Supercontraction forces in spider dragline silk depend on hydration rate

Spider dragline silk is a model biological polymer for biomimetic research due to its many desirable and unusual properties. ‘Supercontraction’ describes the dramatic shrinking of dragline silk fibers when wetted. In restrained silk fibers, supercontraction generates substantial stresses of 40–50 MPa above a critical humidity of 70% relative humidity (RH). This stress may maintain tension in webs under the weight of rain or dew and could be used in industry for robotics, sensor technology, and other applications. Our own findings indicate that supercontraction can generate stress over a much broader range than previously reported, from 10 to 140 MPa. Here we show that this variation in supercontraction stress depends upon the rate at which the environment reaches the critical level of humidity causing supercontraction. Slow humidity increase, over several minutes, leads to relatively low supercontraction stress, while fast humidity increase, over a few seconds, typically results in higher supercontraction stress. Slowly supercontracted fibers take up less water and differ in thermostability from rapidly supercontracted fibers, as shown by thermogravimetric analysis. This suggests that spider silk achieves different molecular configurations depending upon the speed at which supercontraction occurs. Ultimately, rate-dependent supercontraction may provide a mechanism to tailor the properties of silk or biomimetic fibers for various applications.     

Axial and radial filamentous components of the neurofilamentous network

Summary The three-dimensional structure of the neurofilamentous network of the giant axon of the squid has been investigated by stereo electron microscopy and optical image analysis. The neurofilamentous network with its structural associations intact was partially purified by means of extraction of extruded axoplasm in a physiological buffer. The authors employed a double-grid mounting technique for critical point drying of axoplasm which provides high contrast preparations having great depth suitable for the analysis of spatial relations. There is a striking improvement in the perception of the continuity and three-dimensionality of the network, particularly in thin areas produced by teasing apart the specimen prior to mounting. Sidearms and cross-bridges are readily identifiable.In 1-m thick preparations of the extracted axoplasm the neurofilamentous network is composed of two structural entities: (i) long 10-nm wide filaments approximately parallel with the long axis of the extracted axoplasmic cylinder (axial), (ii) and short, finer filaments projecting from them as sidearms or crossbridges (radial). Optical analysis of micrographs of extracted axoplasm indicates that the radial filamentous components of the neurofilamentous network are distributed predominantly in the range of 32° to 67° with respect to the long axis of the axial filaments. We tentatively assign the 220,000 mol wt peptide observed by SDS-PAGE in this preparation to the radial filaments and the 68,000 mol wt peptide to the axial filaments.     

The influenza virus-induced fusion of erythrocyte ghosts does not depend on osmotic forces

The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.     

The effect of lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers. I. Proportionality between the lattice spacing and the fiber width.

Chemically skinned rabbit psoas muscle fibers/bundles were osmotically compressed with a macromolecule dextran T-500 (0-16%, g/100 ml) at 20 degrees C, 200 mM ionic strength, and pH 7.0. The lattice spacing of psoas bundles was measured by equatorial x-ray diffraction studies during relaxation and after rigor induction, and the results were compared with the fiber width measurements by optical microscopy. The purpose of the present study is to determine whether fiber width is a reliable measure of the lattice spacing, and to determine the available spacing for myosin cross-bridges between the thick and thin filaments. We observed that both the lattice spacing and the fiber width decreased with an increase in the dextran concentration during relaxation or after rigor induction, and that the spacing and the fiber width were proportionately related. We further observed that, in the absence of dextran, the lattice spacing (and the fiber width) shrank on a relax-to-rigor transition, whereas in the presence of 16% dextran, the spacing expanded on a relax-to-rigor transition. The cross-over of these plots occurred at the 4-7% dextran concentration. During Ca activation, the fiber width shrank in the absence of dextran, and it slightly expanded in the presence of 14.4% dextran. The degree of expansion was not as large as in the relax-to-rigor transition, and the cross-over occurred at about 11% dextran concentration. We also carried out experiments with dextran T-40 and T-10 to determine the upper limit of the molecular weight that enters the lattice space. We found that the upper limit is about 20 kD.     

1, 0 lattice spacing of skeletal muscle during postnatal growth in mice

1, 0 lattice spacing of extensor digitorum longus muscles of mice (ICR) during growth (2-12 weeks) were measured in situ by X-ray diffraction method. 1, 0 lattice spacing of EDL was 35.4 +/- 0.27 (mean +/- SD, N = 33) nm. No significant difference between spacings in EDL's of each aged mice was observed.     

