Journal: bioRxiv

Article Title: GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer’s disease-like amyloid strain

doi: 10.1101/2023.12.19.572292

Figure Lengend Snippet: a, Domain diagram of full-length tau. The residues phosphorylated by different kinases and detected by mass spectrometry are indicated with red bars. The lengths of the red ticks are adjusted based on the percentage of phosphorylation calculated by dividing the number of peptides detected containing a particular residue by the total number of peptides detected containing the same residue. The black diamond-headed bars refer to previously reported phosphorylation sites that were not detected due to the absence of peptides in the mass spectrometry experiment. b-d , Residue-specific intensity changes observed in 2D 1 H- 15 N HSQC spectra of tau upon phosphorylation by GSK3β (b), CDK5 (c) and ERK2 (d). I and I 0 are the cross-peak intensities of the phosphorylated and unmodified tau, respectively. The location of serine, threonine and tyrosine residues is indicated above. e-g , Signals of the cross-peaks of phosphorylated serine residues in the 1 H- 15 N HSQC spectra of phosphorylated tau appeared due to the phosphorylation by GSK3b (e), CDK5 (f), and ERK2 (g). h , 1 H- 15 N HSQC spectra of either unmodified (black) or GSK3β-phosphorylated (orange) C-terminal fragment of tau comprising residues 369 to 441 (referred to as K26). The cross-peaks of the phosphorylated residues and the residues shifted due to nearby phosphorylation are shown in the spectrum. i , Residue-specific chemical-shift perturbation (CSP) observed in the 1 H- 15 N HSQC spectra of K26 upon phosphorylation by GSK3β. The PHF-1 epitope is marked in the amino acid sequence displayed above.

Article Snippet: 40 µL of 25 µM GSK3β-phosphorylated tau fibrils were pelleted down by centrifugation at 20,000 g using an Eppendorf centrifuge 5424.

Techniques: Mass Spectrometry, Residue, Sequencing

Journal: bioRxiv

Article Title: GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer’s disease-like amyloid strain

doi: 10.1101/2023.12.19.572292

Figure Lengend Snippet: a , Aggregation kinetics of 25 µM unmodified tau and tau phosphorylated by different kinases. Error bars represent the std of three independently aggregated samples. b , ThT-intensity span vs. half time of aggregation (Tm) of unmodified and phosphorylated tau proteins. Error bars represent the std of three independently aggregated samples. c , Amount of protein aggregated vs. half time of aggregation (Tm) of unmodified and phosphorylated tau proteins. The amount of aggregated protein was calculated by comparing the intensity of the supernatant (SN) band (after pelleting down the fibrils) to the tau monomer band as shown in Supplementary Fig. 4. Error bars represent the std of the half-time of aggregation of three independently aggregated samples. d , CD spectrum of fibrils formed by GSK3β-phosphorylated tau at the end of the aggregation assay. e , Negative-stain EM of fibrils formed by GSK3β-phosphorylated tau. Scale bars, 200 nm.

Article Snippet: 40 µL of 25 µM GSK3β-phosphorylated tau fibrils were pelleted down by centrifugation at 20,000 g using an Eppendorf centrifuge 5424.

Techniques: Staining

Journal: bioRxiv

Article Title: GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer’s disease-like amyloid strain

