Equatorial Reflections Research Articles

Fractal carbon nanotube fibers with mesoporous crystalline structure

Macroscopic fibres of carbon nanotubes are hierarchical structures combining long building blocks preferentially oriented along the fibre axis and a large porosity arising from the imperfect packing of bundles. Synchrotron small-angle X-ray scattering (SAXS) measurements show that such structure is a surface fractal with fractal dimension (Ds) of 2.5 for MWCNT fibres and 2.8 for SWCNT fibres. N2 adsorption measurements give similar values of 2.54 and 2.50, respectively. The fractal dimension and deviation from Porod's law are related to density fluctuations associated with the wide distribution of separations between CNTs. These fluctuations are also evident as diffuse wide-angle X-ray scattering (WAXS) from CNTs at distances above turbostratic separation. The structure of CNT fibres produced at different draw ratios is compared in terms of degree of orientation and characteristic lengths parallel and perpendicular to the fibre. Drawing not only increases alignment but also the fraction of graphitic planes forming coherent domains capable of taking part in stress transfer by shear; thus increasing both tensile modulus and strength. The invariant-normalized intensity of the (002) equatorial reflection thus takes the form of a degree of crystallinity closely related to tensile properties.

Strong Intraseasonal Variability of Meridional Currents near 5°N in the Eastern Indian Ocean: Characteristics and Causes

AbstractThis paper reports on strong, intraseasonal, upper-ocean meridional currents observed in the Indian Ocean between the Bay of Bengal (BOB) and the equator and elucidates the underlying physical processes responsible for them. In situ measurements from a subsurface mooring at 5°N, 90.5°E reveal strong intraseasonal variability of the meridional current with an amplitude of ~0.4 m s−1 and a typical period of 30–50 days in the upper 150 m, which by far exceeds the magnitudes of the mean flow and seasonal cycle. Such prominent intraseasonal variability is, however, not seen in zonal current at the same location. Further analysis suggests that the observed intraseasonal flows are closely associated with westward-propagating eddylike sea surface height anomalies (SSHAs) along 5°N. The eddylike SSHAs are largely manifestations of symmetric Rossby waves, which result primarily from intraseasonal wind stress forcing in the equatorial waveguide and reflection of the equatorial Kelvin waves at the eastern boundary. Since the wave signals are generally symmetric about the equator, similar variability is also seen at 5°S but with weaker intensity because of the inclined coastline at the eastern boundary. The Rossby waves propagate westward, causing pronounced intraseasonal SSHA and meridional current in the upper ocean across the entire southern BOB between 84° and 94°E. They greatly weaken in the western Indian Basin, but zonal currents near the equator remain relatively strong.

Hypercontractile mutant of ventricular myosin essential light chain leads to disruption of sarcomeric structure and function and results in restrictive cardiomyopathy in mice.

The E143K (Glu → Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling. The E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts. As a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling.

Physical Properties of Plant Fibers (Sisal, Coir) and Its Composite Material with Tamarind Seed Gum as Low-Cost Housing Material

ABSTRACTIn this study, the physical properties of sisal and coir fibers have been described. Using manual extraction procedures, the sisal fibers were extracted from the sisal plant and coir fibers from the coconut palm. Thermal analysis by differential scanning calorimetry, structural morphology by scanning electron microscopy, and the degree of sharpness of the equatorial reflections of the X-ray fiber diffraction pattern were recorded for the untreated sisal and coir fibers. Tensile strength was determined for the single fiber and the results are correlated with the tensile strength of bundle fibers. Composite material has been prepared with this plant fiber as a filler and tamarind seed gum as a matrix material. The endosperms of roasted tamarind seeds were used for the preparation of tamarind seed gum solution. The different temperature condition maintained for roasting the seeds are 130°C, 160°C, and 180°C. The tensile strength of the prepared composite material is measured and it shows dependency of the roasting temperature condition of the tamarind seed. Scanning electron microscopy and water resistivity test were conducted, and the results were reported for the prepared composite material. Low-cost housing is made using this biodegradable sisal fiber--tamarind seed gum composite material.

