[X-ray interferometry of the axial movement of myosin heads during muscle force generation initiated by T-jump].

The interference fine structure of the M3 reflection on the low-angle x-ray diffraction patterns of muscle fibres is used for the measurements of axial movements of myosin heads with a precision of 0.1-0.2 nm. We have measured changes in the M3 interference profile during tension rise induced by a 5 to 30 degrees C temperature jump in thin bundles of contracting fibers from rabbit skeletal muscle. Interpreting the data with a point diffractor model gives an estimate for the axial movement of the myosin heads during force rise of less than 0.6 nm. Modifications of the point diffractor model are discussed. We show that our experimental data can be explained by a model where myosin heads bind actin in a number of structurally different states.

Structural changes in myosin motors and filaments during relaxation of skeletal muscle.

Structural changes in myosin motors and filaments during relaxation from short tetanic contractions of intact single fibres of frog tibialis anterior muscles at sarcomere length 2.14 mum, 4 degrees C were investigated by X-ray diffraction. Force declined at a steady rate for several hundred milliseconds after the last stimulus, while sarcomere lengths remained almost constant. During this isometric phase of relaxation the intensities of the equatorial and meridional M3 X-ray reflections associated with the radial and axial distributions of myosin motors also recovered at a steady rate towards their resting values, consistent with progressive net detachment of myosin motors from actin filaments. Stiffness measurements confirmed that the fraction of motors attached to actin declined at a constant rate, but also revealed a progressive increase in force per motor. The interference fine structure of the M3 reflection suggested that actin-attached myosin motors are displaced towards the start of their working stroke during isometric relaxation. There was negligible recovery of the intensities of the meridional and layer-line reflections associated with the quasi-helical distribution of myosin motors in resting muscle during isometric relaxation, and the 1.5% increase in the axial periodicity of the myosin filament associated with muscle activation was not reversed. When force had decreased to roughly half its tetanus plateau value, the isometric phase of relaxation abruptly ended, and the ensuing chaotic relaxation had an exponential half-time of ca 60 ms. Recovery of the equatorial X-ray intensities was largely complete during chaotic relaxation, but the other X-ray signals recovered more slowly than force.

Fibrillogenesis in dense collagen solutions: a physicochemical study.

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength=150 mM, 20 degrees C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH approximately 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.

Wormlike micelle formation and flow alignment of a pluronic block copolymer in aqueous solution.

The self-assembly into wormlike micelles of a poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymer Pluronic P84 in aqueous salt solution (2 M NaCl) has been studied by rheology, small-angle X-ray and neutron scattering (SAXS/SANS), and light scattering. Measurements of the flow curves by controlled stress rheometry indicated phase separation under flow. SAXS on solutions subjected to capillary flow showed alignment of micelles at intermediate shear rates, although loss of alignment was observed for high shear rates. For dilute solutions, SAXS and static light scattering data on unaligned samples could be superposed over three decades in scattering vector, providing unique information on the wormlike micelle structure over several length scales. SANS data provided information on even shorter length scales, in particular, concerning "blob" scattering from the micelle corona. The data could be modeled based on a system of semiflexible self-avoiding cylinders with a circular cross-section, as described by the wormlike chain model with excluded volume interactions. The micelle structure was compared at two temperatures close to the cloud point (47 degrees C). The micellar radius was found not to vary with temperature in this region, although the contour length increased with increasing temperature, whereas the Kuhn length decreased. These variations result in an increase of the low-concentration radius of gyration with increasing temperature. This was consistent with dynamic light scattering results, and, applying theoretical results from the literature, this is in agreement with an increase in endcap energy due to changes in hydration of the poly(ethylene oxide) blocks as the temperature is increased.

Cooperative ordering of collagen triple helices in the dense state.

Extracellular matrixes such as bone, skin, cornea, and tendon have ordered structures comprised for the most part of collagen, an elongated protein of well-defined dimensions and composition. Here we show how the cooperative ordering of collagen triple helices in the dense fluid state is exploited to produce dense ordered collagen matrixes. The spontaneous formation of a birefringent phase occurs at critical concentrations that increase from 50-60 to 80-85 mg/mL as the acetic acid concentration of the solvent increases from 5 to 500 mM. We studied by small-angle X-ray scattering (SAXS) the local liquidlike positional order across the isotropic/anisotropic phase transition by unwinding the cholesteric phase with moderate shearing stress. Interparticle scattering gives rise to a broad interference peak. The average distance between triple helices, dav, is thus estimated and decreases linearly as a function of phi-1/2 from 12.7 +/- 0.9 nm (22.5 mg/mL) to 5.0 +/- 0.6 nm (166.4 mg/mL). Equilibrium concentrations and the order parameter of the nematic phase agree reasonably well with theoretical predictions for semiflexible macromolecules. Striated fibrils with a high degree of alignment were obtained by fine-tuning the delicately balanced electrostatic interactions, which yielded strong elastic gels with a hierarchical organization very similar to that of major biological tissues. Typical Bragg reflections corresponding to the 67 nm period characteristic of collagen fibrils in biological tissues were recorded by SAXS with ordered collagen matrixes reconstituted in vitro.