doi: 10.1101/2023.12.19.572292

Figure Lengend Snippet: a , Domain diagram of 2N4R tau. Residues that undergo phosphorylation in the presence of GSK3β, ERK2, CDK5, and C-Abl are marked with green, black, purple, and dark-yellow colored bars, respectively. S396 and S404, which are phosphorylated by GSK3β, form the epitope that is recognized by the phosphorylation-specific antibody PHF-1. b , DIC and fluorescence microscopy of the condensates formed by 25 µM GSK3β-phosphorylated tau at room temperature in the aggregation assay buffer (25 mM HEPES, 10 mM KCl, 5 mM MgCl 2 , 3 mM TCEP, 0.01 % NaN 3 , pH 7.2). A zoomed-in view of the condensate is shown to the right. Micrographs are representative of three independent biological replicates. Scale bar, 10 µm c , Fluorescence recovery after photobleaching (FRAP) experiment of the condensates formed by GSK3β-phosphorylated tau shown in (b). Error bars represent std of averaged three curves for each time point. Representative micrographs of the condensate before bleaching, after bleaching, and at the end of recovery are displayed to the right. Scale bar, 1 µm. d , DIC microscopy of the condensates formed by 25 µM ERK2-phosphorylated tau at room temperature in the aggregation assay buffer. A zoomed-in view of the condensate is shown to the right. Micrographs are representative of three independent biological replicates. Scale bar, 10 µm e,f,g , DIC microscopy of 25 µM CDK5-phosphorylated tau (e), C-Abl-phosphorylated tau (f), and unmodified tau (g) at room temperature in the aggregation assay buffer. Micrographs are representative of three independent biological replicates. Scale bar, 10 µm.

Article Snippet: 40 µL of 25 µM GSK3β-phosphorylated tau fibrils were pelleted down by centrifugation at 20,000 g using an Eppendorf centrifuge 5424.

Techniques: Fluorescence, Microscopy

Journal: bioRxiv

Article Title: GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer’s disease-like amyloid strain

doi: 10.1101/2023.12.19.572292

Figure Lengend Snippet: a , DIC and fluorescence microscopy of tau droplets induced by the addition of 10 % dextran at room temperature in 25 mM HEPES, 5 mM MgCl 2 , pH 7.2 buffer. Fluorescently labeled GSK3β partitioned into the droplets. The partially merged image is due to the mobility of the droplets, i.e., the droplets moved from one place to another place by the time the images were taken using the red and green channel of the microscope. Micrographs are representative of three independent biological replicates. Scale bar, 5 µm. b , DIC and fluorescence microscopy of the tau droplets induced by the addition of 10 % dextran at room temperature in 25 mM HEPES, 5 mM MgCl 2 , pH 7.2 buffer in the presence of 0.02 mg/ml unlabeled GSK3β, and 1 mM ATP. The sample was incubated for 24 hours. A zoomed-in view of the condensate formed after 24 hours is shown. Micrographs are representative of three independent biological replicates. Scale bar, 5 µm. c , FRAP experiment of the larger fused condensates of tau in the presence of GSK3β and ATP after incubation for six hours. Error bars represent std of averaged three curves for each time point. Representative micrographs of the condensate before bleaching, after bleaching, and at the end of recovery are displayed on the top. Scale bar, 5 µm. d , FRAP experiment of the gel-like condensates of tau in the presence of GSK3β and ATP after incubation for one day. Error bars represent std of averaged three curves for each time point. Representative micrographs of the condensate before bleaching, after bleaching, and at the end of recovery are displayed on the top. Scale bar, 5 µm.

Article Snippet: 40 µL of 25 µM GSK3β-phosphorylated tau fibrils were pelleted down by centrifugation at 20,000 g using an Eppendorf centrifuge 5424.

Techniques: Fluorescence, Microscopy, Labeling, Incubation

Journal: bioRxiv

Article Title: GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer’s disease-like amyloid strain

doi: 10.1101/2023.12.19.572292

Figure Lengend Snippet: a , Cryo-electron micrograph of GSK3β-phosphorylated tau fibrils. b, Reconstruction of a GSK3β phosphorylated tau fibril from low-resolution and big box 2D classes for crossover estimation. c, Cross-section of the cryo-EM map of the GSK3β-phosphorylated tau fibril after 3D refinement. d, Cryo-EM density map of GSK3β phosphorylated tau fibrils. e, Cryo-EM density map of GSK3β-phosphorylated tau fibrils (cyan surface) compared with the paired helical filament from sporadic Alzheimer’s disease brain (dark blue isomesh; PDB id: 6HRE).

Article Snippet: 40 µL of 25 µM GSK3β-phosphorylated tau fibrils were pelleted down by centrifugation at 20,000 g using an Eppendorf centrifuge 5424.

Techniques: Cryo-EM Sample Prep