Molecular Mechanisms Involved in Cardioskeletal Dysfunction Caused by Mutations in Myosin RLC Linked to Hypertrophic Cardiomyopathy

The MYL2 gene concurrently expresses the human myosin regulatory light chain (RLC) in the ventricles of the heart and in the slow-twitch skeletal muscle. Mutations in MYL2 are known to cause hypertrophic cardiomyopathy (HCM), but in some cases they also lead to cardioskeletal myopathy in humans. Using transgenic animal models of HCM, we studied the mutation-induced morphological, structural and functional changes and the signaling pathways that trigger pathological remodeling of the HCM heart and slow-twitch skeletal muscle. Electron microscopy data showed severe myofilament disarray, a hallmark of HCM, in the hearts and in the soleus muscle of RLC-mouse models of HCM. Likewise, functional measurements of contractile force in skinned papillary and soleus muscle fibers showed similar direction of changes in HCM mice. Mutation-induced structural abnormalities in papillary and soleus muscles from HCM-R58Q mice were confirmed by high resolution X-ray diffraction showing similar changes in d1,0 (interfilament lattice spacing) and in equatorial reflections’ intensity I1,1/I1,0 ratio in both papillary and soleus muscles. Quantitative proteomic analysis revealed that out of >1560 TMT-labeled proteins identified with high confidence, 372 were different in the hearts of R58Q vs. WT mice, while in the soleus skeletal muscle out of >1100 TMT-proteins, 255 were different in R58Q vs. WT. The two major biological processes affected in both types of muscles in R58Q were the metabolic and cellular processes, with a greater percentage of metabolic processes affected in cardiac muscle. Common metabolic proteins decreased in heart/soleus of R58Q vs. WT mice included the energy production proteins, while the cellular processes included the calcium handling proteins. Our comprehensive data clearly indicated adverse heart/soleus muscle remodeling in HCM mice making them an ideal model to study the molecular, energetic and cellular mechanisms of cardioskeletal myopathy associated with MYL2. Supported by NIH-HL123255 (DSC).

Distinct Lattice Structure Altreations in DCM and HCM Mouse Models Associated with Mutations in Myosin Regulatory Light Chain

In this study we used small angle X-ray diffraction to analyze changes in the interfilament lattice spacing (IFS) and equatorial reflections’ intensity ratio (I1,1/I1,0) due to mutations in myosin RLC shown to cause hypertrophic (HCM) or dilated (DCM) cardiomyopathy. In mice, D166V was found to result in systolic and diastolic dysfunction, reduced contractile force and abnormally high Ca2+-sensitivity, highlighting the severity of the HCM phenotype. The D94A mutation resulted in DCM manifested by increased LV-chamber diameter and reduced ejection fraction with no changes in maximal force production but with D94A-induced decrease in the Ca2+-sensitivity of force. In this report we collected the X-ray data on skinned papillary muscles from D166V-HCM and D94A-DCM vs. WT mice at serial pCa (8, 7, 6, 5.4, 5.2, and 4) and at SL=2.1µm. For D166V-HCM vs. WT-fibers, IFS was significantly increased in pCa8, 6 and 5.4 but significantly decreased at pCa4. For D94A-DCM and for WT, no significant changes in IFS were observed within or between the groups for each pCa. There was a clear correlation between I1,1/I1,0 readings vs. increasing [Ca2+] for all three groups of fibers. A significant increase in I1,1/I1,0 for all pCa solutions (except pCa4) was observed in D166V-HCM vs. WT fibers. These data correlated with D166V-induced changes in force/Ca2+-sensitivity observed in fiber mechanics study. For D94A-DCM, a near significant decrease in I1,1/I1,0 at pCa 5.2 vs. WT was observed (WT:0.43±0.08 vs. D94A: 0.35±0.09 P=0.072). Fitting I1,1/I1,0 –pCa plots yielded pCa50=5.2±0.03 (WT), 4.98±0.05 (D94A) and 5.43±0.10 (D166V), showing a clear correlation in pCa50-values between X-ray and fiber mechanics experiments. Our collective data indicate that RLC mutations lead to HCM or DCM through structural rearrangements of myosin cross-bridge mass with D166V bringing it closer to actin, and D94A moving it farther away from the thin-filaments. Supported by NIH-HL123255 (DSC).

