Results: 5

1.
FIGURE 3.

FIGURE 3. From: Conformational Diversity of Wild-type Tau Fibrils Specified by Templated Conformation Change.

CD analysis of WT, MUT, and WT* Tau fibrils. All compared samples had similar protein concentrations (<10% difference). A, insoluble material from primary WT and MUT fibrillization reactions have distinct CD spectra. B, insoluble material from quaternary WT and WT* fibrillization reactions have distinct CD spectra. Spectra reflect four accumulations each. mdeg, millidegrees.

Bess Frost, et al. J Biol Chem. 2009 February 6;284(6):3546-3551.
2.
FIGURE 4.

FIGURE 4. From: Conformational Diversity of Wild-type Tau Fibrils Specified by Templated Conformation Change.

WT fibrils are more fragile than MUT and WT* fibrils. A, primary WT fibrils were visualized by atomic force microscopy, untreated (U) and after sonication (S). Scale bars = 0.25 μm. B, identically prepared primary MUT fibrils, untreated and after sonication, demonstrate the resistance of fibrils to sonication. C, quaternary WT* fibrils, untreated and after sonication, demonstrate the resistance of fibrils to sonication. D, quantification of the loss of CD signal at 223 nm for WT and MUT primary Tau fibrillization reactions before and after sonication indicates MUT fibrils are stronger than WT fibrils. Loss of signal for MUT was calculated as a fraction of loss of signal for WT, which was set to 100%. *, p = 0.0156 (n = 6). E, quantification of the loss of CD signal at 223 nm for WT and WT* quaternary Tau fibrillization reactions before and after sonication indicates WT* Tau fibrils are stronger than WT fibrils. *, p = 0.0156 (n = 6). Loss of signal for WT* was calculated as a fraction of loss of signal for WT, which was set to 100%.

Bess Frost, et al. J Biol Chem. 2009 February 6;284(6):3546-3551.
3.
FIGURE 5.

FIGURE 5. From: Conformational Diversity of Wild-type Tau Fibrils Specified by Templated Conformation Change.

Negative stain electron microscopy of Tau fibrils. High (A) and low (B) magnification EM images show that quaternary WT fibrils have a paired helical filament morphology. In contrast, high (C) and low (D) magnification EM images show that quaternary MUT fibrils have a distinct, curved morphology that lacks the helical appearance of the WT fibrils. High (E) and low (F) magnification EM images show that quaternary WT* fibrils have a curved morphology similar to MUT Tau. Scale bars in A, C, and E = 1 μm; scale bars in B, D, and F = 0.1 μm. G, shown is the quantification of three separate experiments. 94% of quaternary WT fibrils are paired helical filaments (PHF), whereas 2% of MUT fibrils and 18% of WT* are paired helical filaments. *, p < 10–5.

Bess Frost, et al. J Biol Chem. 2009 February 6;284(6):3546-3551.
4.
FIGURE 1.

FIGURE 1. From: Conformational Diversity of Wild-type Tau Fibrils Specified by Templated Conformation Change.

Fibril seeding. A, arachidonic acid (AA) is used to stimulate monomer fibrillization in the primary reaction. In the secondary reaction, 10% of the primary reaction is used to seed the fibrillization of Tau monomer. In the tertiary reaction, 10% of the secondary reaction is used to seed the fibrillization of Tau monomer. In the quaternary reaction, 10% of the tertiary reaction is used to seed the fibrillization of Tau monomer. For fragility studies, the whole quaternary reaction was used, uncentrifuged. For FTIR, CD, and EM, the quaternary reaction was ultracentrifuged for 1 h at 100,000 × g, and the pellet was used for measurements. B, arachidonic acid is used to stimulate WT or MUT fibrillization in the primary reaction. To generate WT fibrils, 10% of the primary WT reaction is incubated with WT monomer, followed by three serial seeding reactions as described in A. To generate MUT fibrils, 10% of the primary MUT reaction is incubated with MUT monomer, followed by three serial seeding reactions as described in A. To generate WT* fibrils, 10% of the primary MUT reaction is incubated with WT monomer, followed by three serial seeding reactions as described in A. C, after 15 h, primary WT and MUT have comparable degrees of fibrillization as determined by solubility. Primary reactions were ultracentrifuged for 1 h at 100,000 × g. The soluble (S) and insoluble (I) fractions were compared by Coomassie stain. D, shown is quantification of three separate experiments. After 15 h, 81% of WT Tau monomer is insoluble versus 89% of MUT Tau, indicating comparable degrees of fibrillization. E, primary WT and MUT fibrillization reactions were initiated with arachidonic acid and measured over time using ThT fluorescence at ex455/em485. Both reactions proceeded at a similar rate. mon., monomer. F, after 96 h, WT and WT* quaternary reactions have comparable degrees of fibrillization. Quaternary reactions were ultracentrifuged for 1 h at 100,000 × g. The soluble and insoluble fractions were compared by Coomassie stain. G, shown is quantification of three separate experiments. After 96 h, 22% of WT Tau monomer is insoluble versus 21% of WT* Tau, indicating comparable degrees of fibrillization. H, WT monomer, MUT monomer, WT, MUT, and WT* quaternary reactions were monitored for 96 h using ThT fluorescence at ex455/em485. The t½ values for WT, MUT, and WT* are 24, 18, and 13 h, respectively. All reactions reach a plateau by ∼48 h.

Bess Frost, et al. J Biol Chem. 2009 February 6;284(6):3546-3551.
5.
FIGURE 2.

FIGURE 2. From: Conformational Diversity of Wild-type Tau Fibrils Specified by Templated Conformation Change.

FTIR analysis of WT, MUT, and WT* Tau. A, WT monomer (mon.). B, MUT monomer. C–E, quaternary WT, quaternary MUT, and quaternary WT* aggregates, respectively, prepared as described for FTIR. MUT (D) and WT* (E) exhibit distinct spectra from C, WT. WT* aggregates (E) exhibit a red shift of the amide I maximum to 1630 cm–1 compared with WT aggregates (∼1634 cm–1) and MUT aggregates (1632 cm–1). F, a curve fit example demonstrating the achieved accuracy (the calculated sum of band components superimposes the measured amide I band completely) and the five component bands used. These describe (high to low wave number)β-turns, α-helix, random coil, and β-sheet. For β-sheet, a high and a low frequency band were assumed (dashed lines). In A–C and E, six spectra from two preparations are superimposed, and five are superimposed in D. The main reasons for the large variability of the spectra between 1665 and 1645 cm–1 were residual water vapor and a low protein concentration in the samples, especially of WT fibrils. Scale bars = 5 × 10–3 arbitrary units. G, quantification of A–E. The results presented derive from curve fitting and averaging six spectra of two samples for each condition; error bars represent the S.E. *, p < 0.05.

Bess Frost, et al. J Biol Chem. 2009 February 6;284(6):3546-3551.

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