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A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils

Timothy S. Jarvela, View ORCID ProfileKriti Chaplot, View ORCID ProfileIris Lindberg
doi: https://doi.org/10.1101/2020.10.12.335885
Timothy S. Jarvela
Department of Anatomy and Neurobiology, University of Maryland-Baltimore, Baltimore MD 21201
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Kriti Chaplot
Department of Anatomy and Neurobiology, University of Maryland-Baltimore, Baltimore MD 21201
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Iris Lindberg
Department of Anatomy and Neurobiology, University of Maryland-Baltimore, Baltimore MD 21201
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  • ORCID record for Iris Lindberg
  • For correspondence: ilindberg@som.umaryland.edu
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ABSTRACT

Alpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of α-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized α-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between α-synuclein and green fluorescent protein. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, we are able to use protection from TEV cleavage to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted October 12, 2020.
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A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils
Timothy S. Jarvela, Kriti Chaplot, Iris Lindberg
bioRxiv 2020.10.12.335885; doi: https://doi.org/10.1101/2020.10.12.335885
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A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils
Timothy S. Jarvela, Kriti Chaplot, Iris Lindberg
bioRxiv 2020.10.12.335885; doi: https://doi.org/10.1101/2020.10.12.335885

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