Tau hyperphosphorylation impairs cooperative binding to microtubules and perturbs organelle trafficking in neurons
- Department of Bioengineering, McGill University, Canada
- Department of Molecular Physiology and Biophysics, University of Vermont, United States
Figures
Figure 1 with 1 supplement
Tau hyperphosphorylation results in more diffuse microtubule interactions and reduces tau envelope formation in vitro.
(A) Structure of 4R0N tau highlighting 14 disease-associated S/T residues mutated to E in pseudo-hyperphosphorylated (E14) tau or to A in phospho-resistant (AP) tau (Hoover et al., 2010). (B) Schematic illustrating the main steps involved in preparing GFP-tau containing cell extracts and performing reconstitution assays with 500 nM tau (see Supplementary file 1A). Linescans of tau intensity along a microtubule were analyzed using a Gaussian mixture model (GMM) to define intensity thresholds distinguishing envelopes from gaps. (C) Representative images of WT, AP, and E14 GFP-tau (4R0N) on taxol-stabilized microtubules (WT: n = 183, AP: n = 170, E14: n = 170). The indicated n values represent total number of samples over three to four replicates. Below the images, corresponding tau intensity plots along microtubules are shown, with horizontal lines indicating the GMM threshold (orange) and mean background fluorescence intensity (blue). Asterisks mark peaks identified as envelopes. Histograms show the distributions of tau fluorescence intensity fitted with a GMM. Insets show Bayesian Information Criterion (BIC) analyses used to determine whether a unimodal or multimodal distribution best describes the data. Plots quantifying the effects of hyperphosphorylation on (D) tau envelope enrichment intensity, (E) percentage of microtubule length covered by envelopes, and (F) mean width of envelopes. Error bars indicate 95% CI. Statistical significance was assessed using Student’s t-test (***p < 0.0001). Scale bars are 10 µm.
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Figure 1—source data 1
Tau envelope quantification.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Tau hyperphosphorylation results in more diffuse microtubule interactions.
(A) Representative images of WT, AP, and E14 GFP-tau on GMPCPP-polymerized microtubules (WT: n = 180, AP: n = 178, E14: n = 184). The indicated n values represent total number of samples over three to four replicates. Below the images, corresponding tau intensity plots along microtubules are shown, with horizontal lines indicating the Gaussian mixture model (GMM) threshold (orange) and mean background fluorescence intensity (blue). (B, C) Bar plots compare the effects of tau phosphorylation on envelope frequency (B) and the mean tau intensity on microtubules vs in solution using either reversibly stabilized (taxol) or irreversibly stabilized (GMPCPP) microtubules. Error bars indicate 95% CI. Statistical significance was assessed using Student’s t-test (***p < 0.0001). Scale bars are 10 µm.
Figure 2 with 1 supplement
Tau hyperphosphorylation reduces the formation of envelopes in live neurons.
(A) Representative images of mid-axonal segments with tau envelopes present in iPSC-derived MAPT-KO neurons expressing WT (n = 39), AP (n = 39), or E14 (n = 28) GFP-tau (4R0N). Each image is a max projection of 10 frames from live timelapse imaging. Images are all oriented so that the soma is toward the left and the distal axon is toward the right. Below, corresponding tau intensity plots from discrete locations (labeled 1–6) are shown. Horizontal lines indicating the Gaussian mixture model (GMM) threshold (orange) and mean background fluorescence intensity (blue). Histograms of tau fluorescence intensity, fitted with a GMM to define the envelope threshold, are displayed for selected areas (labeled i–iii) marked by magenta ROIs. Insets show Bayesian Information Criterion (BIC) analyses for multimodal distributions. Bar plots comparing (B) tau envelope intensity and (C) the mean envelope width of WT, AP, and E14 tau. Bar plots quantifying tau envelope frequency within discrete locations (D) and the percentage of axons covered by tau envelopes (E). Error bars indicate 95% CI. (F) Timelapse images of fluorescence recovery after photobleaching (FRAP) assays in mid-axons of MAPT-KO neurons expressing WT (n = 20), AP (n = 21), or E14 (n = 20) GFP-tau. Circles indicate the bleached regions. Note that tau envelopes are not clearly evident due to shorter exposure times needed for FRAP experiments and due to selection of higher GFP-tau expressing cells to ensure measurable recovery dynamics. (G) Fluorescence recovery curves of WT (blue), AP (green), and E14 tau (red). Shaded regions indicate SD. The characteristic recovery (τ) and mobile fraction (M) are indicated on the plot for each tau construct. (H) Schematic illustrates how tau phosphorylation influences its dissociation from microtubules. Hyperphosphorylated E14 tau dissociates more readily than WT tau or the phospho-resistant AP tau, which remain more stably bound (Figure 2—figure supplement 1). (I) Plots show how the ratio of tau signal in axons of neurons expressing WT (n = 37), AP (n = 32), and E14 (n = 36) tau over background intensity varies for each tau construct across the proximal, mid, and distal axonal regions. In the bottom plot, blue lines indicate a decrease in the signal from the proximal toward the distal axon, and red lines indicate an increase in tau signal toward the distal axon. The top plot shows the means and gray bars represent SD. Orange bars show means. Wilcoxon signed rank test was used to determine the pairwise comparison of tau intensity between each axonal region as shown, and the p-values are indicated in the above inset. The scale bars are 10 µm (A) and 5 µm (F) (*p < 0.05, **p < 0.001, ***p < 0.0001).
