Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold

Background Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. Results Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. Conclusion Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Elucidation of fibril structure responsible for swimming in Spiroplasma using electron microscopy

Spiroplasma, known pathogens of arthropods and plants, are helical-shaped bacteria lacking the peptidoglycan layer. They swim by alternating between left- and right-handed cell helicity, which is driven by an internal structure called the ribbon. This system is unrelated to flagellar motility that is widespread in bacteria. The ribbon comprises the bacterial actin homolog MreB and fibril, the protein specific to Spiroplasma. Here, we isolated the ribbon and its core, the fibril filament, and using electron microscopy, found that the helicity of the ribbon and the cell is linked to the helicity of the fibril. Single particle analysis using the negative-staining method revealed that the three-dimensional structures of the fibril filament comprise a repeated ring structure twisting along the filament axis. Based on these observations, we propose a scheme for the helicity-switching mechanism in which the twists caused by the conformational changes in the fibril filament are accumulated, transmitted to the ribbon, and then propel the cells by rotating the cell body like a screw.Significance StatementSpiroplasma are widespread globally as pathogens of animals and plants. They are also recognized as male-killing bacteria of insects. Their special swimming mechanism is caused by helicity switching, which could be the simplest swimming mechanism. This mechanism has attracted research attention for many years because of the possible application in the field of nano actuators; however, the details of this mechanism remain to be clarified. Here, we reveal the outline of the swimming mechanism by analyzing the structure of the core of the Spiroplasma ribbon.

Langmuir turbulence and filament frontogenesis in the oceanic surface boundary layer

Submesoscale currents, small-scale turbulence and surface gravity waves co-exist in the upper ocean and interact in complex ways. To expose the couplings, the frontogenetic life cycle of an idealized cold dense submesoscale filament interacting with upper ocean Langmuir turbulence is investigated in large-eddy simulations (LESs) based on the incompressible wave-averaged equations. The simulations utilize large domains and fine meshes with $6.4\times 10^{9}$ grid points. Case studies are made with surface winds or surface cooling with waves oriented in across-filament (perpendicular) or down-filament (parallel) directions relative to the two-dimensional filament axis. The currents $u$, $v$ and $w$ are aligned with the across-filament, down-filament and vertical directions, respectively. Frontogenesis is induced by across-filament Lagrangian secondary circulations in the boundary layer, and it is shown to be strongly impacted by surface waves, in particular the propagation direction relative to the filament axis. In a horizontally heterogeneous boundary layer, surface waves induce both mean and fluctuating Stokes-drift vortex forces that modify a linear, hydrostatic turbulent thermal wind (TTW) approximation for momentum. Down-filament winds and waves are found to be especially impactful, they significantly reduce the peak level of frontogenesis by fragmenting the filament into primary and secondary down-welling sites in a broad frontal zone over a width ${\sim}500~\text{m}$. At peak frontogenesis, opposing down-filament jets $\langle v\rangle$ overlie each other resulting in a vigorous vertical shear layer $\unicode[STIX]{x2202}_{z}\langle v\rangle$ with large vertical momentum flux $\langle v^{\prime }w^{\prime }\rangle$. Filament arrest is induced by a lateral shear instability that generates horizontal momentum flux $\langle u^{\prime }v^{\prime }\rangle$ at low wavenumbers. The turbulent vertical velocity patterns, indicative of coherent Langmuir cells, change markedly across the horizontal domain with both across-filament and down-filament winds under the action of submesoscale currents.

Probing accretion of ambient cloud material into the Taurus B211/B213 filament

Context. Herschel observations have emphasized the role of molecular filaments in star formation. However, the origin and evolution of these filaments are not yet well understood, partly because of the lack of kinematic information. Aims. We confirm from a kinematic viewpoint that the Taurus B211/B213 filament is accreting background cloud material, and we investigate the potential influence of large-scale external effects on the formation of the filament. Methods. To examine whether the B211/B213 filament is accreting background gas because of its gravitational potential, we produced a toy accretion model and compared its predictions to the velocity patterns observed in 12CO (1–0) and 13CO (1–0). We also examined the spatial distributions of Hα, Planck 857 GHz dust continuum, and HI emission to search for evidence of large-scale external effects. Results. We estimate that the depth of the Taurus cloud around the B211/B213 filament is ~0.3–0.7 pc under the assumption that the density of the gas is the same as the critical density of 13CO (1–0). Compared to a linear extent of >10 pc in the plane of the sky, this suggests that the 3D morphology of the cloud surrounding the B211/B213 filament is sheet-like. Position–velocity (PV) diagrams observed in 12CO (1–0) and 13CO (1–0) perpendicular to the filament axis show that the emission from the gas surrounding B211/B213 is redshifted to the northeast of the filament and blueshifted to the southwest, and that the velocities of both components approach the velocity of the B211/B213 filament as the line of sight approaches the crest of the filament. The PV diagrams predicted by our accretion model are in good agreement with the observed 12CO (1–0) and 13CO (1–0) PV diagrams, supporting the previously proposed scenario of mass accretion into the filament. Moreover, inspection of the spatial distribution of the Hα and Planck 857 GHz emission in the Taurus–California–Perseus region on scales up to >200 pc suggests that the B211/B213 filament may have formed as a result of an expanding supershell generated by the Per OB2 association. Conclusions. Based on these results, we propose a scenario in which the B211/B213 filament was initially formed by large-scale compression of HI gas and is now growing in mass by gravitationally accreting molecular gas of the ambient cloud.

