Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier.

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 µm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 µM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Improvement of ultra-small-angle XPCS with the Extremely Brilliant Source.

Recent technical developments and the performance of the X-ray photon correlation spectroscopy (XPCS) method over the ultra-small-angle range with the Extremely Brilliant Source (EBS) at the ESRF are described. With higher monochromatic coherent photon flux (∼1012 photons s-1) provided by the EBS and the availability of a fast pixel array detector (EIGER 500K detector operating at 23000 frames s-1), XPCS has become more competitive for probing faster dynamics in relatively dilute suspensions. One of the goals of the present development is to increase the user-friendliness of the method. This is achieved by means of a Python-based graphical user interface that enables online visualization and analysis of the processed data. The improved performance of XPCS on the Time-Resolved Ultra-Small-Angle X-ray Scattering instrument (ID02 beamline) is demonstrated using dilute model colloidal suspensions in several different applications.

Dynamics of magnetic Janus colloids studied by ultra small-angle X-ray photon correlation spectroscopy.

The orientation behavior and the translational dynamics of spherical magnetic silica-nickel Janus colloids in an external magnetic field have been studied by small-angle X-ray scattering and X-ray photon correlation spectroscopy at ultra small-angles. For weak applied fields and at low volume fractions, the particle dynamics is dominated by Brownian motion even though the net magnetic moments of the individual particles are aligned in the direction of the field as indicated by the anisotropy in the small-angle scattering patterns. For higher fields the magnetic forces result in more complex structural changes with nickel caps of Janus particles pointing predominantly along the applied magnetic field. The alignment ultimately leads to chain-like configurations and the intensity-intensity autocorrelation functions, g2(q,t), show a second slower decay which becomes more pronounced at higher volume fractions. A direction dependent analysis of g2(q,t) revealed a faster than exponential decay perpendicular to the field which is related to the sedimentation of magnetically ordered domains. The corresponding velocity fluctuations could be decoupled from the diffusion of particles by decomposing g2(q,t) into advective and diffusive contributions. Finally, the particle dynamics becomes anisotropic at higher volume fractions and strong magnetic fields. The derived translational diffusion coefficients indicate slower particle dynamics perpendicular to the field as compared to the parallel direction.

High unimer concentration and asymmetric bilayer observed in small unilamellar vesicles of mixed surfactants TDMAO/LiPFO.

The mixed surfactant system of tetradecyldimethylamine oxide (TDMAO) and lithium perfluorooctanoate (LiPFO) is known to spontaneously self-assemble into well-defined small unilamellar vesicles. For a quantitative analysis of small-angle x-ray scattering on this model system, we complemented the measurements with densitometry, conductimetry, and contrast-variation small-angle neutron scattering. The analysis points to two main findings: first, the vesicles formed to contain a much higher mole fraction (0.61-0.64) of TDMAO than the bulk sample (0.43) and predicted by Regular Solution Theory (RST, 0.46). In consequence, the unimer concentration of LiPFO is more than 5 times higher than predicted by RST. Second, the vesicle bilayer is asymmetric with a higher fraction of LiPFO on the outside. These findings on a model system should be of broader relevance for the understanding of similar mixed surfactant vesicle systems and thereby also be of importance for their use in a number of applications.

Myosin filament-based regulation of the dynamics of contraction in heart muscle.

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the "C-zone." Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.

Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments.

Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.

Tube to ribbon transition in a self-assembling model peptide system.

Peptides that self-assemble into ß-sheet rich aggregates are known to form a large variety of supramolecular shapes, such as ribbons, tubes or sheets. However, the underlying thermodynamic driving forces for such different structures are still not fully understood, limiting their potential applications. In the AnK peptide system (A = alanine, K = lysine), a structural transition from tubes to ribbons has been shown to occur upon an increase of the peptide length, n, from 6 to 8. In this work we analyze this transition by means of a simple thermodynamic model. We consider three energy contributions to the total free energy: an interfacial tension, a penalty for deviating from the optimal ß-sheet twist angle, and a hydrogen bond deformation when the ß-sheets adopt a specific self-assembled structure. Whilst the first two contributions merely provide similar constant energy offsets, the hydrogen bond deformations differ depending on the studied structure. Consequently, the tube structure is thermodynamically favored for shorter AnK peptides, with a crossover at n≈ 13. This qualitative agreement of the model with the experimental observations shows, that we have achieved a good understanding of the underlying thermodynamic features within the self-assembling AnK system.

