In cellulo analysis of huntingtin inclusion bodies by cryogenic nanoprobe SAXS

Huntington's disease (HD) is one of nine neurodegenerative disorders associated with an extension of polyglutamine (polyQ) in proteins. In HD, the polyQ tract in the huntingtin protein is extended beyond a threshold of 38 amino acids leading to the formation of amyloidal structures in the cytoplasm and nucleus. We investigated here the structure of Htt (Huntingtin) amyloid fibrils in cellulo with nanoprobe small angle X-ray scattering. As these measurements were performed under cryogenic conditions, the information is obtained on the aggregates in their natural, hydrated environment without the need of staining and chemical fixation. We also could show the presence of oligomer structures not visible in fluorescence microscopy. Structural information on repetitive units inside of Htt inclusion bodies was determined from the SAXS data and compared to electron microscopy images. The results suggest that nanoprobe cryo-SAXS can serve as powerful tool to investigate the kinetics of amyloid aggregate formation inside cells and to understand how fibril formation can be influenced by drugs and other external stimuli

X-ray fluorescence analysis of metal distributions in cryogenic biological samples using large-acceptance-angle SDD detection and continuous scanning at the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III

A new Rococo 2 X-ray fluorescence detector was implemented into the cryogenic sample environment at the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III, DESY, Hamburg, Germany. A four sensor-field cloverleaf design is optimized for the investigation of planar samples and operates in a backscattering geometry resulting in a large solid angle of up to 1.1 steradian. The detector, coupled with the Xspress 3 pulse processor, enables measurements at high count rates of up to 10$^{6}$ counts per second per sensor. The measured energy resolution of ∼129 eV (Mn $\Kappa\alpha$ at 10000 counts s$^{−1}$) is only minimally impaired at the highest count rates. The resulting high detection sensitivity allows for an accurate determination of trace element distributions such as in thin frozen hydrated biological specimens. First proof-of-principle measurements using continuous-movement 2D scans of frozen hydrated HeLa cells as a model system are reported to demonstrate the potential of the new detection system