Single-molecule fluorescence imaging and deep learning reveal highly heterogeneous aggregation of amyloid-β 42

Significance There are various diseases caused by protein aggregation such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. From the diversity in the fibril structure, aggregation is expected to occur via heterogeneous pathways. However, characterization of this heterogeneity is extremely difficult because it requires following individual fibril formation in a mixture from early oligomerization stages. In this work, we investigated aggregation of the 42-residue isoform of amyloid β (Aβ42) using single-molecule fluorescence imaging and deep learning. We could track the growth of individual fibrils, which allows for a quantitative description of heterogeneous fibril formation and discovery of a new fibril nucleation mechanism. Further characterization of heterogeneity involving Aβ42 will be important for better understanding the disease mechanism.


Purification of proteins
Bacteria pellets from 500 mL LB culture were lysed in 20 mL of bacterial protein extraction reagent (B-Per, Thermo Fisher Scientific, Grand Island, NY) with 50 mM benzamidine hydrochloride, 100 g/mL lysozyme (Sigma, St. Louis, MO), and 5 units of benzonase (Novogen, Madison, WI). The pellets were mixed and resuspended in the lysis buffer and incubated at room temperature for 30 minutes. The lysate was transferred to 50 mL spinning tubes and centrifuged at 30000 g for 45 minutes with Sorvall LYNX 4000. The supernatant was removed for electrophoresis and the pellet containing inclusion bodies were resuspended in 30 mL 1 PBS solution with 10 mM DTT and 1% Triton X-100 and sonicated three times for 20 seconds on ice using a sonicator at 100% power (Model Q55, Qsonica, Newtown, CT). The solution was then centrifuged at 30000 g for 30 minutes at 4C. The supernatant was discarded and the remaining pellet was resuspended in the same PBS buffer used in the previous sonication step. One molar sodium chloride was added to remove DNA and RNA from pellets. The mixture was sonicated as in the previous step and centrifuged at 30000 g for 30 minutes at 4C. Resuspension, sonication, and centrifugation were repeated in 1 PBS. The pellet containing inclusion bodies was dissolved in 5 mL of 50 mM Tris-HCl with 6 M guanidine hydrochloride (GdmCl) and 10 mM DTT and kept at room temperature overnight for complete extraction of A proteins. The solution was then centrifuged at 30000 g at 4C for 45 minutes to remove the insoluble pellet. The supernatant was collected for further purification. The supernatant was loaded on PhastSystem (Pharmacia, Baltimore, MD) gels. Gels were stained with Phastgel Blue R (Pharmacia, Baltimore, MD) then washed until protein bands were clearly shown. A proteins with and without AviTag and linker appeared at 8 kDa and 5 kDa on gels, respectively, and these were the smallest proteins presenting in the inclusion body. The protein solutions (200 μL) were loaded onto the AKTA pure FPLC system equipped with a Superdex TM 75 10/300GL size exclusion column (GE Healthcare, Chicago, IL). The separation was run with 50 mM Tris-HCl, 4 M GdmCl solution at a flow rate of 0.8 mL/min. The fractions containing 5 kDa or 8 kDa proteins identified by Phastgel were collected and concentrated using Amicon Ultra centrifugal filters (EMD Millipore, Billerica, MA) and then subjected to the second round of FPLC purification.

Dye-labeling and purification
We labeled Avi-C-A42 with Alexa Fluor 488 maleimide (Alexa 488, A10254, Thermo Fisher Scientific, Carlsbad, CA) and C-A42 with Alexa Fluor 594 maleimide (Alexa 594, A10256, Thermo Fisher Scientific, Carlsbad, CA). A (~ 0.2 mg) in 4 M GdmCl Tris buffer solution (pH 8) was concentrated to 100 L in 6 M GdmCl using Amicon centrifugal filters and pH was adjusted to 7.0 by acetate buffer. 100 L of protein solution was mixed with 0.1 mg of Alexa 488 or 594 pre-dissolved in 5L of DMSO. We incubated the mixture at room temperature overnight. Then the reaction was quenched by adding 4 L of -mercaptoethanol. The reaction mixture was fractionated on a Superdex TM 75 10/300GL size exclusion column equilibrated with 50 mM Tris-HCl, pH 8.0, 4 M GdmCl to remove the excess free dye. The peptide labeled with the dyes showed overlapping peaks of absorbance monitored at 280, 494 nm for Alexa 488, and 280, 594 nm for Alexa 594. The labeled protein concentration was determined by the absorbance at 494 nm or 594 nm measured by Cary 8454 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA). Purified samples were aliquoted into 10L and kept at -80C for future experiments.