Structural changes in the myosin filament and cross-bridges during active force development in single intact frog muscle fibres: stiffness and X-ray diffraction measurements.

Structural and mechanical changes occurring in the myosin filament and myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4 degrees C and 2.15 microm sarcomere length, using sarcomere level mechanics and X-ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 myosin-based reflections were recorded with 5 ms frames using the gas-filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T(0)) with a half-time of 40 ms, and the spacings of the M3 and M6 reflections (S(M3) and S(M6)) increase by 1.5% from their resting values, with time courses that lead that of force by approximately 10 and approximately 20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by approximately 10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half-sarcomere)(-1) shortening at the velocity (V(0)) that maintains zero force. Shortening at V(0) transiently reduces S(M3) following the latent period and delays the subsequent increase in S(M3), but only delays the S(M6) increase without a transient decrease. Shortening at V(0) imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (I(M3)), whereas the intensity of the M6 reflection (I(M6)) is only slightly reduced. The changes in half-sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the myosin heads attached to the actin filament and the M6 reflection originates mainly from a fixed structure in the myosin filament signalling myosin filament length changes during the tetanus rise.

Probing fast kinetics in complex fluids by combined rapid mixing and small-angle X-ray scattering.

Stopped-flow mixing coupled to small-angle X-ray scattering (SAXS) is an established technique for investigating structural kinetics in solution down to the millisecond range. More recently, the emphasis has shifted to the sub-millisecond range using continuous flow microfluidic mixing devices. The aim of this article is to review the present status and limitations when applying mixing techniques to a wide range of soft matter and biological systems. In the case of SAXS, special consideration of the mixing quality is necessary for a quantitative description of the scattered intensity. This is demonstrated through two representative examples involving protein refolding and micellar self-assembly.

The structural basis of the increase in isometric force production with temperature in frog skeletal muscle.

X-ray diffraction patterns were recorded from isolated single fibres of frog skeletal muscle during isometric contraction at temperatures between 0 and 17 degrees C. Isometric force was 43 +/- 2% (mean +/- S.E.M., n = 10) higher at 17 degrees C than 0 degrees C. The intensity of the first actin layer line increased by 57 +/- 18% (n = 5), and the ratio of the intensities of the equatorial 1,1 and 1,0 reflections by 20 +/- 7% (n = 10), signalling radial or azimuthal motions of the myosin head domains. The M3 X-ray reflection from the axial repeat of the heads along the filaments was 27 +/- 4% more intense at 17 degrees C, suggesting that the heads became more perpendicular to the filaments. The ratio of the intensities of the higher and lower angle peaks of the M3 reflection (R(M3)) was 0.93 +/- 0.02 (n = 5) at 0 degrees C and 0.77 +/- 0.02 at 17 degrees C. These peaks are due to interference between the two halves of each myosin filament, and the R(M3) decrease shows that heads move towards the midpoint of the myosin filament at the higher temperature. Calculations based on a crystallographic model of the heads indicated that the observed R(M3) change corresponds to tilting of their light-chain domains by 9 deg, producing an axial displacement of 1.4 nm, which is equal to that required to strain the actin and myosin filaments under the increased force. We conclude that the higher force generated by skeletal muscle at higher temperature can be accounted for by axial tilting of the myosin heads.

Dynamics of the self-assembly of unilamellar vesicles.

We have studied the transient stages in the formation of unilamellar vesicles with millisecond time resolution. The self-assembly was initiated by rapid mixing of equimolar amounts of anionic and zwitterionic micelles and the transient micellar entities were probed by time-resolved small-angle x-ray scattering. Within the mixing time, original micelles transformed to disklike micelles which evolved further to a critical size and then closed to form monodisperse unilamellar vesicles within a second. Subsequent growth led to an unexpected broadening of the vesicle size distribution.

Physical properties of aqueous suspensions of goethite (alpha-FeOOH) nanorods. Part I: In the isotropic phase.

