Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates

Abstract

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

Keywords: Aggregation or Oligomerization; Fluorescence; Microscopy; Proteins; Single-Molecule.

Figure 1

TCCD detection of antibody‐tagged α‐syn aggregates. a) Schematic of the experimental setup. b) Representative TCCD single‐molecule data taken from α‐syn aggregate‐containing solutions, showing intensity from AF488‐tagged antibodies (green) and AF647‐tagged antibodies (red). Stars show example coincident bursts corresponding to protein aggregates.

Figure 2

TCCD measurement of a concentration series of unlabeled α‐syn aggregates. a) Representative TCCD data for samples containing 0 pM, 10 pM, 10 nM, and 1 μM of α‐syn subjected to conditions favoring aggregation for 24 h at a higher concentration. Example coincident bursts are starred. b) Plots of coincident event rate per second and c) association quotient Q shown for varying concentrations of α‐syn. Data are shown as mean±SD, n=3.

Figure 3

TCCD approach is specific to α‐syn aggregates. a) Representative ThT SAVE images of 500 nM α‐syn, amyloid‐β, tau, and TDP‐43. Scale bars are 5 μm and 1 μm in length for the full field of views and insets, respectively. b) Quantification of ThT‐active species detected for each amyloid protein using single‐molecule confocal microscopy. c) TCCD detection of amyloid proteins. Data shown as mean±SD, n=3. **** P<0.0001, One‐way ANOVA with Tukey multiple comparisons test.

Figure 4

The TCCD approach effectively tracks the aggregation of unlabeled α‐syn. a) Representative SAVE images of 500 nM α‐syn aggregates collected at multiple timepoints. Scale bars are 5 μm and 1 μm in length for full field of view and insets respectively. b) ThT detection of 500 nM α‐syn aggregates using the single‐molecule confocal microscope. c) TCCD event rate from a 10 nM α‐syn sample. d) Association quotient for each timepoint. e) The fraction of TCCD events which were also ThT active. f) Intensity distributions (summed from both channels) from aggregates detected at different timepoints. All data in b–e plotted as mean±SD, n=3.

Figure 5

Detection of α‐syn aggregates in human CSF. CSF was spiked with 500 pM sonicated α‐syn aggregates subjected to aggregating conditions for 120 h. To account for increased background fluorescence events thresholds were increased to 11 and 19 photons bin−1 for the blue and red channel, respectively. Mean±SD, n=3, unpaired student t test (** P<0.005).