Myosin filament activation in the heart is tuned to the mechanical task.

The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors.

Development of a crystal collimation system for high-resolution ultra-small-angle X-ray scattering applications.

Crystal collimation offers a viable alternative to the commonly used pinhole collimation in small-angle X-ray scattering (SAXS) for specific applications requiring highest angular resolution. This scheme is not affected by the parasitic scattering and diffraction-limited beam broadening. The Darwin width of the rocking curve of the crystals mainly defines the ultimate beam divergence. For this purpose, a dispersive Si-111 crystal collimation set-up based on two well conditioned pseudo channel-cut crystals (pairs of well polished, independent parallel crystals) using a higher-order reflection (Si-333) has been developed. The gain in resolution is obtained at the expense of flux. The system has been installed at the TRUSAXS beamline ID02 (ESRF) for reducing the horizontal beam divergence in high-resolution mesurements. The precise mechanics of the system allows reproducible alignment of the Bragg condition. The high resolution achieved at a sample-detector distance of 31 m is demonstrated by ultra-small-angle X-ray scattering measurements on a model system consisting of micrometre-sized polystyrene latex particles with low polydispersity.

Stabilizing Colloidal Particles against Salting-out by Shortening Surface Grafts.

A dramatic improvement is reported in the stability of colloidal particles when stabilizing surface grafts are systematically shortened from small polymers to single monomers. The colloidal dispersions consist of fluorinated latex particles, exhibiting a weak van der Waals attraction, with grafted steric layers of poly(ethylene glycol) (PEG) of different chain lengths. Using an effective salting-out electrolyte, Na2CO3, particle aggregates are detected above a threshold salt concentration that is independent of the particle concentration. The results are interpreted in terms of a sudden onset of nondispersibility of single particles, triggered by the solvent not completely wetting particle surfaces. By decreasing the PEG chain length, the threshold salt concentration is found to increase sharply. For grafts with just a single ethylene glycol group, dispersions remain stable up to exceedingly high concentrations of Na2CO3. However, on removal of the surface coverage altogether, the classical stability behavior of charge-stabilized dispersions is recovered. The behavior can be captured by a simple model that incorporates effective polymer-solvent interactions in the presence of an electrolyte.

The force and stiffness of myosin motors in the isometric twitch of a cardiac trabecula and the effect of the extracellular calcium concentration.

KEY POINTS: Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6 pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07 pN nm-1 ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. ABSTRACT: The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (Tp ) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+ ]o ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07 ± 0.09 pN nm-1 , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8 pN nm-1 ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca2+ ]o from 1 to 2.5 mm at sarcomere length ∼2.2 µm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08 ± 0.01 to 0.12 ± 0.02, proportional to Tp . Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of Tp and [Ca2+ ]o . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.

Anomalous Dynamics of Magnetic Anisotropic Colloids Studied by XPCS.

The influence of an applied magnetic field on the collective dynamics of novel anisotropic colloidal particles whose shape resembles peanuts is reported. Being made up of hematite cores and silica shells, these micrometer-sized particles align in a direction perpendicular to the applied external magnetic field, and assemble into chains along the field direction. The anisotropic dynamics of these particles is investigated using multispeckle ultrasmall-angle X-ray photon correlation spectroscopy (USA-XPCS). The results indicate that along the direction of the magnetic field, the particle dynamics strongly depends on the length scale probed. Here, the relaxation of the intermediate scattering function follows a compressed exponential behavior at large distances, while it appears diffusive at distances comparable or smaller than the particle size. Perpendicular to the applied field (and along the direction of gravity), the experimental data can be quantitatively reproduced by a combination of an advective term originating from sedimentation and a purely diffusive one that describes the thermal diffusion of the assembled chains and individual particles.

Microstructure and dynamics of Janus particles in a phase separating medium.

