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Ultrasensitive Measurement of Ca 2+ Influx into Lipid Vesicles Induced by Protein Aggregates
Author(s) -
Flagmeier Patrick,
De Suman,
Wirthensohn David C.,
Lee Steven F.,
Vincke Cécile,
Muyldermans Serge,
Knowles Tuomas P. J.,
Gandhi Sonia,
Dobson Christopher M.,
Klenerman David
Publication year - 2017
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201700966
Subject(s) - vesicle , chemistry , total internal reflection fluorescence microscope , biophysics , protein aggregation , fluorescence , lipid vesicle , fluorescence microscope , membrane , chaperone (clinical) , biochemistry , biology , pathology , medicine , physics , quantum mechanics
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.