Ultrasensitive Measurement of Ca(2+) Influx into Lipid Vesicles Induced by Protein Aggregates.
Angewandte Chemie - International Edition
John Wiley & Sons Ltd.
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Flagmeier, P., De, S., Wirthensohn, D., Lee, S., Vincke, C., Muyldermans, S., Knowles, T., et al. (2017). Ultrasensitive Measurement of Ca(2+) Influx into Lipid Vesicles Induced by Protein Aggregates.. Angewandte Chemie - International Edition, 56 (27), 7750-7754. https://doi.org/10.1002/anie.201700966
To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca(2+) entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca(2+) sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca(2+) influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.
Alzheimer's disease, fluorescence imaging, nano-scale biophysics, neurodegeneration, protein aggregation
ECH2020 EUROPEAN RESEARCH COUNCIL (ERC) (669237)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (701013)
External DOI: https://doi.org/10.1002/anie.201700966
This record's URL: https://www.repository.cam.ac.uk/handle/1810/265901