Tau forms oligomeric complexes on microtubules that are distinct from tau aggregates

Abstract

Tau is a microtubule-associated protein, which promotes neuronal microtubule assembly and stability. Accumulation of tau into insoluble aggregates known as neurofibrillary tangles (NFTs) is a pathological hallmark of several neurodegenerative diseases. The current hypothesis is that small, soluble oligomeric tau species preceding NFT formation cause toxicity. However, thus far, visualizing the spatial distribution of tau monomers and oligomers inside cells under physiological or pathological conditions has not been possible. Here, using single-molecule localization microscopy, we show that tau forms small oligomers on microtubules ex vivo. These oligomers are distinct from those found in cells exhibiting tau aggregation and could be precursors of aggregated tau in pathology. Furthermore, using an unsupervised shape classification algorithm that we developed, we show that different tau phosphorylation states are associated with distinct tau aggregate species. Our work elucidates tau's nanoscale composition under nonaggregated and aggregated conditions ex vivo.

Keywords: protein aggregation; super-resolution microscopy; tau.

Fig. 1.

Microtubule-associated tau is partially oligomeric under nonaggregated conditions. (A) Super-resolution image of tau in Clone 4.0 cells expressing 4R-P301L-GFP tau stained with GFP nanobody conjugated to Alexa Fluor 647 after overnight Dox induction of tau expression. (Inset) Pictured together with corresponding Voronoi segmentation. Segmented images are pseudo color coded with different colors corresponding to different segmented nanoclusters. (B) Super-resolution image of tau in stable BSC-1 cells constitutively expressing 3R-WT-GFP tau stained with GFP nanobody conjugated to Alexa Fluor 647. (Inset) Pictured together with corresponding Voronoi segmentation. Segmented images are pseudo color coded with different colors corresponding to different segmented nanoclusters. (C) Super-resolution image of oligomeric tau detected by the tau oligomer-specific T22 antibody in Clone 4.0 cells expressing 4R-P301L-GFP tau after overnight Dox induction of tau expression. (Inset) Pseudo color-coded Voronoi segmentation of nanoclusters. (D) Violin plots showing the number of localizations per nanocluster segmented with Voronoi segmentation in the different cell lines used in this study (green: Clone 4.0 cells expressing 4R-P301L-GFP tau after overnight Dox induction of tau expression; yellow: stable BSC-1 cells constitutively expressing 3R-WT-GFP tau; and cyan: rat hippocampal neurons). Plots for A and B correspond to the quantification of tau nanoclusters stained and imaged with a GFP nanobody. Plot for H corresponds to the quantification of tau nanoclusters stained and imaged with a Tau-5 antibody. Plots for C and I correspond to the quantification of tau nanoclusters stained and imaged with the oligomeric T22 antibody. The dashed lines indicate the median, and the dotted lines indicate the 25th and 75th percentile. (A) n = 15 cells, n = 3 experiments. (B) n = 15 cells, n = 2 experiments. (C) n = 19 cells, n = 3 experiments. (H) n = 3 cells. (I) n = 3 cells. (E) Two-color super-resolution images of α-tubulin (magenta), total tau (cyan), and overlay in Clone 4.0 cells expressing 4R-P301L-GFP tau after overnight Dox induction of tau expression. The results of the colocalization analysis are shown in which tau nanoclusters colocalized with α-tubulin are color coded in magenta, and isolated tau nanoclusters are shown in yellow. (F) Two-color super-resolution images of oligomeric tau detected by tau oligomer-specific T22 antibody (magenta), total tau (cyan), and overlay in Clone 4.0 cells expressing 4R-P301L-GFP tau after overnight Dox induction of tau expression. The results of the colocalization analysis are shown in which tau nanoclusters colocalized with T22 are color coded in magenta, and isolated tau nanoclusters are shown in yellow. (G) Violin plots showing the percentage of tau nanoclusters colocalized with α-tubulin, T22, Thr231, and AT8 antibodies in Clone 4.0 cells expressing 4R-P301L-GFP tau after overnight Dox induction of tau expression (green) and Clone 4.1 cells maintained in Dox and expressing 4R-P301L-GFP tau (pink). (Tubulin in Clone 4.0: n = 9 cells, n = 2 experiments; Tubulin in Clone 4.1: n = 7 cells, n = 2 experiments; T22 in Clone 4.0: n = 9 cells, n = 3 experiments; T22 in Clone 4.1: n = 6 cells, n = 2 experiments; Thr231 in Clone 4.1: n = 6 cells, n = 2 experiments; and AT8 in Clone 4.1: n = 6 cells, n = 3 experiments). (H) Super-resolution image of tau in rat hippocampal neurons, stained with Tau-5 antibody, which detects all tau isoforms. (Inset) Pictured together with corresponding Voronoi segmentation. Segmented images are pseudo color coded with different colors corresponding to different segmented nanoclusters. (I) Super-resolution image of oligomeric tau detected by oligomer tau-specific T22 antibody in rat hippocampal neurons. (Inset) Pseudo color-coded Voronoi segmentation of nanoclusters.

Fig. 2.

