TY - JOUR
T1 - Ultrasensitive Measurement of Ca(2+) Influx into Lipid Vesicles Induced by Protein Aggregates
AU - Flagmeier, Patrick
AU - De, Suman
AU - Wirthensohn, David C
AU - Lee, Steven F
AU - Vincke, Cécile
AU - Muyldermans, Serge
AU - Knowles, Tuomas P J
AU - Gandhi, Sonia
AU - Dobson, Christopher M
AU - Klenerman, David
N1 - © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2017/6/26
Y1 - 2017/6/26
N2 - 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.
AB - 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.
U2 - 10.1002/anie.201700966
DO - 10.1002/anie.201700966
M3 - Article
C2 - 28474754
VL - 56
SP - 7750
EP - 7754
JO - Angewandte Chemie International Edition
JF - Angewandte Chemie International Edition
SN - 1433-7851
IS - 27
ER -