Flagmeier, P; De, S; Wirthensohn, DC; Lee, SF; Vincke, C; Muyldermans, S; Knowles, TPJ; ... Klenerman, D; + view all Flagmeier, P; De, S; Wirthensohn, DC; Lee, SF; Vincke, C; Muyldermans, S; Knowles, TPJ; Gandhi, S; Dobson, CM; Klenerman, D; - view fewer (2017) Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates. Angewandte Chemie - International Edition , 56 (27) pp. 7750-7754. 10.1002/anie.201700966.

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Abstract

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 Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ 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 Ca2+ 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.

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