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Figure 2—source data 1
Bootstrapped means of tau envelope fluorescence intensity vs gap fluorescence intensity.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig2-data1-v1.xlsx
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Figure 2—source data 2
Tau envelope width data in neurons.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig2-data2-v1.xlsx
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Figure 2—source data 3
Frequency and coverage of tau envelopes in axons.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig2-data3-v1.xlsx
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Figure 2—source data 4
FRAP data in neurons.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig2-data4-v1.xlsx
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Figure 2—source data 5
Mean tau fluorescence intensities by axonal region.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig2-data5-v1.xlsx
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Figure 2—source data 6
FRAP data in Cos7 cells.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig2-data6-v1.xlsx
Figure 2—figure supplement 1
Characterization of GFP-tau phospho-variants in neurons and COS-7 cells.
(A) Full-field images of MAPT-KO neurons expressing WT, AP, and E14 GFP-tau. Yellow boxes indicate the axonal regions shown as cropped images in Figure 2A. (B) Timelapse images of fluorescence recovery after photobleaching assays of COS-7 cells expressing WT (n = 15), AP (n = 16), or E14 (n = 15) GFP-tau. Circles indicate the bleached regions. Fluorescence recovery curves of WT (blue), AP (green), and E14 tau (red). Shaded regions indicate SD. The characteristic recovery (t) and mobile fraction (M) are indicated on the plot for each tau construct. Scale bars are 10 µm (A) and 5 µm (B).
Figure 3 with 1 supplement
Tau phosphorylation differentially regulates kinesin-1 and kinesin-3 motility.
Kymographs of constitutively active Janelia Fluor 554 (JFX554)-labeled (A) KIF5C(1–560)-JFX554 and (C) KIF1A(1–393)-JFX554 motors moving along taxol-stabilized microtubules incubated with mock cell lysate or lysates containing 500 nM WT, AP, or E14 GFP-tau (magenta). KIF5C (mock: n = 1016, WT: n = 792, AP: n = 1078, E14: n = 1203) and KIF1A (mock: n = 864, WT: n = 173, AP: n = 218, E14: n = 305) over three to four replicates. (B, D) Bar graphs show the fraction of time that kinesin motors are paused or exhibit processive motility. Error bars indicate 95% CI. Bar graph shows the bootstrapped mean run length and mean velocity of KIF5C (E) and KIF1A (F) ± WT, AP, and E14 GFP-tau. Error bars indicate SEM. (G) Schematic illustrating the approach used to assess the impact of tau on kinesin-microtubule attachment and detachment. Motors were observed attaching to or detaching from microtubules either within tau envelopes or outside of them. (H) Paired sample plots show the attachment and detachment frequencies of KIF5C (top) and KIF1A (bottom). Red lines indicate increased frequency inside envelopes compared to outside and blue lines indicate decreased frequencies inside of envelopes. Insets for KIF1A show a zoomed-in view of the attachment and detachment frequencies. Blue bars indicate 95% CI, orange lines mark mean values, and gray bars denote SD. (I) Schematic illustrating the impact of tau on the detachment kinetics of kinesin motors. KIF5C is weakly inhibited by WT and phospho-resistive tau compared to hyperphosphorylated tau that has a similar dissociation rate compared to KIF5C in mock conditions. Conversely, KIF1A is more strongly inhibited by tau hyperphosphorylation and dissociates at a faster rate compared to WT and AP tau. (*p < 0.05, **p < 0.001, ***p < 0.0001). Horizontal scale bars are 5 µm, vertical scale bars are 5 s.