Depending on volume fraction, aqueous suspensions of goethite (alpha-FeOOH) nanorods form a liquid-crystalline nematic phase (above 8.5%) or an isotropic liquid phase (below 5.5%). In this article, we investigate by small-angle X-ray scattering, magneto-optics, and magnetometry the influence of a magnetic field on the isotropic phase. After a brief description of the synthesis and characterisation of the goethite nanorod suspensions, we show that the disordered phase becomes very anisotropic under a magnetic field that aligns the particles. Moreover, we observe that the nanorods align parallel to a small field (< 350 mT), but realign perpendicular to a large enough field (> 350 mT). This phenomenon is interpreted as due to the competition between the influence of the nanorod permanent magnetic moment and a negative anisotropy of magnetic susceptibility. Our interpretation is supported by the behaviour of the suspensions in an alternating magnetic field. Finally, we propose a model that explains all experimental observations in a consistent way.

Physical properties of aqueous suspensions of goethite (alpha-FeOOH) nanorods. Part II: In the nematic phase.

At volume fractions larger than 8.5%, aqueous suspensions of lath-like goethite (alpha-FeOOH) nanorods form a lyotropic nematic phase. In this article, we first discuss the nematic ordering within statistical-physics models of the isotropic/nematic phase transition. We then describe the influence of a magnetic field on the nematic phase. Because the nanorods bear permanent magnetic moments, the nematic suspensions have dipolar order and very low Frederiks thresholds. Moreover, the nematic phase aligns parallel to a small magnetic field but realigns perpendicular to a high field because of a competition between the permanent moments of the nanorods and their negative anisotropy of magnetic susceptibility. This magneto-optical study of the nematic phase is completely consistent with that of the isotropic phase of the same suspensions published in Part I (this issue, p. 291). Besides, we demonstrate the field-induced biaxiality of a nematic single domain aligned perpendicular to the field. We also describe here preliminary experiments where an a.c. electric field is applied to the nematic phase. Both field amplitude and frequency were found to control the alignment direction and homeotropic-to-planar alignment transitions were observed. From this data, simple models were used to estimate some physical constants of the nematic phase.

Magnetic-field-induced nematic-columnar phase transition in aqueous suspensions of goethite (alpha-FeOOH) nanorods.

Colloidal aqueous suspensions of goethite lath-shaped nanorods form nematic and isotropic phases. We show that they also display a 2D rectangular (c2mm) columnar phase at volume fractions phi larger than 15%. Interestingly, the nematic-columnar first-order transition can also be triggered by applying to the nematic phase a magnetic field of intensity decreasing with phi (1 T at 8.5%; 0.5 T at 12%). Single domains of the columnar phase were thus produced and their structure investigated by synchrotron x-ray scattering. This magnetic-field-induced transition is fully reversible and reproducible.

Structure and rheology during shear-induced crystallization of a latex suspension.

Microstructure and rheology of a concentrated sterically stabilized colloidal suspension undergoing flow-induced ordering was studied by combined small-angle x-ray scattering and rheometry. This system is known to form bundlelike structures at high stress values in continuous shear flow. Under large amplitude oscillatory flow, hexagonal close-packed crystalline domains are formed within 1 sec of the inception of shear. In the intermediate range of frequencies and amplitudes, a nearly perfect hexagonally close-packed layer structure was observed after the cessation of flow. Lower frequencies or stress amplitudes resulted in polycrystals and, on the other hand, high frequencies or stress amplitudes led to partial melting of the layered structure. During the oscillatory flow, the intensity of the Bragg peaks showed pronounced oscillations.

Outstanding magnetic properties of nematic suspensions of goethite (alpha-FeOOH) nanorods.

Aqueous suspensions of goethite (alpha-FeOOH) nanorods form a mineral lyotropic nematic phase that aligns in a very low magnetic field (20 mT for samples 20 microm thick). The particles orient along the field direction at intensities smaller than 350 mT, but they reorient perpendicular to the field beyond 350 mT. This outstanding behavior is also observed in the isotropic phase which has a very strong magnetic-field induced birefringence that could be interesting for applications. We interpret these magnetic effects as resulting from a competition between a nanorod remanent magnetic moment and a negative anisotropy of its magnetic susceptibility.

A novel method for the cross-sectional TEM preparation of thin films deposited onto water-soluble substrates.

A preparational method was developed solving the problem of cross-sectional TEM preparation of thin films and layer systems deposited onto water-soluble substrates. The technique is based on the replacement of the sample onto steady substrate, followed by mechanical and ion beam thinning. Cross-sectional TEM micrographs of Ag and Ag/Ag2Se layers are shown presenting the efficiency of this novel technique.