The evolution of interactions and dynamics of Janus colloidal particles suspended in quasi-binary liquid mixtures undergoing phase separation is presented. The experimental system consisted of silica-nickel Janus particles dispersed in mixtures of 3-methylpyridine, water, and heavy water. Colloidal microstructure and dynamics were probed by ultra-small-angle X-ray scattering and ultra-small-angle X-ray photon correlation spectroscopy, respectively. The observed static and dynamic behaviors are significantly different from those found for Stöber silica colloids in this mixture. The Janus particles manifest a slow aggregation below the coexistence temperature and become strongly attractive upon phase separation of the solvent mixture. In the two-phase region, particles tend to display surfactant-like behavior with silica and nickel surfaces likely preferring water and 3-methylpyridine rich phases, respectively. While the onset of diffusiophoretic motion is evident in the dynamics, it is gradually suppressed by particle clustering at the investigated colloid volume fractions.

Effective interactions and dynamics of small passive particles in an active bacterial medium.

This article presents an investigation of the interparticle interactions and dynamics of submicron silica colloids suspended in a bath of motile Escherichia coli bacteria. The colloidal microstructure and dynamics were probed by ultra-small-angle x-ray scattering and multi-speckles x-ray photon correlation spectroscopy, respectively. Both static and hydrodynamic interactions were obtained for different colloid volume fractions and bacteria concentrations as well as when the interparticle interaction potential was modified by the motility buffer. Results suggest that motile bacteria reduce the effective attractive interactions between passive colloids and enhance their dynamics at high colloid volume fractions. The enhanced dynamics under different static interparticle interactions can be rationalized in terms of an effective viscosity of the medium and unified by means of an empirical effective temperature of the system. While the influence of swimming bacteria on the colloid dynamics is significantly lower for small particles, the role of motility buffer on the static and dynamic interactions becomes more pronounced.

Self-assembly of smallest magnetic particles.

The assembly of tiny magnetic particles in external magnetic fields is important for many applications ranging from data storage to medical technologies. The development of ever smaller magnetic structures is restricted by a size limit, where the particles are just barely magnetic. For such particles we report the discovery of a kind of solution assembly hitherto unobserved, to our knowledge. The fact that the assembly occurs in solution is very relevant for applications, where magnetic nanoparticles are either solution-processed or are used in liquid biological environments. Induced by an external magnetic field, nanocubes spontaneously assemble into 1D chains, 2D monolayer sheets, and large 3D cuboids with almost perfect internal ordering. The self-assembly of the nanocubes can be elucidated considering the dipole-dipole interaction of small superparamagnetic particles. Complex 3D geometrical arrangements of the nanodipoles are obtained under the assumption that the orientation of magnetization is freely adjustable within the superlattice and tends to minimize the binding energy. On that basis the magnetic moment of the cuboids can be explained.

The Closed State of the Thin Filament Is Not Occupied in Fully Activated Skeletal Muscle.

Muscle contraction is powered by actin-myosin interaction controlled by Ca2+ via the regulatory proteins troponin (Tn) and tropomyosin (Tpm), which are associated with actin filaments. Tpm forms coiled-coil dimers, which assemble into a helical strand that runs along the whole ∼1 µm length of a thin filament. In the absence of Ca2+, Tn that is tightly bound to Tpm binds actin and holds the Tpm strand in the blocked, or B, state, where Tpm shields actin from the binding of myosin heads. Ca2+ binding to Tn releases the Tpm from actin so that it moves azimuthally around the filament axis to a closed, or C, state, where actin is partially available for weak binding of myosin heads. Upon transition of the weak actin-myosin bond into a strong, stereo-specific complex, the myosin heads push Tpm strand to the open, or O, state allowing myosin binding sites on several neighboring actin monomers to become open for myosin binding. We used low-angle x-ray diffraction at the European Synchrotron Radiation Facility to check whether the O- to C-state transition in fully activated fibers of fast skeletal muscle of the rabbit occurs during transition from isometric contraction to shortening under low load. No decrease in the intensity of the second actin layer line at reciprocal radii in the range of 0.15-0.275 nm-1 was observed during shortening suggesting that an azimuthal Tpm movement from the O- to C-state does not occur, although during shortening muscle stiffness is reduced compared to the isometric state, and the intensities of other actin layer lines demonstrate a ∼2-fold decrease in the fraction of myosin heads strongly bound to actin. The data show that a small fraction of actin-bound myosin heads is sufficient for supporting the O-state and, therefore the C-state is not occupied in fully activated skeletal muscle that produces mechanical work at low load.

