The mutational landscape of a prion-like domain, bioRxiv, 2019-04-02

AbstractSpecific insoluble protein aggregates are the hallmarks of many neurodegenerative diseases1–5. For example, cytoplasmic aggregates of the RNA-binding protein TDP-43 are observed in 97% of cases of Amyotrophic Lateral Sclerosis (ALS)6,7. However, it is still unclear for ALS and other diseases whether it is the insoluble aggregates or other forms of the mutated proteins that cause these diseases that are actually toxic to cells8–13. Here we address this question for TDP-43 by systematically mutating14 the protein and quantifying the effects on cellular toxicity. We generated >50,000 mutations in the intrinsically disordered prion-like domain (PRD) and observed that changes in hydrophobicity and aggregation potential are highly predictive of changes in toxicity. Surprisingly, however, increased hydrophobicity and cytoplasmic aggregation actually reduce cellular toxicity. Mutations have their strongest effects in a central region of the PRD, with variants that increase toxicity promoting the formation of more dynamic liquid-like condensates. The genetic interactions in double mutants reveal that specific structures exist in this ‘unstructured’ region in vivo. Our results demonstrate that deep mutagenesis is a powerful approach for probing the sequence-function relationships of intrinsically disordered proteins as well as their in vivo structural conformations. Moreover, we show that aggregation of TDP-43 is not harmful but actually protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

biorxiv genetics 200-500-users 2019

ezTrack An open-source video analysis pipeline for the investigation of animal behavior, bioRxiv, 2019-03-30

AbstractTracking small animal behavior by video is one of the most common tasks in the fields of neuroscience and psychology. Although commercial software exists for the execution of this task, commercial software often presents enormous cost to the researcher, and can also entail purchasing specific hardware setups that are not only expensive but lack adaptability. Moreover, the inaccessibility of the code underlying this software renders them inflexible. Alternatively, available open source options frequently require extensive model training and can be challenging for those inexperienced with programming. Here we present an open source and platform independent set of behavior analysis pipelines using interactive Python (iPythonJupyter Notebook) that researchers with no prior programming experience can use. Two modules are described. One module can be used for the positional analysis of an individual animal across a session (i.e., location tracking), amenable to a wide range of behavioral tasks including conditioned place preference, water maze, light-dark box, open field, and elevated plus maze, to name but a few. A second module is described for the analysis of conditioned freezing behavior. For both modules, a range of interactive plots and visualizations are available to confirm that chosen parameters produce results that conform to the user’s approval. In addition, batch processing tools for the fast analysis of multiple videos is provided, and frame-by-frame output makes aligning the data with neural recording data simple. Lastly, options for cropping video frames to mitigate the influence of fiberopticelectrophysiology cables, analyzing specified portions of time in a video, and defining regions of interest, can be implemented with ease.

biorxiv neuroscience 200-500-users 2019

 

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