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Ultrasensitive Measurement of Ca²⁺ Influx into Lipid Vesicles Induced by Protein Aggregates.

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 A?42 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 A? peptide. We show that the assay can be used to study aggregates from other proteins, such as ?-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.

SUBMITTER: Flagmeier P

PROVIDER: S-EPMC5615231 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates.

Flagmeier Patrick P De Suman S Wirthensohn David C DC Lee Steven F SF Vincke Cécile C Muyldermans Serge S Knowles Tuomas P J TPJ Gandhi Sonia S Dobson Christopher M CM Klenerman David D

Angewandte Chemie (International ed. in English) 20170505 27

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 reflectio ...[more]

PMID: 28474754

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Ultrasensitive Measurement of Ca²⁺ Influx into Lipid Vesicles Induced by Protein Aggregates.

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Ultrasensitive Measurement of Ca<sup>2+</sup> Influx into Lipid Vesicles Induced by Protein Aggregates.

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