal end and thus reflects the trend of mfp-1 in the foot. Inductively coupled plasma analysis indicates that certain metal ions including iron follow the trend in Dopa along the thread. Energy-dispersive x-ray spectrometry showed that iron, when present, was concentrated in the cuticle of the threads but sparse in the core. The axial iron gradient appears most closely correlated with the Dopa gradient. The direct incubation of mussels and byssal threads in Fe(3+) supplemented seawater showed that byssal threads are unable to sequester iron from the seawater. Instead, particulate/soluble iron is actively taken up by mussels during filter feeding and incorporated into byssal threads during their secretion. Our results suggest that mussels may exploit the interplay between Dopa and metals to tailor the different parts of threads for specific mechanical properties.