シリコーンゴムにおけるメゾ相の形成と結晶化

Silicone rubber consists of cross-linked poly(dimethyl siloxane)(PDMS). Formation of the mesomorphic phase was found in silicone rubber that is highly stretched at room temperature. The mesomorphic phase is characterized by a pair of sharp equatorial reflections in the wide-angle X-ray diffraction (WAXD) pattern. At low temperature, the stretched silicone rubber crystallizes. The WAXD patterns from the crystallized silicone rubber exhibit totally different features depending on the draw ratio. The author successfully obtained single crystals of linear PDMS. Electron diffraction patterns of the single crystals indicate that at least four different crystal forms exist. The observed WAXD patterns and their peculiar transition scheme could be explained by the combination of the crystal forms.

The supramolecular structure of bone: X-ray scattering analysis and lateral structure modeling.

The evolution of vertebrates required a key development in supramolecular evolution: internally mineralized collagen fibrils. In bone, collagen molecules and mineral crystals form a nanocomposite material comparable to cast iron in tensile strength, but several times lighter and more flexible. Current understanding of the internal nanoscale structure of collagen fibrils, derived from studies of rat tail tendon (RTT), does not explain how nucleation and growth of mineral crystals can occur inside a collagen fibril. Experimental obstacles encountered in studying bone have prevented a solution to this problem for several decades. This report presents a lateral packing model for collagen molecules in bone fibrils, based on the unprecedented observation of multiple resolved equatorial reflections for bone tissue using synchrotron small-angle X-ray scattering (SAXS; ∼1 nm resolution). The deduced structure for pre-mineralized bone fibrils includes features that are not present in RTT: spatially discrete microfibrils. The data are consistent with bone microfibrils similar to pentagonal Smith microfibrils, but are not consistent with the (nondiscrete) quasi-hexagonal microfibrils reported for RTT. These results indicate that collagen fibrils in bone and tendon differ in their internal structure in a manner that allows bone fibrils, but not tendon fibrils, to internally mineralize. In addition, the unique pattern of collagen cross-link types and quantities in mineralized tissues can be can be accounted for, in structural/functional terms, based on a discrete microfibril model.

Hydrothermal Transformation of Wood Cellulose Crystals into Pseudo-Orthorhombic Structure by Cocrystallization.

The ultrastructural transformation of wood cellulose crystals by hydrothermal treatment was followed by synchrotron and standard X-ray scattering experiments. When treated at 200 °C for 2 h in the presence of an excess of water, a significant sharpening of the equatorial reflections of crystalline cellulose was observed, and the average crystallite size, estimated from the X-ray line broadening, was twice as large as that of untreated wood cellulose. During the treatment, the cellulose structure was converted from the native monoclinic form of cellulose I into a pseudo-orthorhombic system, coined as cellulose I', a transformation occurring only with an excess of water, above 180 °C and after more than half an hour. In situ experiments indicated that the increase of crystallite size was likely due to cocrystallization of individual crystallites rather than to the crystallization of the amorphous domains of cellulose.

Knockdown of fast skeletal myosin-binding protein C in zebrafish results in a severe skeletal myopathy.