Tau oligomers in cells harboring tau aggregates are distinct from tau oligomers in cells modeling a nonaggregated tau state. (A) Super-resolution image of tau in Clone 4.1 cells maintained in Dox and expressing 4R-P301L-GFP tau. Zoomed in regions after Voronoi segmentation are shown. Segmented images are pseudo color coded with different colors corresponding to different segmented objects. Nanoclusters (magenta circles), fibrillary structures (green circles), branched fibrils (yellow circles), and conglomerate NFT-like structures (white circles) are visible in the segmented images. (B) Violin plots showing the number of localizations per Voronoi-segmented tau nanocluster in Clone 4.0 cells expressing 4R-P301L-GFP tau after overnight Dox induction of tau expression (green) and Voronoi-segmented tau object in Clone 4.1 cells maintained in Dox and expressing 4R-P301L-GFP tau (pink). The dashed lines indicate the median, and the dotted lines indicate the 25th and 75th percentile (Clone 4.0: n = 15 cells, n = 3 experiments; Clone 4.1: n = 20 cells, n = 3 experiments). ****P < 0.0001. (C) Two-color super-resolution images of α-tubulin (magenta), total tau (cyan), and overlay in Clone 4.1 cells maintained in Dox and expressing 4R-P301L-GFP tau. The results of the colocalization analysis are shown in which tau aggregates colocalized with α-tubulin are color coded in magenta, and isolated aggregates are shown in yellow. (D) Two-color super-resolution images of oligomeric tau detected by tau oligomer-specific T22 antibody (magenta), total tau (cyan), and overlay in Clone 4.1 maintained in Dox and expressing 4R-P301L-GFP tau. The results of the colocalization analysis are shown in which tau aggregates colocalized with T22 are color coded in magenta, and isolated aggregates are shown in yellow. (E) Violin plots showing the number of localizations per Voronoi-segmented tau nanocluster that colocalizes with α-tubulin in Clones 4.0 cells expressing 4R-P301L-GFP tau after overnight Dox induction of tau expression (green) and Clone 4.1 cells maintained in Dox and expressing 4R-P301L-GFP tau (pink). The dashed lines indicate the median and the dotted lines indicate the 25th and 75th percentile (4.0: n = 9 cells, n = 2 experiments; 4.1: n = 7 cells, n = 2 experiments). ****P < 0.0001.

Fig. 3.

Shape classification reveals distinct classes of tau aggregates in Clone 4.1 cells. (A–C and F–H) Ellipse plots showing the different tau classes found in Clone 4.1 cells maintained in Dox and expressing 4R-P301L-GFP tau. They are showing either low (green) or high (magenta) levels of tau aggregation. Each ellipse represents a separate class. The plot axes represent the number of localizations per tau aggregate and area of tau aggregates in square micrometer. Each ellipse is placed on the plot to represent the average number of localizations and area of the tau aggregates within that class. The size of the ellipses is scaled within each plot to represent the proportion of tau aggregates contained in that particular class, but the size is not comparable between the different plots. Green represents the proportion of tau aggregates from low tau aggregation cells, whereas magenta represents the proportion of tau aggregates from high tau aggregation cells. The bar charts next to the ellipse plots represent the percentage of tau aggregates from each category (low in green and high in magenta) found in classes in the specified number of localizations and area range. Ellipses represent average numbers, not extreme outliers. (D, E, I, and J) Representative super-resolution images of tau aggregates from some of the most prominent classes in each plot. Shapes from cells showing low levels of tau aggregation are colored green, whereas those from cells showing high levels of tau aggregation cells are colored magenta. (Scale bars: 500 nm.)

Fig. 4.

Branched tau fibrils and long tau fibrils are the predominant tau structures recognized by Thr231 and AT8 antibodies, respectively. (A) Super-resolution image of tau labeled with the Thr231 antibody in Clone 4.1 cells maintained in Dox and expressing 4R-P301L-GFP tau. Zoomed-in regions after Voronoi segmentation are shown. Segmented images are pseudo color coded with different colors corresponding to different segmented objects. Yellow circles highlight branched tau fibril-like structures. (B) Super-resolution image of tau labeled with AT8 antibody in QBI cells, Clone 4.1, expressing 4R-P301L-GFP tau. Zoomed in regions after Voronoi segmentation are shown. Segmented images are pseudo color coded with different colors corresponding to different segmented objects. Yellow circles highlight long tau fibril-like structures.

Fig. 5.

Shape classification differentiates higher-order aggregates based on phosphorylation status. (A–C and F–H) Ellipse plots showing the different tau classes found in Thr231- (magenta), AT8- (green), or GFP nanobody- (blue) labeled Clone 4.1 cells maintained in Dox and expressing 4R-P301L-GFP tau. Ellipse representation is the same as in Fig. 3. (D, E, I, and J) Representative super-resolution images of tau aggregates from some of the most prominent classes in each plot. Shapes from Thr231-stained cells are colored magenta, those from AT8-stained cells are colored green, and those from the GFP nanobody staining are colored blue. N/A indicates not available. (Scale bars: 500 nm.)