Structural conversion of the spidroin C-terminal domain during assembly of spider silk fibers.

The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to ß-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into ß-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.

Insights into the material properties of dragline spider silk affecting Schwann cell migration.

Dragline silk of Trichonephila spiders has attracted attention in various applications. One of the most fascinating uses of dragline silk is in nerve regeneration as a luminal filling for nerve guidance conduits. In fact, conduits filled with spider silk can measure up to autologous nerve transplantation, but the reasons behind the success of silk fibers are not yet understood. In this study dragline fibers of Trichonephila edulis were sterilized with ethanol, UV radiation, and autoclaving and the resulting material properties were characterized with regard to the silk's suitability for nerve regeneration. Rat Schwann cells (rSCs) were seeded on these silks in vitro and their migration and proliferation were investigated as an indication for the fiber's ability to support the growth of nerves. It was found that rSCs migrate faster on ethanol treated fibers. To elucidate the reasons behind this behavior, the fiber's morphology, surface chemistry, secondary protein structure, crystallinity, and mechanical properties were studied. The results demonstrate that the synergy of dragline silk's stiffness and its composition has a crucial effect on the migration of rSCs. These findings pave the way towards understanding the response of SCs to silk fibers as well as the targeted production of synthetic alternatives for regenerative medicine applications.

Mesoscale Confinement in Bagworm Silk: A Hidden Structural Organization.

While silk fibers produced by silkworms and spiders are frequently described as a network of amorphous protein chains reinforced by crystalline ß-sheet nanodomains, the importance of higher-order, self-assembled structures has been recognized for advanced modeling of mechanical properties. General acceptance of hierarchical structural models is, however, currently limited by lack of experimental results. Indeed, X-ray scattering studies of spider's dragline-type fibers have been particularly limited by low crystallinities. Here we are reporting on probing the local structure of exceptionally crystalline bagworm silk fibers by X-ray nanobeam scattering. Probing the comparable thickness of cross sections with an X-ray nanobeam allows removing the strong scattering background from the outer sericin layer and reveals a hidden structural organization due to a radial gradient in diameters of mesoscale nanofibrillar bundles in the fibroin phase. Our results provide direct support for lateral interactions between nanofibrils.

Mesoscale Ordering of Phycocyanin Molecules in Langmuir-Blodgett Multilayers.

Phycocyanin molecules, which are part of light-harvesting complexes in cyanobacteria, can be assembled into mesoscale multilayer nanofilms by the Langmuir-Blodgett technique. Results obtained by quartz crystal microbalance and atomic force microscopy confirm the homogeneity and reproducibility of phycocyanin Langmuir-Blodgett multilayer deposition. We show by cryo-electron microdiffraction that amorphous phycocyanin Langmuir-Blodgett multilayers form, after annealing at 150 °C and cooling to room temperature, a layered nanofibrillar lattice with rotational disorder. Scanning X-ray nanodiffraction suggests that structural transformation is not homogeneous through the film but limited to patches of up to about 10 µm diameter.

Size-Exclusion Particle Separation Driven by Micro-Flows in a Quasi-Spherical Droplet: Modelling and Experimental Results.

Aqueous solution droplets are supported quasi contact-free by superhydrophobic surfaces. The convective flow in evaporating droplets allows the manipulation and control of biological molecules in solution. In previous works, super-hydrophobic drops on nano-patterned substrates have been used to analyze otherwise undetectable species in extremely low concentration ranges. Here, we used particle image velocimetry (PIV) for studying the flow field in water droplets containing polystyrene particles on a pillared silicon super-hydrophobic chip. The particles describe vortex-like motions around the droplet center as long as the evaporating droplet maintains a spherical shape. Simulations by a Finite Element Method (FEM) suggest that the recirculating flow is due to the temperature gradient along the droplet rim, generating a shear stress. Notably, the characteristics of the internal flow can be modulated by varying the intensity of the temperature gradient along the drop. We then used the flow-field determined by experiments and an approximate form of the Langevin equation to examine how particles are transported in the drop as a function of particle size. We found that larger particles with an average size of µ36 µm are preferentially transported toward the center of the substrate, differently from smaller particles with a 10-fold lower size that are distributed more uniformly in the drop. Results suggest that solutions of spherical particles on a super-hydrophobic chip can be used to separate soft matter and biological molecules based on their size, similarly to the working principle of a time-of-flight (ToF) mass analyzer, except that the separation takes place in a micro-sphere, with less space, less time, and less solution required for the separation compared to conventional ToF systems.

Mesoscale structures in amorphous silks from a spider's orb-web.