Velocity Fluctuations in Sedimenting Brownian Particles.

We report a gradual transition of dynamics in sedimenting suspensions of charge stabilized Brownian particles prior to the onset of the macroscopic sedimentation front. Using multispeckle ultrasmall-angle x-ray photon correlation spectroscopy (USA-XPCS), we show that well-defined advective motions dominate the colloid dynamics during the early stages of sedimentation. With elapsing time, these advective currents decay and diffusive motions become the dominating contribution in the dynamics. Probing the temporal development of these fluctuations at smaller Peclet numbers (<1) provides a new perspective for the mechanism determining the transient nature of velocity fluctuations in sedimentation and demonstrates new experimental capabilities enabled by multispeckle USA-XPCS.

Protein Immobilization onto Cationic Spherical Polyelectrolyte Brushes Studied by Small Angle X-ray Scattering.

The immobilization of bovine serum albumins (BSA) onto cationic spherical polyelectrolyte brushes (SPB) consisting of a solid polystyrene (PS) core and a densely grafted poly(2-aminoethyl methacrylate hydrochloride) (PAEMH) shell was studied by small-angle X-ray scattering (SAXS). The observed dynamics of adsorption of BSA onto SPB by time-resolved SAXS can be divided into two stages. In the first stage (tens of milliseconds), the added proteins as in-between bridge instantaneously caused the aggregation of SPB. Then BSA penetrated into the brush layer driven by electrostatic attractions, and reached equilibrium in the second stage (tens of seconds). The amount of BSA immobilized onto brush layer reached the maximum when pH was increased to about 6.1 and BSA concentration to 10 g/L. The cationic SPB were confirmed to provide stronger adsorption capacity for BSA compared to anionic ones.

Tropomyosin movement is described by a quantitative high-resolution model of X-ray diffraction of contracting muscle.

Contraction of skeletal and cardiac muscle is controlled by Ca2+ ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) associated with the thin actin filaments in sarcomeres. In the absence of Ca2+, Tn-C binds actin and shifts the Tpm strand to a position where it blocks myosin binding to actin, keeping muscle relaxed. According to the three-state model (McKillop and Geeves Biophys J 65:693-701, 1993), upon Ca2+ binding to Tn, Tpm rotates about the filament axis to a 'closed state' where some myosin heads can bind actin. Upon strong binding of myosin heads to actin, Tpm rotates further to an 'open' position where neighboring actin monomers also become available for myosin binding. Azimuthal Tpm movement in contracting muscle is detected by low-angle X-ray diffraction. Here we used high-resolution models of actin-Tpm filaments based on recent cryo-EM data for calculating changes in the intensities of X-ray diffraction reflections of muscle upon transitions between different states of the regulatory system. Calculated intensities of actin layer lines provide a much-improved fit to the experimental data obtained from rabbit muscle fibers in relaxed and rigor states than previous lower-resolution models. We show that the intensity of the second actin layer line at reciprocal radii from 0.15 to 0.3 nm-1 quantitatively reports the transition between different states of the regulatory system independently of the number of myosin heads bound to actin.

Phoretic motion of colloids in a phase separating medium.

The enhanced motion of dispersed particles driven by a concentration gradient is the basis for diffusiophoresis. Here we present the dynamics of colloids in a phase separating medium probed by X-Ray Photon Correlation Spectroscopy (XPCS) in the ultra-small angle scattering range. Charge stabilized silica colloids suspended in a binary mixture of 3-methylpyridine and water/heavy water are preferentially wetted by 3-methylpyridine and consequently display a phoretic motion towards that phase upon demixing. This activity lasts for hundreds of seconds before the phase separation is complete and the enhanced motion is arrested as the colloids return to normal diffusive dynamics.