Plate reader experiment for monitoring Aβ aggregation
A protein sample (20 -50 L) was loaded into 96-well half-bottom none binding surface polystyrene plates (REF 3881, Corning, Kennebunk, ME) for plate reader recording (Spark, TECAN, Switzerland). To monitor the aggregation of unlabeled Aβ42, A42 stock solution (30 M) was diluted into 6 M thioflavin T (ThT, Sigma, St. Louis, MO) in 1 PBS (pH 7.4) to the final concentration of 2 M and volume of 50 L. All wells were sealed with a piece of parafilm to prevent evaporation in the bottom reading mode. The aggregation was monitored at 37ºC for several hours. The fluorescence signal was recorded every 5 min. Samples were excited (50 flashes) at 420 nm (ThT) and 575 nm (Alexa 594) and fluorescence was detected at 480 nm (ThT) and 627 nm (Alexa 594) with a 20 nm bandwidth. The focus along z-axis was set manually at 29500 m and the automatic gain regulation feature was used.

Electron microscopy experiment
The fibrils of unlabeled A42 and Alexa 594-Aβ42, prepared by incubating 5 M proteins at 37ºC overnight, were imaged with FEI Morgagni microscope, which was operated at 80 kV and equipped with an AMT Advantage HR CCD camera. The samples were adsorbed to glowdischarged carbon films on lacey-carbon-coated copper mesh grids for 1 min, rinsed with deionized water, stained with 3% uranyl acetate for 1 min, and dried in air for imaging.
We immobilized Alexa 488-labeled Avi-D-A42 on a biotin-embedded, polyethyleneglycol-coated glass coverslip (Bio_01, Microsurfaces Inc.) via a biotin (surface)-NeutrAvidin-biotin (protein) linkage (2) and incubated with 500 nM of A-A42 in 1 PBS, pH 7.5, including a cocktail of 100 mM β-mercaptoethanol, 10 mM Cystamine (3), 2 mM 4nitrobenzyl alcohol (NBA), 2 mM cyclooctatetraene (COT), and 2 mM Trolox (4,5) to reduce photoblinking and photobleaching of dyes. The purpose of this approach was to search for stepwise oligomer formation or fibril growth from immobilized donor-labeled A42 monomer, but this was not observed. This finding is expected because the number of immobilized molecules is so much smaller than the number in solution, so the chance of stable oligomer formation originating from immobilized monomers is extremely low. Therefore, detected oligomers and fibrils contain only acceptor fluorophores (Alexa 594), which emit fluorescence after direct excitation (no energy transfer from Alexa 488) at the excitation wavelength, 485 nm.
In FLIM experiments, a region of 10 × 10 m 2 was raster scanned in the pulsed mode of the laser at 0.2 W. The scan was repeated for 16 (4 × 4) -36 regions (6 × 6). After finishing one round of scans (24 -54 min), the stage was moved back to the first region and the scan was repeated, which results in movies of each region of 14 -24 hours.
Since each region was scanned multiple times over a period of many hours, there were lateral drifts and defocusing. As seen in the movies, frame-by-frame movements of fibrils by several pixels were observed, which can result from both lateral drifts and actual movement on the surface. There was no experimental correction for these lateral movements. However, training of FNet was performed using the movies of overlapping fibrils synthesized by random samplings of the manually separable fibrils (i.e., non-overlapping fibrils) extracted from the experimental data. Therefore, the effect of lateral movements of fibrils are accounted for by the neural network model. Defocusing is more serious problem because it makes the image dimmer and blurry. To avoid this problem, the stage was re-focused along the axial direction prior to each raster scan.