Journal: Nature Communications

Article Title: Tubulin transforms Tau and α-synuclein condensates from pathological to physiological

doi: 10.1038/s41467-026-69618-3

Figure Lengend Snippet: a Time-resolved (0.5, 4 and 72 hr) confocal microscopy of Tau, αSyn, and Tubulin mixtures at varying concentrations (0–20 µM) in LLPS buffer. Mixtures also included fluorescently labeled Tau-A488, αSyn-A647, and Rhodamine-Tubulin (Rh-Tub) at labeled to unlabeled protein concentration ratios of 1:40, 1:50 and 1:50, respectively. Scale bars = 50 µm. b Representative conditions from ‘a’ showing morphological difference between droplets and Tubulin-rich tactoids. c Quantification of shape eccentricity in select conditions (20 µM Tau and 10 µM αSyn ± 10 µM Tubulin after 4 hr). n = 3 images with at least 80 droplets/image. Statistical analysis: one-way ANOVA: **** p = 0.0001, ns = not significant. d FRAP data of Tau/αSyn ± Tubulin condensates from b . n = 3 independently prepared samples, Statistical analysis: one-way ANOVA; * p = 0.05, ** p = 0.01, ns = not significant. Error bars = SEM. e Partition efficiency (ratio of mean fluorescence intensity inside condensates vs. in the dilute phase) of Tau/αSyn/Tubulin mixtures (represented in ‘ a ’). n = 4 independently prepared samples, statistical analysis: one-way ANOVA multiple comparisons; * p = 0.05, ** p = 0.01, *** p = 0.001, **** p = 0.0001, ns = not significant. f Average partition efficiency (across all Tau and αSyn concentrations in graphs shown in ‘ e ’) of Tau (green) and αSyn (blue) at different Tubulin concentrations. n = 3 independent samples including the various concentrations of Tau and αSyn, statistical analysis: one-way ANOVA; * p = 0.05, ** p = 0.01, ns = not significant. Error bars = SEM. g Cartoon representation of Tau/αSyn/Tubulin ternary system (morphological diagram). The diagram depicts phase and morphological transitions based on confocal images ‘ a ’, western blots of oligomers (Fig. ) and dot blot amyloid assays (Fig. ).

Article Snippet: Solution mixtures with Tau, αSyn, and Tubulin (Cytoskeleton, Inc.) or combinations of the three were prepared by diluting the proteins to their respective concentrations in LLPS buffer.

Techniques: Confocal Microscopy, Labeling, Protein Concentration, Fluorescence, Western Blot, Dot Blot

Journal: Nature Communications

Article Title: Tubulin transforms Tau and α-synuclein condensates from pathological to physiological

doi: 10.1038/s41467-026-69618-3

Figure Lengend Snippet: Representative western blots (anti-Tau, a ; anti-αSyn, b ) of Tau/αSyn/Tubulin samples (pellet fraction) incubated for 0, 16, and 36 hr in LLPS buffer. Orange asterisks (*) and *TS refer to Tau/αSyn heterodimers. c – e Quantification of bands in blots represented in ‘ a ’ and ‘ b ’ ( n = 3 wells from independently prepared samples), showing Tau HMW oligomers (larger than Tau trimer), αSyn HMW oligomers (larger than monomer), and Tau/αSyn (TS) heterodimers, respectively. Statistical analysis: one-way ANOVA multiple comparisons, * p = 0.05, ** p = 0.01, ns = not significant. Error bars = SEM. f Representative ( n = 3) amyloid dot blot assay (anti-OC) of Tau, αSyn, and Tubulin in different combinations (20 µM each), incubated for ~20 days. 2 µM Amyloid beta (Aβ) was used as reference. g Quantitation of dot blots ( n = 3 dotted regions from independently prepared samples) normalized to Aβ intensity. Statistical analysis: one-way ANOVA multiple comparisons; *** p = 0.001, ns = not significant. Error bars = SEM.

Article Snippet: Solution mixtures with Tau, αSyn, and Tubulin (Cytoskeleton, Inc.) or combinations of the three were prepared by diluting the proteins to their respective concentrations in LLPS buffer.