The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke

Mucus propelling cilia are excitable by many stimulants, and have been shown to increase their beating frequency up to threefold, by physiological extracellular stimulants, such as adenosine-triphosphate, acetylcholine, and others. This is thought to represent the evolutionary adaptation of mucociliary systems to the need of rapid and efficient cleansing the airways of foreign particles. However, the mucus transport velocity depends not only on the beat frequency of the cilia, but on their beat pattern as well, especially in the case of mucus bearing cilia that beat in a complex, three-dimensional fashion. In this study, we directly measured the force applied by live ciliary tissues with an atomic force microscope, and found that it increases linearly with the beating frequency. This implies that the arc swept by the cilia during their effective stroke remains unchanged during frequency increase, thus leading to a linear dependence of transport velocity on the beat frequency. Combining the atomic force microscope measurements with optical measurements, we have indications that the recovery stroke is performed on a less inclined plane, leading to an effective shortening of the overall path traveled by the cilia tip during this nontransporting phase of their beat pattern. This effect is observed to be independent of the type of stimulant (temperature or chemical), chemical (adenosine-triphosphate or acetylcholine), or concentration (1 μM-100 μM), indicating that this behavior may result from internal details of the cilium mechanical structure.     

Cell locomotion forces versus cell contraction forces for collagen lattice contraction: an in vitro model of wound contraction

Cultured human dermal fibroblasts suspended in a rapidly polymerizing collagen matrix produce a fibroblast-populated collagen lattice. With time, this lattice will undergo a reduction in size referred to as lattice contraction. During this process, two distinct cell populations develop. At the periphery of the lattice, highly oriented sheets of cells, morphologically identifiable as myofibroblasts, show cell-to-cell contacts and thick, actin-rich staining cytoplasmic stress fibers. It is proposed that these cells undergoing cell contraction produce a multicellular contractile unit which reorients the collagen fibrils associated with them. The cells in the central region, referred to as fibroblasts, are randomly oriented, with few cell-to-cell contacts and faintly staining actin cytoplasmic filaments. In contrast it is proposed that cells working as single units use cell locomotion forces to reorient the collagen fibrils associated with them. Using this model, we sought to determine which of these two mechanisms, cell contraction or cell locomotion, is responsible for the force that contracts collagen lattices. Our experiments showed that fibroblasts produce this contractile force, and that the mechanism for lattice contraction appears to be related to cell locomotion. This is in contrast to a myofibroblast; where the mechanism for contraction is based upon cell contractions. Fibroblasts attempting to move within the collagen matrix reorganize the surrounding collagen fibrils; when these collagen fibrils can be organized no further and cell-to-cell contacts develop, which occurs at the periphery of the lattice first, these cells can no longer participate in the dynamic aspects of lattice contraction.     

The effect of the lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers. II. Elementary steps affected by the spacing change.

The actin-myosin lattice spacing of rabbit psoas fibers was osmotically compressed with a dextran T-500, and its effect on the elementary steps of the cross-bridge cycle was investigated. Experiments were performed at the saturating Ca (pCa 4.5-4.9), 200 mM ionic strength, pH 7.0, and at 20 degrees C, and the results were analyzed by the following cross-bridge scheme: [formula: see text] where A = actin, M = myosin head, S = MgATP, D = MgADP, and P = Pi = phosphate. From MgATP and MgADP studies on exponential process (C) and (D), the association constants of cross-bridges to MgADP (K0), MgATP (K1a), the rate constants of the isomerization of the AM S state (k1b and k-1b), and the rate constants of the cross-bridge detachment step (k2 and k-2) were deduced. From Pi study on process (B), the rate constants of the cross-bridge attachment (power stroke) step (k4- and k-4) and the association constant of Pi ions to cross-bridges (K5) were deduced. From ATP hydrolysis measurement, the rate constant of ADP-isomerization (rate-limiting) step (k6) was deduced. These kinetic constants were studied as functions of dextran concentrations. Our results show that nucleotide binding, the ATP-isomerization, and the cross-bridge detachment steps are minimally affected by the compression. The rate constant of the reverse power stroke step (k-4) decreases with mild compression (0-6.3% dextran), presumably because of the stabilization of the attached cross-bridges in the AM*DP state. The rate constant of the power stroke step (k4) does not change with mild compression, but it decreases with higher compression (> 6.3% dextran), presumably because of an increased difficulty in performing the power stroke. These results are consistent with the observation that isometric tension increases with a low level of compression and decreases with a high level of compression. Our results also show that the association constant K5 of Pi with cross-bridge state AM*D is not changed with compression. Our result further show that the ATP hydrolysis rate decreased with compression, and that the rate constants of the ADP-isomerization step (k6) becomes progressively less with compression. The effect of compression on the power stroke step and rate-limiting step implies that a large-scale molecular rearrangement in the myosin head takes place in these two slow reaction steps.     