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Figure 3—source data 1
KIF5C processive motility data.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig3-data1-v1.xlsx
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Figure 3—source data 2
KIF1A processive motility data.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig3-data2-v1.xlsx
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Figure 3—source data 3
KIF5C and KIF1A attachment and detachment frequencies.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig3-data3-v1.xlsx
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Figure 3—source data 4
Kinesin and tau pixel intensity correlation data.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig3-data4-v1.xlsx
Figure 3—figure supplement 1
Tau phosphorylation differentially regulates kinesin-1 and kinesin-3 motility.
Histograms display the distributions of run lengths, dwell times, and velocities for KIF5C (A–C) and KIF1A (D–F) in the presence of mock lysate or lysates containing WT, AP, or E14 GFP-tau. Run length distributions (A, D) and dwell time distributions (B, E) were fitted with a single exponential function to extract the characteristic run length and the dissociation rate constants (koff), respectively. For visual comparison, distributions from tau-containing conditions are shown as black-outlined transparent bars, overlaid on light gray bars representing the control (mock lysate) distribution. Red lines indicate exponential fits for control conditions, and blue lines represent fits for each tau condition. The extracted run lengths and koff values are displayed next to each plot, with 95% CI shown in brackets. (G, H) Heat maps show the correlation between tau pixel intensities along microtubules with standard deviation projections of kinesin intensity, where bright pixels indicate higher dynamics. Spearman’s correlation analysis was performed showing that there is no relationship between kinesin dynamics and tau intensities (rS values indicated above each plot). Colors represent the fraction of pixels for each bin. Dashed magenta lines are present to highlight the subtle differences of KIF5C and KIF1A dynamics in regions of higher tau intensities across tau phospho-variants.
Figure 4 with 1 supplement
Tau perturbations impact the trafficking of lysosomes in neurons.
(A) Images show mid-axonal GFP-tau signal and max projections of lysosomes (lys) in control, MAPT-KO, and MAPT-KO neurons expressing WT, AP, and E14 GFP-tau. Below, kymographs show anterograde and retrograde lysosome transport for each condition. The number of cells analyzed for each condition are indicated in Supplementary file 1B. Below each set of images, plots show the total number of lysosome trajectories mapped as position over time. Histograms next to each plot show the frequency distribution of lysosome travel distances for each condition. Positive values indicate anterograde transport (toward distal axon), while negative values indicate retrograde transport (toward soma). Trajectories are color-coded based on GFP-tau expression level: light blue (1.1–2× background), orange (2–5×), and red (>5×). Corresponding travel distance histograms are color-coded and overlaid with control data (gray) for comparison. (B) Plots show the mean absolute displacements for each condition. (C) Bar plot shows the fraction of anterograde and retrograde long-distance trajectories for control (CTL), MAPT-KO (KO), and MAPT-KO neurons expressing WT, AP, or E14 GFP-tau. Above the plot, asterisks represent statistical significance tested for anterograde (black) and retrograde (red) trajectories (*p < 0.05, **p < 0.001, ***p < 0.0001). (D) Plots show the frequency of lysosomes along axons in the proximal, mid, and distal axon for each condition. Proximal regions were defined as ~50 µm from the soma, mid-axonal regions as ~halfway along the axon, and distal regions as ~50 µm from the axon terminal. Statistical significance is indicated by asterisks above the at the top of plots (*p < 0.05, **p < 0.001, ***p < 0.0001). For B and D, blue bars indicate 95% CI, orange lines mark mean values, and gray bars denote SD. Scale bars are 10 µm.
