Robin Schubert

Exosomal cellular prion protein drives fibrillization of amyloid beta and counteracts amyloid beta-mediated neurotoxicity.

Alzheimers disease is a common neurodegenerative, progressive, and fatal disorder. Generation and deposition of amyloid beta (Aβ) peptides associate with its pathogenesis and small soluble Aβ oligomers show the most pronounced neurotoxic effects and correlate with disease initiation and progression. Recent findings showed that Aβ oligomers bind to the cellular prion protein (PrPC) eliciting neurotoxic effects. The role of exosomes, small extracellular vesicles of endosomal origin, in Alzheimers disease is only poorly understood. Besides serving as disease biomarkers they may promote Aβ plaque formation, decrease Aβ‐mediated synaptotoxicity, and enhance Aβ clearance. Here, we explore how exosomal PrPC connects to protective functions attributed to exosomes in Alzheimers disease. To achieve this, we generated a mouse neuroblastoma PrPC knockout cell line using transcription activator‐like effector nucleases. Using these, as well as SH‐SY5Y human neuroblastoma cells, we show that PrPC is highly enriched on exosomes and that exosomes bind amyloid beta via PrPC. Exosomes showed highest binding affinity for dimeric, pentameric, and oligomeric Aβ species. Thioflavin T assays revealed that exosomal PrPC accelerates fibrillization of amyloid beta, thereby reducing neurotoxic effects imparted by oligomeric Aβ. Our study provides further evidence for a protective role of exosomes in Aβ‐mediated neurodegeneration and highlights the importance of exosomal PrPC in molecular mechanisms of Alzheimers disease.

Reliably distinguishing protein nanocrystals from amorphous precipitate by means of depolarized dynamic light scattering

Crystallization of biological macromolecules such as proteins implies several prerequisites, for example, the presence of one or more initial nuclei, sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process. The initiation of a crystallization process itself is a stochastic event, forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters. The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable. However, early identification of the occurrences of unit cells is the key to nanocrystal detection. The optical properties of a crystal lattice offer a potential signal with which to detect whether a transition from disordered to ordered particles occurs, one that has so far not been tested in nanocrystalline applications. The ability of a lattice to depolarize laser light depends on the different refractive indices along different crystal axes. In this study a unique experimental setup is used to detect nanocrystal formation by application of depolarized scattered light. The results demonstrate the successful detection of nano-sized protein crystals at early stages of crystal growth, allowing an effective differentiation between protein-dense liquid cluster formation and ordered nanocrystals. The results are further verified by complementary methods like X-ray powder diffraction, second harmonic generation, ultraviolet two-photon excited fluorescence and scanning electron microscopy.

A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

In situ crystallization using a Kapton sandwich assembly allows diffraction data to be recorded from multiple protein crystals at room temperature with millisecond temporal resolution at high-brilliance synchrotron X-ray radiation sources.

X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography

Multi-crystal serial crystallography data can be used for UV and X-ray radiation-damage-induced phasing.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 µm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase – the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

X-Ray Diffraction Data Cubic Insulin And Thaumatin Ssx For Rip Phasing

Diffraction images collected on dectris Pilatus. Format of images in *.cbf.nnImages collected with Mesh and Collect strategy at ESRF.nnThaumatin : contain 6 sets of exposure reflecting 6 increasing Dose (from 1 to 6)nnCubic Insulin : contain 3 sets of sub-data sets : Before_1 and Before_2 (means before UV irradiation). and After : (after UV irradiation).nnFor full information refer to material and method in : ( submit, will be update )