The relation between Z--disk lattice spacing and sarcomere length in sartorius muscle fibres from Hyla cerulea.

Sartorius muscles from the green tree frog Hyla cerulea were set at variety of muscle lengths and fixed for electron microscopy using acrolein followed by osmium tetroxide. The sarcomere length, s, was determined in thick sections using laser-diffraction. The Z-disk lattice spacing, z, was measured in electron micrographs of thin sections from the same muscles. The Z-disk lattice was found to expand as sarcomere length decreased such that the quantity sz2 was constant at 1-05 X 10(6) nm3 for all sarcomere lengths in the range 1-9-2-9 mum. Thus, the sarcomere length dependency of the Z-disk lattice is similar to that of the myosin filament lattice. The density of thin filaments per unit cross section of fibril leaving the Z-disk is less than their density in the A band. Thus, fibrils have a smaller cross section in the I band, leaving more inter-fibrillar space there. This may explain why more mitochondria and lipid droplets are located in the I bands than in the A bands. It is suggested that the Z-disk may contributed to the short range elasticity of muscle fibres.     

Mesoscopic analysis of motion and conformation of cross-bridges

The orientation of a cross-bridge is widely used as a parameter in determining the state of muscle. The conventional measurements of orientation, such as that made by wide-field fluorescence microscopy, electron paramagnetic resonance (EPR) or X-ray diffraction or scattering, report the average orientation of 10¹²–10⁹ myosin cross-bridges. Under conditions where all the cross-bridges are immobile and assume the same orientation, for example in normal skeletal muscle in rigor, it is possible to determine the average orientation from such global measurements. But in actively contracting muscle, where a parameter indicating orientation fluctuates in time, the measurements of the average value provide no information about cross-bridge kinetics. To avoid problems associated with averaging information from trillions of cross-bridges, it is necessary to decrease the number of observed cross-bridges to a mesoscopic value (i.e. the value affected by fluctuations around the average). In such mesoscopic regimes, the averaging of the signal is minimal and dynamic behavior can be examined in great detail. Examples of mesoscopic analysis on skeletal and cardiac muscle are provided.     

Consequences of the number and spacing of pregnancies on outcome,and of pregnancy outcome on spacing

Abstract

This study is based on 3,098 once‐married women in Abderdeen, Scotland, who had a total of 10,825 pregnancies, which resulted in wastages of 285 infant deaths, 173 stillbirths, 712 involuntary abortions, and 200 voluntary terminations. Wastage varies by pregnancy number, particularly after the third pregnancy. There is, however, a selective factor operating here in that women who have a wastage are more likely to continue on to the next higher pregnancy number, and those who have a wastage at one pregnancy number are more likely to have a wastage at the next pregnancy outcome also. Wastage tends to be cumulative. Women who enter the reproductive cycle at the younger ages have a larger number of pregnancies and a higher wastage rate than women who postpone their first pregnancy until the older ages. Women who experience a wastage at any given pregnancy number are not only more likely to have another pregnancy, but they do so over a shorter time interval than those whose last pregnancy resulted in a live birth. Except for terminations, wastage is highest among women who closely space their pregnancy     

Filament lattice of frog striated muscle. Radial forces, lattice stability, and filament compression in the A-band of relaxed and rigor muscle.

Repulsive pressure in the A-band filament lattice of relaxed frog skeletal muscle has been measured as a function of interfilament spacing using an osmotic shrinking technique. Much improved chemical skinning was obtained when the muscles were equilibrated in the presence of EGTA before skinning. The lattice shrank with increasing external osmotic pressure. At any specific pressure, the lattice spacing in relaxed muscle was smaller than that of muscle in rigor, except at low pressures where the reverse was found. The lattice spacing was the same in the two states at a spacing close to that found in vivo. The data were consistent with an electrostatic repulsion over most of the pressure range. For relaxed muscle, the data lay close to electrostatic pressure curves for a thick filament charge diameter of approximately 26 nm, suggesting that charges stabilizing the lattice are situated about midway along the thick filament projections (HMM-S1). At low pressures, observed spacings were larger than calculated, consistent with the idea that thick filament projections move away from the filament backbone. Under all conditions studied, relaxed and rigor, at short and very long sarcomere lengths, the filament lattice could be modeled by assuming a repulsive electrostatic pressure, a weak attractive pressure, and a radial stiffness of the thick filaments (projections) that differed between relaxed and rigor conditions. Each thick filament projection could be compressed by approximately 5 or 2.6 nm requiring a force of 1.3 or 80 pN for relaxed and rigor conditions respectively.     

Response of equatorial x-ray reflections and stiffness to altered sarcomere length and myofilament lattice spacing in relaxed skinned cardiac muscle

Low angle x-ray diffraction measurements of myofilament lattice spacing (D(1,0)) and equatorial reflection intensity ratio (I(1,1)/I(1,0)) were made in relaxed skinned cardiac trabeculae from rats. We tested the hypothesis that the degree of weak cross-bridge (Xbr) binding, which has been shown to be obligatory for force generation in skeletal muscle, is modulated by changes in lattice spacing in skinned cardiac muscle. Altered weak Xbr binding was detected both by changes in I(1,1)/I(1,0) and by measurements of chord stiffness (chord K). Both measurements showed that, similar to skeletal muscle, the probability of weak Xbr binding at 170-mM ionic strength was significantly enhanced by lowering temperature to 5 degrees C. The effects of lattice spacing on weak Xbr binding were therefore determined under these conditions. Changes in D(1,0), I(1,1)/I(1,0), and chord K by osmotic compression with dextran T500 were determined at sarcomere lengths (SL) of 2.0 and 2.35 micro m. At each SL increasing [dextran] caused D(1,0) to decrease and both I(1,1)/I(1,0) and chord K to increase, indicating increased weak Xbr binding. The results suggest that in intact cardiac muscle increasing SL and decreasing lattice spacing could lead to increased force by increasing the probability of initial weak Xbr binding.     

Stretch activation and nonlinear elasticity of muscle cross-bridges.

When active insect fibrillar flight muscle is stretched, its ATPase rate increases and it develops "negative viscosity," which allows it to perform oscillatory work. We use a six-state model for the cross-bridge cycle to show that such "stretch activation" may arise naturally as a nonlinear property of a cross-bridge interacting with a single attachment site on a thin filament. Attachment is treated as a thermally activated process in which elastic energy must be supplied to stretch or compress the cross-bridge spring. We find that stretch activation occurs at filament displacements where, before the power stroke, the spring is initially in compression rather than in tension. In that case, pulling the filaments relieves the initial compression and reduces the elastic energy required for attachment. The result is that the attachment rate is enhanced by stretching. The model also displays the "delayed tension" effect observed in length-step experiments. When the muscle is stretched suddenly, the power stroke responds very quickly, but there is a time lag before dissociation at the end of the cycle catches up with the increased attachment rate. This lag is responsible for the delayed tension and hence also for the negative viscosity.     

Orientational disorder and motion of weakly attached cross-bridges.

In a relaxed muscle fiber at low ionic strength, the cross-bridges may well be in states comparable to the one that precedes the cross-bridge power stroke (Schoenberg, M. 1988. Adv. Exp. Med. Biol. 226:189-202). Using electron paramagnetic resonance (EPR) and (saturation transfer) electron paramagnetic resonance (ST-EPR) techniques on fibers labeled with maleimide spin label, under low ionic strength conditions designed to produce a majority of weakly-attached heads, we have established that (a) relaxed labeled fibers show a speed dependence of chord stiffness identical to that of unlabeled, relaxed fibers, suggesting similar rapid dissociation and reassociation of cross-bridges; (b) the attached relaxed heads at low ionic strength are nearly as disordered as in relaxation at physiological ionic strength where most of the heads are detached from actin; and (c) the microsecond rotational mobility of the relaxed heads was only slightly restricted compared to normal ionic strength, implying great motional freedom despite attachment. The differences in head mobility between low and normal ionic strength scale with filament overlap and are thus due to acto-myosin interactions. The spectra can be modeled in terms of two populations: one identical to relaxed heads at normal ionic strength (83%), the other representing a more oriented population of heads (17%). The spectrum of the latter is centered at approximately the same angle as the spectrum in rigor but exhibits larger (40 degrees) axial probe disorder with respect to the fiber axis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Geometrical factors influencing muscle force development. I. The effect of filament spacing upon axial forces.

The influence of geometry on the force and stiffness measured during muscle contraction at different sarcomere lengths is examined by using three specific models of muscle cross-bridge geometry which are based upon the double-hinge model of H. E. Huxley (Science [Wash. D.C.]. 1969, 164:1356-1366) extended to three dimensions. The force generated during muscle contraction depends upon the orientation of the individual cross-bridge force vectors and the distribution of the cross-bridges between various states. For the simplest models, in which filament separation has no effect upon cross-bridge distribution, it is shown that changes in force vectors accompanying changes in myofilament separation between sarcomere lengths 2.0 and 3.65 microgram in an intact frog skeletal muscle fiber have only a small effect upon axial force. The simplest models, therefore, produce a total axial force proportional to the overlap between the actin and myosin filaments and independent of filament separation. However, the analysis shows that it is possible to find assumptions that produce a cross-bridge model in which the axial force is not independent of filament spacing. It is also shown that for some modes of attachment of subfragment-1 (S1) to actin the azimuthal location of the actin site is important in determining the axial force. A mode of S1 attachment to actin similar to that deduced by Moore et al. (J. Mol. Biol., 1970, 50:279-294), however, exhibits rather constant cross-bridge behavior over a wide range of actin site location.     

Lateral forces in the filament lattice of vertebrate striated muscle in the rigor state.

The repulsive pressure between filaments in the lattice of skinned rabbit and frog striated muscle in rigor has been measured as a function of interfilament spacing, using the osmotic pressure generated by solutions of large, uncharged polymeric molecules (dextran and polyvinylpyrrolidone). The pressure/spacing measurements have been compared with theoretically derived curves for electrostatic pressure. In both muscles, the major part of the experimental curves (100-2,000 torr) lies in the same region as the electrostatic pressure curves, providing that a thick filament charge diameter of approximately 30 nm in rabbit and approximately 26 nm in frog is assumed. In chemically skinned or glycerol-extracted rabbit muscle the fit is good; in chemically skinned frog sartorius and semitendinosus muscle the fit is poor, particularly at lower pressures where a greater spacing is observed than expected on theoretical grounds. The charge diameter is much larger than the generally accepted value for thick filament backbone diameter. This may be because electron microscope results have underestimated the amount of filament shrinkage during sample preparation, or because most of the filament charge is located at some distance from the backbone surface, e.g., on HMM-S2. Decreasing the ionic strength of the external solution, changing the pH, and varying the sarcomere length all give pressure/spacing changes similar to those expected from electrostatic pressure calculations. We conclude that over most of the external pressure range studied, repulsive pressure in the lattice is predominantly electrostatic.     

Cooperative effects of rigor and cycling cross-bridges on calcium binding to troponin C

The effects of rigor and cycling cross-bridges on distributions of calcium (Ca) bound within sarcomeres of rabbit psoas muscle fibers were compared using electron probe x-ray microanalysis. Calcium in the overlap region of rigor fibers, after correction for that bound to thick filaments, was significantly higher than in the I-band at all pCa levels tested between 6.9 and 4.8, but the difference was greatest at pCa 6.9. With addition of MgATP, differences were significant at high levels of activation (pCa 5.6 and 4.9); near and below the threshold for activation, Ca was the same in I-band and overlap regions. Comparison of Ca and mass profiles at the A-I junction showed elevation of Ca extending 55-110 nm (up to three regulatory units) into the I-band. Extraction of TnC-reduced I-band and overlap Ca in rigor fibers at pCa 5.6 to the same levels found in unextracted fibers at pCa 8.9, suggesting that variations reported here reflect changes in Ca bound to troponin C (TnC). Taken together, these observations provide evidence for near-neighbor cooperative effects of both rigor and cycling cross-bridges on Ca(2+) binding to TnC.