Myosin-binding protein C (MyBPC) in the muscle sarcomere interacts with several contractile and structural proteins. Mutations in the cardiac isoform (MyBPC-3) in humans, or animal knockout, are associated with cardiomyopathy. Function of the fast skeletal isoform (MyBPC-2) in living muscles is less understood. This question was addressed using zebrafish models, combining gene expression data with functional analysis of contractility and small-angle x-ray diffraction measurements of filament structure. Fast skeletal MyBPC-2B, the major isoform, was knocked down by >50% using morpholino antisense nucleotides. These morphants exhibited a skeletal myopathy with elevated apoptosis and up-regulation of factors associated with muscle protein degradation. Morphant muscles had shorter sarcomeres with a broader length distribution, shorter actin filaments, and a wider interfilament spacing compared with controls, suggesting that fast skeletal MyBPC has a role in sarcomere assembly. Active force was reduced more than expected from the decrease in muscle size, suggesting that MyBPC-2 is required for optimal force generation at the cross-bridge level. The maximal shortening velocity was significantly increased in the MyBPC-2 morphants, but when related to the sarcomere length, the difference was smaller, reflecting that the decrease in MyBPC-2B content and the resulting myopathy were accompanied by only a minor influence on filament shortening kinetics. In the controls, equatorial patterns from small-angle x-ray scattering revealed that comparatively few cross-bridges are attached (as evaluated by the intensity ratio of the 11 and 10 equatorial reflections) during active contraction. X-ray scattering data from relaxed and contracting morphants were not significantly different from those in controls. However, the increase in the 11:10 intensity ratio in rigor was lower compared with that in controls, possibly reflecting effects of MyBPC on the cross-bridge interactions. In conclusion, lack of MyBPC-2 results in a severe skeletal myopathy with structural changes and muscle weakness.

X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

Rare Cardiomyopathy Phenotypes Associated with Mutations in Myosin Light Chains

We report here on two rare phenotypes of dilated (DCM) and restrictive (RCM) cardiomyopathy linked to mutations in myosin light chains. The D94A (Aspartic acid→Alanine)-DCM mutation was found in the regulatory (RLC), and E143K (Glutamic acid→Lysine)-RCM in the essential (ELC) light chains. 5 mo-old D94A mice showed increased LV diameter but only D94A-males (M) displayed lower ejection fraction (35±0.6%) compared with WT-RLC-M (63±8%). Invasive hemodynamic evaluation showed lower cardiac output in and higher Ea (arterial elastance, mmHg/µl) in D94A-M mice. X-ray studies on papillary muscles from D94A-mice showed reduced I1,1/I1,0 (equatorial reflections’ ratio at SL=2.3µm: 0.30±0.02) vs. WT-RLC (0.46±0.07) indicating repositioning of D94A-cross-bridges farther away from actin. Contractile force measurements in skinned muscle fibers revealed a decreased Ca-sensitivity in D94A-RLC vs. WT-RLC, confirming X-ray results and indicating that higher [Ca2+] is needed for activation by D94A-cross-bridges. The E143K-RCM hearts did not show hypertrophic morphology compared with WT-ELC hearts. However, a severe RCM phenotype was observed in Masson's trichrome stained heart sections from E143K-ELC mice with profound fibrosis and sarcomere irregularities (Z-line streaming and M-line vanishing) in 11 mo-old E143K mice by transmission electron microscopy. RT-qPCR revealed upregulation of ANF, BNP and collagen-type1&3, but α-MHC gene expression was downregulated. In vivo examination of E143K-hearts showed higher end-diastolic-pressure and Tau (relaxation time) and lower dP/dtmin (relaxation velocity), indicating diastolic dysfunction in the mutant vs. WT-ELC mice. In addition, we noted slightly elevated contractile force, lower Ca2+-sensitivity and reduced myofibrillar-ATPase activity in E143K vs. WT-ELC mice. Higher maximal force was paralleled by decreased lattice spacing in E143K vs. WT-ELC fibers in X-ray experiments. In conclusion, both mutations in RLC and ELC cause disruption of sarcomere structure followed by impairment of cardiac function. Supported by NIH-HL108343 (DSC) and AHA-15PRE23020006 (CCY).

