Biology subjects

Rees, E. J.

Publications and source records attributed to Rees, E. J..

5 recordsLinked to original sources

bioRxiv2020

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled repeats were stronger than any of the natural repeats, suggesting balancing of relative properties in BRC repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.44 kCal/mol compared to the strongest natural repeat, BRC4. Crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended {beta}-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear foci after ionizing radiation.

biochemistry↗

bioRxiv2019

Concepts for structured illumination microscopy with extended axial resolution through mirrored illumination

Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the resolution limit both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field microscopy. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5M microscopy, have improved the axial resolution beyond that of the lateral, but at the cost of a significantly more complex optical setup. Here, we investigate a simpler dual-objective scheme which we propose can be easily added to an existing 3D-SIM microscope, providing lateral and axial resolutions in excess of 125 nm with conventional fluorophores and without the need for interferometric detection.

bioengineering↗

bioRxiv2019

OptiJ: Open-source optical projection tomography of large organ samples

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 m resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples.

biophysics↗

bioRxiv2019

An Open-Hardware sample mounting solution for inverted light-sheet microscopes with large detection objective lenses

Implementations of light-sheet microscopes are often incompatible with standard methods of sample mounting. Light-sheet microscopy uses orthogonal illumination and detection to create a thin sheet of light which does not illuminate the sample outside of the depth of field of the detection axis. Typically, this configuration involves a pair of orthogonal objectives which constrains the positioning of a length of coverslips or microscopes in range of the detection objective. We present an open-hardware (1, 2) sample mounting system for light-sheet microscopes using large detection objectives.

cell biology↗

bioRxiv2019

Intra-mitochondrial protein degradationalleviates alpha-synuclein seeding and Abeta 42 aggregation

Mitochondria have long been implicated in Parkinsons disease (PD), however, it is not clear how mitochondrial impairment and alpha-synuclein pathology are coupled. We report here that intra-mitochondrial protein homeostasis plays a major role in alpha-synuclein fibril elongation, as interference with intra-mitochondrial proteases and mitochondrial protein import significantly aggravate alpha-synuclein aggregation. In contrast, direct inhibition of mitochondrial complex I, increase in intracellular calcium concentration or formation of reactive oxygen species (ROS), all of which have been associated with mitochondrial stress, did not affect alpha-synuclein pathology. We further demonstrate that similar mechanisms are involved in Amyloid {beta} 1-42 (A{beta}42) aggregation, suggesting that mitochondria are directly capable of influencing cytosolic protein homeostasis of aggregation-prone proteins.

neuroscience↗