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.


Conditions for α-synuclein aggregation
Monomeric α-synuclein was incubated at a concentration of 70 μM in 25 mM Tris-HCl with 100 mM NaCl (pH 7.4) with constant shaking at 200 rpm for 5 h at 37 °C, conditions shown previously to result in the formation of oligomeric species [6] .

CSF Sample
The CSF sample was collected from a healthy individual (aged 65 years) by lumbar puncture. Standardized protocols for the collection and storage of CSF (www.neurochem.gu.se/TheAlzAssQCProgram) were followed. In short, the lumbar puncture was performed between 9 a.m. and 12 noon to collect 15 mL of CSF in sterile polypropylene tubes. The sample was divided into 1 mL aliquots that were frozen on dry ice and stored at −80 °C in Sarstedt 2mL tube. The time between sample collection, centrifugation, and freezing was maximum 1 h.

Preparation of the nanobody Nb3 and clusterin
Nb3 was prepared as previously described [7][8][9] . Briefly, it was recombinantly expressed in Escherichia coli [9] and purified using immobilized metal affinity chromatography and size-exclusion chromatography [7] . The concentration was measured by UV absorbance spectroscopy using a molecular extinction coefficient, which was calculated based on the sequence of the protein at 280 nm of 21,555 M −1 cm −1 . Clusterin was obtained as previously described [10,11] , and purified from human serum by IgG affinity chromatography or by affinity chromatography using MAb G7 [12] .

Optimization of the dye filled vesicle preparation
Initially we screened a series of different dye molecules for this assay. To ensure that we could attach vesicles to the surface and for probing surface coating protocols we used the dye rhodamine (Rh6G) for encapsulation.
Thereafter, we tested the Ca 2+ -sensitive dyes Fluo-4, Fluo-8 and Cal-520 (Stratech Scientific Ltd, Newmarket, UK) and found that we detected the strongest increase in localized fluorescence intensity using the dye Cal-520.
We tested vesicles of varying sizes (50, 100 and 200, 400 nm) and found that all these vesicles can be used in this assay. We probed vesicles containing varying concentrations of incorporated dye,1-100 μM, and found that improved signals can be detected at higher dye concentrations. Higher concentrations of the dye were found to be preferable for focusing of the instrument on samples incubated in L15 medium or samples that did not induce Ca 2+ influx. However, the incorporation of higher concentrations of dye molecules into the vesicles resulted in the surrounding solution containing a high concentration of free dye, we therefore performed size exclusion chromatography in order to remove free dye molecules from the surrounding solution. We tested both nonpurified and purified vesicle samples and found that we observed considerably less background signal using purified vesicles.
Finally, based on these optimizations and our calculations (see Supporting Information Note 1 and 2, Supporting Information Fig. 1) we performed our experiments using purified vesicles composed of POPC with an average size of 200 nm containing Cal-520 at a concentration of 100 μM. To separate non-incorporated dye molecules from the vesicles, size-exclusion chromatography was performed in buffer using a Superdex TM 200 Increase 10/300 GL column attached to an AKTA pure system (GE Life Sciences) with a flow rate of 0.5 mL/min (Supporting Information Fig. 3).

Preparation of PEGylated slides and immobilization of single vesicles
Initially we screened a variety of surface treatment protocols [13][14][15][16][17][18] and for our experiments we optimized and followed a previously described protocol [18] with slight modifications to perform the actual experiments.

Imaging using Total Internal Reflection Fluorescence Microscope
Imaging was performed using a homebuilt Total Internal Reflection Fluorescence Microscope (TIRFM) based on an inverted Olympus IX-71 microscope. This imaging mode restricts the detected fluorescence signal to within 100-150 nm from the glass-water interface. A 488 nm laser (Toptica, iBeam smart, 200 mW, Munich, Germany) was used to excite the sample. The expanded and collimated laser beam was focused using two Plano-convex lens onto the back-focal plane of the 60X, 1.49NA oil immersion objective lens (APON60XO TIRF, Olympus, product number N2709400) to a spot of adjustable diameter. The fluorescence signal was collected by the same objective and was separated from the excitation beam by a dichroic (Di01-R405/488/561/635, Semrock). The emitted light was passed through an appropriate set of filters (BLP01-488R, Semrock and FF01-520/44-25, Semrock) ( Figure S14). The fluorescence signal was then passed through a 2.5x beam expander and imaged onto a 512 × 512 pixel EMCCD camera (Photometrics Evolve, E VO-512-M-FW-16-AC-110). Images were acquired with a 488nm laser (~10 W/cm 2 ) for 50 frames with a scan speed of 20 Hz and bit depth of 16 bits. Each pixel corresponds to 100 nm. All the measurements were carried out under ambient conditions (T=295K). The open source microscopy manager software Micro Manager 1.4 was used to control the microscope hardware and image acquisition [19,20] .

