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.

Keywords: Alzheimer's disease; fluorescence imaging; nano-scale biophysics; neurodegeneration; protein aggregation.

Figure 1

Quantitative high‐throughput fluorescence imaging of Ca²⁺ ion influx into individual surface‐tethered vesicles imaged using TIRF microscopy. a) Individual vesicles filled with the fluorescent dye Cal‐520 are immobilized on a polymer‐passivated (PLL‐g‐PEG/PLL‐g‐PEG‐biotin) glass cover slide through biotin‐neutravidin tethering. Addition of membrane‐disrupting species (e.g. protein aggregates) leads to Ca²⁺ influx into vesicles resulting in an increase in the localized fluorescence intensity. b) Identical positions of the coverslides are imaged under three different conditions, shown schematically (i)–(iii) and as TIRF images (iv)–(vi). Images are acquired in the presence of only Ca²⁺ buffer [(i) and (iv)], followed by the addition of protein aggregates [(ii) and (v)], and then the addition of the ionophore ionomycin [(iii) and (vi)]. The TIRF images were averaged over 50 frames with an exposure time of 50 ms each without further image processing. Individual vesicles containing Cal‐520 dye molecules appear as localized bright spots under 488‐nm illumination. In the presence of Ca²⁺ buffer alone, the intensity of a vesicle is comparable to that of the background due to no or minimal Ca²⁺ influx into the vesicles. Addition and incubation with protein aggregates causes a significant increase in the fluorescence of some of the vesicles and subsequent addition of ionomycin results in saturation of all vesicles by Ca²⁺, causing detection of a maximum value of the fluorescence signal. All the images are shown with equal contrast. The scale bar: 3 μm. c) Ca²⁺ influx into 13 individual vesicles as shown in (b) (iv)–(vi). The percentage of Ca²⁺ influx in each vesicle was calculated using: (F _aggregate−F _blank)*100/ (F _ionomycin−F _aggregate), where F _blank, F _aggregate, F _ionomycin represent the fluorescence in the presence of Ca²⁺ containing buffer, a solution containing protein samples (e.g. aggregates), and ionomycin, respectively. d) Histogram showing the distribution of the percentage of Ca²⁺ influx into 744 individual vesicles after the addition of aliquots taken from an aggregation reaction of Aβ42 with an average Ca²⁺ influx of 19.44 %.

Figure 2

Ca²⁺ influx caused by aggregates of the Aβ42 peptide. a) Kinetic profile of Aβ42 aggregation under quiescent conditions at a concentration of 2 μm protein. The time points correspond to the start of the aggregation reaction (t₁), the end of the lag‐phase (t₂), and the plateau phase (t₃), at each of which an aliquot was taken and diluted to a concentration of 5 nm (monomer equivalents). b) Ca²⁺ influx induced by an aggregation mixture at time points t₁, t₂, and t₃; a Ca²⁺ influx of 100 % corresponds to the Ca²⁺ influx caused by ionomycin. c) Size exclusion chromatography of an aliquot taken at t₂ and the degree of Ca²⁺ influx induced by different elution volumes was then tested. Oligomeric forms of the protein were found to elute at volumes of 7 to 12 mL and monomeric protein at a later elution volume of 14 to 17 mL. d) The dependence of Ca²⁺ influx on the total concentration of Aβ42 (in monomer equivalents) measured by serial dilution of an aliquot taken at t₂. The error bars represent variations between different fields of view and the data correspond to averages over measurements of more than 700 vesicles. Inset: expansion of the low concentration region.

Figure 3

Testing the inhibition of aggregate‐induced Ca²⁺ influx resulting from the treatment of mixtures with binding proteins of aliquots of an Aβ42 aggregation reaction corresponding to time t₂. a) Inhibition of Ca²⁺ influx resulting from Aβ42 aggregates by the chaperone clusterin (at 100 nm), the nanobody Nb3 (at 100 nm), and an anti‐GFP antibody (P<0.05 between Aβ42 and Aβ42 + antiGFP antibody). b) Concentration dependence of the inhibition by clusterin and c) by Nb3. Insets in (b) and (c) show the normalized Ca²⁺ influx with respect to the Ca²⁺ influx in the absence of clusterin and Nb3, respectively. Lines are guides to the eye. The error bars represent variations between different fields of view and the data correspond in each case to averages over measurements of more than 700 vesicles.