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. Author manuscript; available in PMC: 2021 May 4.
Published in final edited form as: Cell Rep. 2021 Mar 16;34(11):108843. doi: 10.1016/j.celrep.2021.108843

Figure 4. AD tau core seeds polymerization of full-length wild-type tau.

Figure 4.

(A) The AD tau core and full-length tau were incubated alone or together for 3 days at 37°C with constant agitation. The assembly reactions were centrifuged to obtain the supernatant (S) and pellet (P) fractions, which were evaluated by SDS-PAGE followed by Coomassie blue staining or immunoblotting for the tau antibodies E1 or ADC1.

(B) Following the 3-day incubation, aggregation was monitored by thioflavin positivity in the assembly reactions. Data are shown as the mean ± SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; ****p < 0.0001.

(C and D) Following co-incubation of the AD tau core with full-length wild-type tau, assembly reactions were evaluated by immuno-EM. Filaments are labeled with E1 (C) and MC1 (D) detected with gold particles. Scale bar represents 0.2 μm.

(E) The sarkosyl-insoluble fraction obtained from the frontal cortex of a patient with AD (AD P3) was added to the tau biosensor cell line, and the induction of FRET relative to buffer-treated cells was quantified by flow cytometry. Data are represented as mean ± SEM.

(F) Recombinant AD tau core assembled by incubating at 37°C with constant agitation (220 rpm) for the indicated time points was added to the tau biosensor cell line. After 3 days, cells were collected, and the induction of FRET was measured by flow cytometry. Data are represented as mean ± SEM.

(G–L) Confocal microscopy was utilized to examine inclusion formation of the YFP-tagged tau construct expressed in the biosensor cell line following treatment with assembly mixtures after 0 (G–I) or 3 (J–L) days of incubation. Assembly mixtures contained full-length 4R0N tau alone (G and J), AD tau core alone (H and K), or full-length 4R0N and the AD tau core (I and L). Scale bar represents 10 μm.

(M) Tau biosensor cells were exposed to assembly mixtures on day 0 or following the 3-day incubation, with cell lysates collected 48 h later and FRET quantified by flow cytometry. Data are shown as the mean ± SEM and were analyzed by two-way ANOVA with Sidak’s multiple comparisons test. ****p < 0.0001.