Techniques: Western Blot, Incubation, Dot Blot, Quantitation Assay

Journal: Nature Communications

Article Title: Tubulin transforms Tau and α-synuclein condensates from pathological to physiological

doi: 10.1038/s41467-026-69618-3

Figure Lengend Snippet: a Tau and αSyn domain organization highlighting the locations of the Alexa Fluor dyes (cyan and yellow spheres) for FRET detection. CryoEM structures of Tau amyloids (5O3L), αSyn amyloids (2N0A) and extended Tau on microtubules (EMD-7522). b Representative confocal and FLIM images of Tau (C290/380-A488/594, ~ 20 nM) in the presence of αSyn, without Tubulin. Dilute (D) and condensate (C) images were generated by intensity thresholding, with donor lifetime histograms shown. Scale bars = 10 µm c Representative confocal and FLIM images of αSyn (C7/84-A488/594, ~ 20 nM) in the presence of Tau, without Tubulin. D and C images were generated by intensity thresholding, with donor lifetime histograms shown. d Violin plot of FLIM donor lifetimes of Tau (C291/380- A488/594) (left) or αSyn (C7/84- A488/594) (right) for various conditions (Tau ± αSyn ± Tubulin, Supplemental Fig. ). D and C represent measurements made in the dilute and condensed phases, respectively. n = 5, statistical analysis: One-way ANOVA; * p = 0.05, ** p = 0.01, *** p = 0.001, **** p = 0.0001, ns = not significant. Violin spread = SD, center of violin = mean, width of violin = distribution. Scale bars = 10 µm e FRET-FLIM images of αSyn (C7/84- A488/594) in conditions with and without Tubulin. FRET-FLIM histograms show distinct αSyn conformations. f Phasor analysis of the dual-labeled FRET αSyn. Confocal images were collected for each of the 5 independently prepared samples. Scale bars = 10 µm g FRET-FLIM images of Tau (C291/380- A488/594) in conditions with and without Tubulin. FRET-FLIM histograms show distinct Tau conformations. Confocal images were collected for each of the 5 independently prepared samples. Scale bars = 10 µm h, Phasor analysis of the dual-labeled FRET Tau mixtures with and without Tubulin. For both phasor plots, numbers within clusters indicate mean phase lifetimes ( τ Ψ) . i Schematic diagram for how the presence of Tubulin transforms Tau/αSyn pathological to Tau/αSyn/Tubulin physiological condensates with corresponding favored Tau and αSyn conformations.

Article Snippet: Solution mixtures with Tau, αSyn, and Tubulin (Cytoskeleton, Inc.) or combinations of the three were prepared by diluting the proteins to their respective concentrations in LLPS buffer.

Techniques: Generated, Labeling

Journal: Nature Communications

Article Title: Tubulin transforms Tau and α-synuclein condensates from pathological to physiological

doi: 10.1038/s41467-026-69618-3

Figure Lengend Snippet: a Western blots (anti-α-Tubulin, -Tau, -pTau, -αSyn and -GAPDH; top to bottom) of undifferentiated mouse N2a cells with and without Tubulin siRNA transfection. Quantification of α-Tubulin, αSyn, Tau, and pTau oligomer levels (for αSyn, only oligomers above 100 kDa were considered) normalized to GAPDH (α-Tubulin siRNA transfection, hashed bars vs. untransfected cells, unhashed bars). n = 3 biological replicates loaded into independent wells, statistical analysis: One-way ANOVA; * p = 0.05, ** p = 0.01, ns = not significant. Error bars = SEM. b Tau optogenetic construct (OptoTau: Tau-mCherry-Cry2, red) stably expressed in undifferentiated human neuroblastoma SH-SY5Y cells. Cells were treated with the oxidative stress agent okadaic acid (OA; 25 nM, 18 hr). Blue light activation (458 nm laser, 100% power, 20 min) was performed in localized target regions of interest (ROIs) ( ~ 5 µm ROI; yellow dash lines; performed on 3 independent replicates). Scale bars = 1 μm. c Cry2-Tau colocalization with microtubules in both the absence and presence of blue light in SH-SY5Y cells, performed on 3 independent replicates, treated with 25 nM OA to model ND-related stress Cry2- mcherry-Tau (red), Sir-Tubulin (green). Scale bar = 10 μm. d Confocal microscopy images of N2a cells stably co-expressing OptoTau (red) and GFP-αSyn (green). Cells were either transfected with α-Tubulin siRNA or treated with OA (25 nM, 18 hr). Plot of % neurites (ratio of number of neurites vs. total cells, n = 3 biological replicates). Statistical analysis: One-way ANOVA; * p = 0.05. Error bars = SD. Scale bars = 20 µm. d Representative images of N2a cells stably co-expressing OptoTau and GFP-αSyn stressed with 25 nM OA for 18 hr. Cell boundaries are outlined in yellow dashes. Blue light activation (20 min) resulted in enhanced colocalization of OptoTau and GFP-αSyn (yellow). Error bars = positive SD Scale bars = 2 μm.

Article Snippet: Solution mixtures with Tau, αSyn, and Tubulin (Cytoskeleton, Inc.) or combinations of the three were prepared by diluting the proteins to their respective concentrations in LLPS buffer.

Techniques: Western Blot, Transfection, Construct, Stable Transfection, Activation Assay, Confocal Microscopy, Expressing