Browsing by Subject "Stress Granules"

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    Development of High-Throughput and High-Content Analysis Assays for Neurodegeneration-Related Intrinsically Disorderd Proteins
    (2020) Nathan, Noah
    We developed a FRET-based protein-protein biosensor of Fused in Sarcoma (FUS), an Amyotrophic Lateral Sclerosis and Frontotemporal Dementia related protein. The FUS biosensor had a robust signal, yielding a FRET efficiency of 7.61% with low signal to noise ratio. Based on high-throughput FRET measurements, we determined a standard deviation of 0.0158 ns for the donor/acceptor fluorescent lifetime. In future drug screens for compounds that modulate FUS aggregation, the threshold for hits will be set at 2.45 ± 0.0474 ns (3 SD). In addition, we implemented a MATLAB script that quantifies the ratio of cytoplasmic to nuclear FUS-rich stress granules from fluorescent images of FUS-GFP-expressing N2a cells. We showed that sorbitol, which has been shown to cause FUS mislocalization via hypertonic stress, caused a shift in the cytoplasmic to nuclear ratio of FUS as compared to untreated cells. We implemented a second MATLAB automated algorithm that quantifies total neurite outgrowth from neurospheres expressing the Parkinson's disease-related protein alpha-synuclein. We found that the mutant aSyn A53T caused a reduction of 41% in total neurites as compared to WT aSyn-expressing neurospheres. This result was validated by counting neurites manually with ImageJ, which yielded a reduction in neurites of 49%.