Effect of Active Shortening and Stretching on Lattice Spacing and Cross-Bridge Binding in Skinned Muscle Fibres

When a tetanized skeletal muscle is actively shortened or stretched the resulting steady-state force is different from the purely isometric force at the corresponding length. Active shortening reduces steady-state force (termed force depression-FD), while active lengthening increases steady-state force (termed force enhancement-FE). According to the cross-bridge theory, FD and FE can occur if myofilament lattice spacing was modified after active shortening and stretching leading to a change in the proportion of attached cross-bridges, or if the kinetics of cross-bridges were modified leading to a change in the proportion of attached cross-bridges and/or the average force per cross-bridge. Here, we determined the effect of active shortening and stretching on the myofilament lattice spacing and the proximity of the myosin heads to the thin filaments using small angle X-ray diffraction.Lattice spacing (d1,0) was calculated from the spacing between the 1,0 and 1,1 equatorial reflections. The ratio of the intensities of the 1,1 and 1,0 equatorial reflections (I1,1:I1,0) was used as an estimate of the degree of association of myosin heads with the thin filaments.d1,0 increased (38.9±0.1 versus 38.1±0.2nm, p 0.05) between the FE state and its reference state, and I1,1:I1,0 was increased (2.5±0.2 versus 1.3±0.1, p<0.05) in the FE state. These results suggest that the proportion of attached cross-bridges might be increased after active stretch and that this increase is not due to a decrease in lattice spacing but rather a change in cross-bridge kinetics.

Functional and Electrical Integration of Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Myocardial Infarction Rat Heart.

In vitro expanded beating cardiac myocytes derived from induced pluripotent stem cells (iPSC-CMs) are a promising source of therapy for cardiac regeneration. Meanwhile, the cell sheet method has been shown to potentially maximize survival, functionality, and integration of the transplanted cells into the heart. It is thus hypothesized that transplanted iPSC-CMs in a cell sheet manner may contribute to functional recovery via direct mechanical effects on the myocardial infarction (MI) heart. F344/NJcl-rnu/rnu rats were left coronary artery ligated (n = 30), followed by transplantation of Dsred-labeled iPSC-CM cell sheets of murine origin over the infarct heart surface. Effects of the treatment were assessed, including in vivo molecular/cellular evaluations using a synchrotron radiation scattering technique. Ejection fraction and activation recovery interval were significantly greater from day 3 onward after iPSC-CM transplantation compared to those after sham operation. A number of transplanted iPSC-CMs were present on the heart surface expressing cardiac myosin or connexin 43 over 2 weeks, assessed by immunoconfocal microscopy, while mitochondria in the transplanted iPSC-CMs gradually showed mature structure as assessed by electron microscopy. Of note, X-ray diffraction identified 1,0 and 1,1 equatorial reflections attributable to myosin and actin-myosin lattice planes typical of organized cardiac muscle fibers within the transplanted cell sheets at 4 weeks, suggesting cyclic systolic myosin mass transfer to actin filaments in the transplanted iPSC-CMs. Transplantation of iPSC-CM cell sheets into the heart yielded functional and electrical recovery with cyclic contraction of transplanted cells in the rat MI heart, indicating that this strategy may be a promising cardiac muscle replacement therapy.

The molecular structure of the silk fibers from Hymenoptera aculeata (bees, wasps, ants).

Silks from the Hymenoptera aculeata (bees, wasps, ants) contain ropes with four α-helical strands, rather than the more usual two strands found, for example, in α-keratin and myosin molecules. Extensive studies of the chemical structure of the silks have shown that each of the four chains in the molecule contains a central coiled-coil rod domain. However, little progress has been made in modeling the three-dimensional structure. X-ray diffraction data on honeybee silk (Apis mellifera), recorded by Rudall and coworkers, has been re-examined in detail and possible structures developed for the various types of filament seen in the silk glands, and for the packing arrangement in the spun fibers. The original X-ray data were re-collected by scanning figures in the original publications, de-screening and averaging perpendicular to the direction of interest, thereby reducing the graininess of the original images. Sufficient numbers of equatorial and meridional reflections were collected to define the axial projection of the base of the unit cell in fibers drawn from the contents of the silk glands, and to suggest that the axial period is different from that suggested by Rudall and coworkers. Models for two types of filament of increasing diameter are developed based on the node-internode packing scheme observed in protein crystals containing four-strand α-helical ropes. The central domains of the four component chains in the molecule are enclosed by N- and C-terminal domains with widely different lengths and compositions. The fibers thus have a composite filament-matrix texture, and possible locations for the matrix are discussed.

Thermal characterisation of thermotropic nematic liquid-crystalline elastomers

ABSTRACTNematic liquid-crystalline elastomers (LCEs) are weakly cross-linked polymeric networks that exhibit rubber elasticity and liquid-crystalline orientational order due to the presence of mesogenic groups. Three end-on side-chain nematic LCEs were investigated using real-time synchrotron wide-angle X-ray scattering (WAXS), differential scanning calorimetry (DSC), and thermogravimetry (TG) to correlate the thermal behaviour with structural and chemical differences among them. The elastomers differed in cross-linking density and mesogen composition. Thermally reversible glass transition temperature, Tg, and nematic-to-isotropic transition temperature, Tni, were observed upon heating and cooling. By varying the heating rate, Tg0 and Tni0 were determined at zero heating rate. The temperature dependence of the orientational order parameter was determined from the anisotropic azimuthal angular distribution of equatorial reflections seen during real-time WAXS. Results show that the choice of cross-linking unit, its shape, density, and structure of co-monomers, all influence the temperature range over which the thermal transitions take place. Including multi-ring aromatic groups as cross-linkers increased the effective stiffness of the cross-linking, resulting in a higher glass transition temperature. The nematic-to-isotropic transition temperature increased in the presence of multi-ring aromatic structures, as either cross-linkers or mesogens, particularly when the multi-ring structures were larger than the low-molar-mass mesogen common to all three samples.

X-Ray Fiber Diffraction Recordings from Oriented Demembranated Chlamydomonas Flagellar Axonemes

The high homology of its axonemal components with humans and a large repertoire of axonemal mutants make Chlamydomonas a useful model system for experiments on the structure and function of eukaryotic cilia and flagella. Using this organism, we explored the spatial arrangement of axonemal components under physiological conditions by small-angle x-ray fiber diffraction. Axonemes were oriented in physiological solution by continuous shear flow and exposed to intense and stable x rays generated in the synchrotron radiation facility SPring-8, BL45XU. We compared diffraction patterns from axonemes isolated from wild-type and mutant strains lacking the whole outer arm (oda1), radial spoke (pf14), central apparatus (pf18), or the α-chain of the outer arm dynein (oda11). Diffraction of the axonemes showed a series of well-defined meridional/layer-line and equatorial reflections. Diffraction patterns from mutant axonemes exhibited a systematic loss/attenuation of meridional/layer-line reflections, making it possible to determine the origin of various reflections. The 1/24 and 1/12 nm−1 meridional reflections of oda1 and oda11 were much weaker than those of the wild-type, suggesting that the outer dynein arms are the main contributor to these reflections. The weaker 1/32 and 1/13.7 nm−1 meridional reflections from pf14 compared with the wild-type suggest that these reflections come mainly from the radial spokes. The limited contribution of the central pair apparatus to the diffraction patterns was confirmed by the similarity between the patterns of the wild-type and pf18. The equatorial reflections were complex, but a comparison with electron micrograph-based models allowed the density of each axonemal component to be estimated. Addition of ATP to rigor-state axonemes also resulted in subtle changes in equatorial intensity profiles, which could report nucleotide-dependent structural changes of the dynein arms. The first detailed description of axonemal reflections presented here serves as a landmark for further x-ray diffraction studies to monitor the action of constituent proteins in functional axonemes.

Aberrant post-translational modifications compromise human myosin motor function in old age.

Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

Titin-Based Modulation of Sarcomere Structure as Revealed by Equatorial X-Ray Diffraction

Myofilament Length Dependent Activation (LDA) is the cellular mechanism underlying the Frank-Starling Law of the Heart. LDA is defined as an increase in force for a given amount of calcium when the muscle is stretched to a longer sarcomere length. Despite its physiological importance, the sensor that underlies LDA is not known. To address this issue we use X-ray diffraction of intact twitching rat papillary muscle where muscle initially at maximum sarcomere length (Lmax) is quickly released to slack length (Slack). X-ray patterns are taken is a 10 ms window just prior to force generation.Previous analyses of the data did not support the hypothesis that there was a radially outward movement of myosin heads at Lmax requiring some other explanation. The equatorial portion of our X-ray patterns show 5 pairs of well-resolved diffraction spots from the 1,0 to the 3, 0 equatorial reflection. This allowed calculation of two dimensional electron density maps (ED maps) comparing Lmax and Slack from both wild type rats and mutant rats with exceptionally long titin (J Mol Cell Cardiol. 44:983-91 2008) where the passive tension generated at Lmax, as well as LDA, is much reduced. Difference ED maps (Lmax-Slack) from WT rats indicate a better localization of both thick and thin filaments at Lmax plus the existence of bridging density joining the thick and thin filaments at Lmax and not at slack. In difference ED maps from the titin mutant rats, the bridging structures are absent and only the thick filaments appeared better localized. These results support a model where a small number of crossbridges transmit titin-based strain from the thick filament to the thin filament as part of the mechanism of LDA. Supported by NIH R01HL075494 and 9 P41 GM103622.

Hugh Esmor Huxley (1924–2013)

Hugh Huxley on the Boston North Shore. Hugh Esmor Huxley was born on February 25th, 1924 and raised in Birkenhead, Cheshire, England. He gained admittance to Christ’s College Cambridge in 1941 and studied physics. Britain was at war; Hugh elected to interrupt his studies and join the Royal Air Force, working on the development of radar. He was honored for his substantial contributions by being awarded membership of the Order of the British Empire (MBE). Hugh returned to Cambridge in 1947, his enthusiasm for nuclear physics irrevocably diminished by the horrors of Hiroshima. For his doctoral research at the Cavendish Laboratory, rather than pursue nuclear physics Hugh joined Max Perutz’s group working on the X-ray analysis of crystalline proteins, where he became John Kendrew’s first research student. Hugh had come across Dick Bear’s X-ray diffraction patterns from air-dried muscle and, moreover, was amazed to discover no one really knew how muscle works. Hugh set about building an X-ray camera and X-ray generator capable of resolving spacing of 300–400 A to take diffraction photographs of wet “living” muscle. He saw for the first time the equatorial reflections arising from a hexagonal array of filaments and somewhat prophetically guessed that these arose from filaments of myosin on the hexagonal lattice points, with actin filaments in between. Hugh also suggested the filaments were linked by “cross bridges.” In 1952 Hugh went to the Massachusetts Institute of Technology (MIT) as a postdoctorate scholar in Frank Schmitt’s department to learn electron microscopy. Soon, Hugh’s tranverse sections of fixed muscle showed the arrangement of filaments he had postulated from his X-ray studies, together with a hint of cross-bridges connecting the filaments. The arrival of Jean Hanson at MIT provided further impetus. Jean was experienced in phase-contrast microscopy. The units of … [↵][1]1E-mail: holmes{at}mpimf-heidelberg.mpg.de. [1]: #xref-corresp-1-1