Performing the Ca 2+ influx assay using TIRFM
Single vesicles tethered to PLL-PEG coated borosilicate glass coverslides (VWR International, 22x22 mm, product number 63 1-0122) were placed on an oil immersion objective mounted on an inverted Olympus IX-71 microscope. Each coverslide was affixed at Frame-Seal incubation chambers and was incubated with 50 µL of HEPES buffer of pH 6.5. Just before the imaging, the HEPES buffer was replaced with 50 µL Ca 2+ containing buffer solution L-15. 16 (4×4) images of the coverslide were recorded under three different conditions (background, in the presence of Aβ42 and after addition of ionomycin (Cambridge Bioscience Ltd, Cambridge, UK), respectively). The distance between each field of view was set to 100 μm, and was automated (bean-shell script, Micromanager) to avoid any user bias ( Figure S3). After each measurement the script allowed the stage (Prior H117, Rockland, MA, USA) to move the field of view back to the start position such that identical fields of view could be acquired for the three different conditions. We screened surface treatment protocols, PEG: biotin-PEG ratios, vesicle size, different encapsulate Ca 2+ binding dyes and their concentrations to maximize the sensitivity of this assay.
Images of the background were acquired in the presence of L15 buffer. For each field of view 50 images were taken with an exposure time of 50 ms. Thereafter, 50 µL of the aggregation reaction, diluted to a concentration of twice the targeted value, was added and incubated for 10 min. Importantly we made sure that the glass coverslides were not moved during the addition of samples and then images were recorded. Next, 10 µL of a solution containing 1 mg/mL of ionomycin (Cambridge Bioscience Ltd, Cambridge, UK) was added and incubated for 5 min and subsequently images of Ca 2+ saturated single vesicles in the same fields of view were acquired.

Experiments with recombinant Aβ42 in CSF
To study the influence of the presence of a complex environment on the Ca 2+ influx, we have taken samples of recombinant Aβ42 aggregation reactions corresponding to t2 and serially diluted it in the CSF to measure the concentration dependence of the Ca 2+ influx. Firstly, we imaged the coverslides in presence of 15 µL of L15 buffer. Then aliquots of recombinant Aβ42 were diluted in 15 µL of CSF which was added to the coverslides and incubated for 10 min before images were acquired as described previously. Thereafter, we added ionomycin to the sample and imaged the identical fields of view using automatic stage movement to determine the Ca 2+ influx.

Data analysis and quantification of the extent of Ca 2+ influx
The recorded images were analyzed using ImageJ [21,22] to determine the fluorescence intensity of each spot under the three different conditions, namely background (Fbackground), in the presence of an aggregation mixture (Faggregate), and after the addition of ionomycin (FIonomycin). The relative influx of Ca 2+ into an individual vesicle due to aggregates of Aβ42 peptide was then determined using the following equation: The average degree of Ca 2+ influx was calculated by averaging the Ca 2+ influx into individual vesicles.

Supporting Information Note 1: Calculation of the concentration of an individual dye molecule entering into a vesicle
The volume of a single vesicle with a diameter d can be calculated using equation (1).
We used vesicles with diameters of approximately 200 nm, which have a volume of 4.186 x 10 -18 L. For a single molecule, the number of moles can be calculated using Avogadro's number (Navogadro = 6.023 x 10 23 ). Using this value we can determine the concentration of a single molecule that enters a vesicle using equation (2).
Thus, the concentration of a single molecule entering a vesicle with a diameter of 200 nm is 396 nM (Supporting Information Fig. 1).

Supporting Information Note 2: Rationalization for using vesicles with a diameter of 200nm
The effectiveness of our single vesicle assay is primarily determined by two parameters with different dependencies on the size of a vesicle -(i) high dynamic range and (ii) high sensitivity (Supporting Information Fig.   1).
High dynamic range is the capacity to detect differences in fluorescence intensity over a range of varying amounts of Ca 2+ influx within individual vesicles, without reaching saturation. The dynamic range of the assay described here is related to the maximum amount of measurable Ca 2+ influx (e.g. when all dye molecules within a single vesicle are saturated with Ca 2+ ), which is directly proportional to the volume of a vesicle. For example, using vesicles with a larger volume enables the encapsulation of more Cal-520 dye molecules, therefore reaching saturation of the maximum fluorescence intensity at larger amounts of Ca 2+ influx compared to a vesicle of a smaller volume with less Cal-520 dye molecules incorporated.