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Figure 4—source data 1
Mean-lysosome displacement data.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig4-data1-v1.xlsx
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Figure 4—source data 2
Long-range lysosome trajectory data.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig4-data2-v1.xlsx
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Figure 4—source data 3
Lysosome frequency along axon by region.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
Tau perturbations impact the trafficking of lysosomes in different axonal regions.
(A) Schematic of a neuron indicating the regions where tau’s effects on lysosome transport were assessed. Lysosome transport was recorded in proximal, mid, and distal axonal regions. Proximal regions were defined as ~50 µm from the soma, mid-axonal regions as ~halfway along the axon, and distal regions as ~50 µm from the axon terminal. Images are all oriented so that the soma is toward the left and the distal axon is toward the right. (B) Maximum intensity projections show lysosomes (LysoTracker) in the proximal, mid, and distal regions of the axon in DIV 7–8 iPSC-derived control neurons, MAPT-KO neurons, and MAPT-KO neurons expressing WT, AP, or E14 GFP-tau. Images are all oriented so that the soma is toward the left and the distal axon is toward the right. Below each set of images, plots show the total number of lysosome trajectories mapped as position over time. Histograms next to each plot show the frequency distribution of lysosome travel distances for each condition. Positive values indicate anterograde transport (toward distal axon), while negative values indicate retrograde transport (toward soma). Trajectory plots are color-coded based on GFP-tau expression level: light blue (1.1–2× background), orange (2–5×), and red (>5×). Corresponding travel distance histograms are color-coded and overlaid with control data (gray) for comparison. Scale bars are 10 µm. (C) Bar plot shows how tau expression levels affect the fraction of anterograde and retrograde long-distance trajectories for control (CTL), MAPT-KO (KO), and MAPT-KO neurons expressing WT, AP, or E14 GFP-tau in the proximal, mid, and distal axon.
Figure 5
Tau hyperphosphorylation relieves inhibition of processive lysosome motility in neurons.
(A) Shown are examples of lysosome trajectories segmented into stationary (red), diffusive (blue), and processive (green) motility based on run length. Stationary segments were defined as periods between two reversal events with a run length (RL)<0.16 µm; diffusive segments as those with run lengths between 0.16 and 1.2 µm; and processive segments as those >1.2 µm. (B) Plots show the fraction of time lysosomes exhibited processive, diffusive, or stationary phases of motility for control (CTL), MAPT-KO (KO), and MAPT-KO neurons expressing WT, AP, and E14 GFP-tau. Error bars represent 95% CI. Statistical significance is indicated above the plots for comparison of the fraction of processive (proc; green), diffusive (diff; blue), and stationary (stat; red) time for each condition. Bar plots show (C) the mean reversal frequency of lysosomes, (D) and the fraction of time of anterograde or retrograde directed processive motility. Bar plots show (E) the mean run lengths and (F) mean velocities of processive runs of lysosomes for each condition. Error bars in (C, E, F) indicate SEM and in (D) indicate 95% CI. The number of trajectories and cells analyzed under each condition are indicated in Supplementary file 1B. (G) Schematic summarizing the impact of tau hyperphosphorylation on bidirectional lysosome transport in neurons. Low tau phosphorylation reduces processive anterograde transport but has no significant impact on retrograde transport, whereas hyperphosphorylated tau relieves inhibition of anterograde transport and enhances retrograde transport, similar to the effects observed in tau knockout conditions. Statistical significance is shown above each plot, where red asterisks indicate comparisons of retrograde transport and black asterisks indicate comparisons of anterograde transport. (*p < 0.05, **p < 0.001, ***p < 0.0001).
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Figure 5—source data 1
Fraction of time of processive, diffusive, and stationary lysosome motility in neurons.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig5-data1-v1.xlsx
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Figure 5—source data 2
Processive lysosome motility data in neurons.
- https://cdn.elifesciences.org/articles/110011/elife-110011-fig5-data2-v1.xlsx
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/110011/elife-110011-mdarchecklist1-v1.docx
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Supplementary file 1
Overview of tau concentrations and neuronal sample sizes.
- https://cdn.elifesciences.org/articles/110011/elife-110011-supp1-v1.xlsx
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Tau hyperphosphorylation impairs cooperative binding to microtubules and perturbs organelle trafficking in neurons
eLife 15:RP110011.
https://doi.org/10.7554/eLife.110011.3