Of the 7-8 silk fibers making up an orb-web only the hierarchical structural organization of semicrystalline radial fibers -composed of major ampullate silk- has been studied in detail, given its fascinating mechanical features. While major ampullate silk's nanofibrillar morphology is well established, knowhow on mesoscale (> 50-100 nm) assembly and its contribution to mechanical performance is limited. Much less is known on the hierarchical structural organization of other, generally less crystalline fibers contributing to an orb-webs' function. Here we show by scanning X-ray micro&nanodiffraction that two fully amorphous, fine silk fibers from the center of an orb-web have different mesoscale features. One of the fibers has a fibrillar composite structure resembling stiff egg case silk. The other fiber has a skin-core structure based on a nanofibrillar ribbon wound around a disordered core. A fraction of nanofibrils appears to have assembled into mesoscale fibrils. This fiber becomes readily attached to the coat of major ampullate silk fibers. We observe that a detached fiber has ripped out the glycoprotein skin-layer containing polyglycine II nanocrystallites. The anchoring of the fiber in the coat suggests that it could serve for strengthening the tension and cohesion of major ampullate silk fibers.

Exploiting Sphingo- and Glycerophospholipid Impairment to Select Effective Drugs and Biomarkers for CMT1A.

In Charcot-Marie-Tooth type 1A (CMT1A), Schwann cells exhibit a preponderant transcriptional deficiency of genes involved in lipid biosynthesis. This perturbed lipid metabolism affects the peripheral nerve physiology and the structure of peripheral myelin. Nevertheless, the identification and functional characterization of the lipid species mainly responsible for CMT1A myelin impairment currently lack. This is critical in the pathogenesis of the neuropathy since lipids are many and complex molecules which play essential roles in the cell, including the structural components of cellular membranes, cell signaling, and membrane trafficking. Moreover, lipids themselves are able to modify gene transcription, thereby affecting the genotype-phenotype correlation of well-defined inherited diseases, including CMT1A. Here we report for the first time a comprehensive lipid profiling in experimental and human CMT1A, demonstrating a previously unknown specific alteration of sphingolipid (SP) and glycerophospholipid (GP) metabolism. Notably, SP, and GP changes even emerge in biological fluids of CMT1A rat and human patients, implying a systemic metabolic dysfunction for these specific lipid classes. Actually, SP and GP are not merely reduced; their expression is instead aberrant, contributing to the ultrastructural abnormalities that we detailed by X-ray diffraction in rat and human internode myelin. The modulation of SP and GP pathways in myelinating dorsal root ganglia cultures clearly sustains this issue. In fact, just selected molecules interacting with these pathways are able to modify the altered geometric parameters of CMT1A myelinated fibers. Overall, we propose to exploit the present SP and GP metabolism impairment to select effective drugs and validate a set of reliable biomarkers, which remain a challenge in CMT1A neuropathy.

Nanoscale Structural Features in Major Ampullate Spider Silk.

Spider major ampullate silk is often schematically represented as a two-phase material composed of crystalline nanodomains in an amorphous matrix. Here we are interested in revealing its more complex nanoscale organization by probing Argiope bruennichi dragline-type fibers using scanning X-ray nanodiffraction. This allows resolving transversal structural features such as an about 1 µm skin layer composed of around 100 nm diameter nanofibrils serving presumably as an elastic sheath. The core consists of a composite of several nm size crystalline nanodomains with poly(l-alanine) microstructure, embedded in a polypeptide network with short-range order. Stacks of nanodomains separated by less ordered nanosegments form nanofibrils with a periodic axial density modulation which is particularly sensitive to radiation damage. The precipitation of larger ß-type nanocrystallites in the outer core-shell is attributed to MaSp1 protein molecules.

Superhydrophobic Surfaces Boost Fibril Self-Assembly of Amyloid ß Peptides.

Amyloid ß (Aß) peptides are the main constituents of Alzheimer's amyloid plaques in the brain. Here we report how the unique microfluidic flows exerted by droplets sitting on superhydrophobic surfaces can influence the aggregation mechanisms of several Aß fragments by boosting their fibril self-assembly. Aß(25-35), Aß(1-40), and Aß(12-28) were dried both on flat hydrophilic surfaces (contact angle (CA) = 37.3°) and on nanostructured superhydrophobic ones (CA = 175.8°). By embedding nanoroughened surfaces on top of highly X-ray transparent Si3N4 membranes, it was possible to probe the solid residues by raster-scan synchrotron radiation X-ray microdiffraction (µXRD). As compared to residues obtained on flat Si3N4 membranes, a general enhancement of fibrillar material was detected for all Aß fragments dried on superhydrophobic surfaces, with a particular emphasis on the shorter ones. Indeed, both Aß(25-35) and Aß(12-28) showed a marked crystalline cross-ß phase with varying fiber textures. The homogeneous evaporation rate provided by these nanostructured supports, and the possibility to use transparent membranes, can open a wide range of in situ X-ray and spectroscopic characterizations of amyloidal peptides involved in neurodegenerative diseases and for the fabrication of amyloid-based nanodevices.

Methodological challenges of optical tweezers-based X-ray fluorescence imaging of biological model organisms at synchrotron facilities.

Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.