Interaction of the massive cluster system Abell 3016/3017 embedded in a cosmic filament

The galaxy cluster system RXCJ0225.9-4154 with the two sub-clusters A3016 and A3017 is embedded in a large-scale structure filament with signatures of filamentary accretion. In a Chandra observation of this system at a redshift of z = 0.2195 we detect both clusters in X-rays. In addition we detect a filament of X-ray emission connecting the two clusters and a galaxy group therein. The main cluster, A3017, shows indications of shocks most probably from a recent interaction with cluster components along the filament axis as well as a cold front at about 150 kpc from the cluster centre. The filament between the two clusters is likely to be heated by the accretion shocks of the clusters. We discuss two scenarios for the origin of the X-ray filament between the two clusters. In the first scenario the material of the filament has been ripped off of A3017 during the fly-by of A3016 and is now trailing the latter sub-cluster. Support for this scenario is a gas deficit on the eastern side of A3017. In the second scenario the filament between the two clusters does not come from either of them, but a significant contribution could come from the galaxy group located inside and the entire structure is on its first collapse. We favour the second explanation as the gas mass in the filament seems to be too large to be supplied by the interaction of the two Abell clusters. The paper describes many properties of the components of this cluster merger system that are used to assist the interpretation of the observed configuration.

Horizontal flow below solar filaments

Context. Observations of the internal fine structures of solar filaments indicate that the threads of filaments follow magnetic field lines. The magnetic field inside the filament has a strong axial component. Some models of magnetic fields suggest that the field structure in filaments could be caused by the horizontal plasma velocity field near both sides below the filament, where observable shearing effects from the axial component are expected. Aims. The horizontal velocity field in the vicinity of polarity inversion lines is measured in order to determine, if it exhibits a systematic movement that induces shear along the filament axis and convergence perpendicular to the axis. Methods. The horizontal velocity was obtained from the displacement of supergranules, which were derived from Doppler measurements in the solar photosphere. Dopplergrams corrected for rigid rotation and p-mode oscillations were further analyzed by local correlation tracking. Results. Vector fields of the horizontal velocities were measured in 16 areas during 8 time intervals in the years 2000–2002 on both solar hemispheres, each for a few consecutive days. For 64 selected filaments the nearby horizontal velocity vectors were split up into a component along the filament axis and a perpendicular component. Conclusions. Differences between the axial velocities on both sides of the filaments were calculated. In almost all cases the velocity gradient corresponds to the inclination of the threads observed in Hα images. The average transverse velocity does not show any preferred tendency towards a divergence or convergence to the filament axis. Testing the horizontal velocity for the creation of the differential rotation profile in the photosphere reveals a strong dependence of the averaging process on the scale of our velocities.

Dynamic changes in nitric oxide synthase expression are involved in seawater acclimation of rainbow trout Oncorhynchus mykiss

Recent research has shown that nitric oxide (NO) produced by nitric oxide synthases (NOS) is an inhibitor of ion transporter activity and a modulator of epithelial ion transport in fish, but little is known on changes in the NOS/NO system during osmotic stress. We hypothesized that the NOS/NO system responds to salinity changes as an integrated part of the acclimation process. Expression and localization of nos1/Nos1 and nos2/Nos2 were investigated in gill, kidney, and intestine of freshwater (FW)- and seawater (SW)-transferred trout using quantitative PCR, Western blotting, and immunohistochemistry, along with expressional changes of major ion transporters in the gill. The classical branchial ion transporters showed expected expressional changes upon SW transfer, there among a rapid decrease in Slc26a6 mRNA, coding a branchial Cl−/[Formula: see text] exchanger. There was a major downregulation of nos1/ nos2/Nos2 expression in the gill during SW acclimation. A significant decrease in plasma nitrite supported an overall decreased Nos activity and NO production. In the middle intestine, Nos1 was upregulated during SW acclimation, whereas no changes in nos/Nos expression were observed in the posterior intestine and the kidney. Nos1 was localized along the longitudinal axis of the gill filament, beneath smooth muscle fibers of the intestine wall and in blood vessel walls of the kidney. Nos2 was localized within the epithelium adjacent to the gill filament axis and in hematopoietic tissues of the kidney. We conclude that downregulation of branchial NOS is integrated to the SW acclimation process likely to avoid the inhibitory effects of NO on active ion extrusion.

Frontogenesis and frontal arrest of a dense filament in the oceanic surface boundary layer

The evolution of upper ocean currents involves a set of complex, poorly understood interactions between submesoscale turbulence (e.g. density fronts and filaments and coherent vortices) and smaller-scale boundary-layer turbulence. Here we simulate the lifecycle of a cold (dense) filament undergoing frontogenesis in the presence of turbulence generated by surface stress and/or buoyancy loss. This phenomenon is examined in large-eddy simulations with resolved turbulent motions in large horizontal domains using${\sim}10^{10}$grid points. Steady winds are oriented in directions perpendicular or parallel to the filament axis. Due to turbulent vertical momentum mixing, cold filaments generate a potent two-celled secondary circulation in the cross-filament plane that is frontogenetic, sharpens the cross-filament buoyancy and horizontal velocity gradients and blocks Ekman buoyancy flux across the cold filament core towards the warm filament edge. Within less than a day, the frontogenesis is arrested at a small width,${\approx}100~\text{m}$, primarily by an enhancement of the turbulence through a small submesoscale, horizontal shear instability of the sharpened filament, followed by a subsequent slow decay of the filament by further turbulent mixing. The boundary-layer turbulence is inhomogeneous and non-stationary in relation to the evolving submesoscale currents and density stratification. The occurrence of frontogenesis and arrest are qualitatively similar with varying stress direction or with convective cooling, but the detailed evolution and flow structure differ among the cases. Thus submesoscale filament frontogenesis caused by boundary-layer turbulence, frontal arrest by frontal instability and frontal decay by forward energy cascade, and turbulent mixing are generic processes in the upper ocean.

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin’s catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures.