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 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.

Optical Structural Analysis of Individual α‐Synuclein Oligomers

“…We imaged individual liposomes in the presence of Ca 2+ buffer (blank), followed by the addition of an aliquot of α‐synuclein aggregates at a concentration of 50 n m and subsequent addition of ionomycin. In the presence of only Ca 2+ buffer, the fluorescence intensity of the vesicles was low and comparable to that of background noise due to minimal Ca 2+ presence within the vesicle 26. After incubation (for 10 minutes) with α‐synuclein samples, we detected an increase in the localised fluorescence intensity of the vesicles showing that Ca 2+ ions could enter the vesicles as a consequence of the aggregates’ induced membrane permeability.…”

“…In the presence of only Ca 2+ buffer, the fluorescence intensity of the vesicles was low and comparable to that of background noise due to minimal Ca 2+ presence within the vesicle. [26] After incubation (for 10 minutes) with a-synuclein samples,w ed etected an increase in the localised fluorescence intensity of the vesicles showing that Ca 2+ ions could enter the vesicles as ac onsequence of the aggregates induced membrane permeability. Subsequent addition of ionomycin, an ionophore enabling Ca 2+ to enter the vesicles,r esults in the saturation of all vesicles with Ca 2+ ions,a llowing us to quantify the extent of membrane disruption (Figure 4c).…”

“…To correlate the structural information of the aggregated species with the ability of these aggregates to generate toxic effects in cells we evaluated their capability to disrupt membranes with at echnique that uses vesicles filled with aC a 2+ sensitive dye. [26] Upon the interaction of ap rotein aggregate with the vesiclesm embrane,C a 2+ ions enter the vesicle from the surrounding solution and hence becomes fluorescent. This change in fluorescence can be detected using TIRF microscopy.W ei maged individual liposomes in the presence of Ca 2+ buffer (blank), followed by the addition of an aliquot of a-synuclein aggregates at ac oncentration of 50 nm and subsequent addition of ionomycin.…”

“…To correlate the structural information of the aggregated species with the ability of these aggregates to generate toxic effects in cells we evaluated their capability to disrupt membranes with a technique that uses vesicles filled with a Ca 2+ sensitive dye 26. Upon the interaction of a protein aggregate with the vesicle's membrane, Ca 2+ ions enter the vesicle from the surrounding solution and hence becomes fluorescent.…”

Optical Structural Analysis of Individual α‐Synuclein Oligomers

“…To correlate the structural information of the aggregated species with the ability of these aggregates to generate toxic effects in cells we evaluated their capability to disrupt membranes with a technique that uses vesicles filled with a Ca 2+ sensitive dye . Upon the interaction of a protein aggregate with the vesicle's membrane, Ca 2+ ions enter the vesicle from the surrounding solution and hence becomes fluorescent.…”