Skip to main content
. 2023 Jun 30;12:e86936. doi: 10.7554/eLife.86936

Figure 5. DnaJC7 regulates tau seeding in multiple experimental approaches.

(A) Rescue of DnaJC7 knockout (KO) in tau biosensor cells with either wildtype (WT C7, green) or HPQ mutant (HPQ C7, dark blue) DnaJC7 constructs. The Ruby fluorophore alone (Ruby Control, orange) and a vehicle control (Vehicle Control, purple) were also added to the DnaJC7 KO cells. (B) Overexpression of DnaJC7 constructs in control tau biosensor cells. The cells were transfected with the same constructs as in (A). (C) Model of DnaJC7 with domains colored as follows: TPR1, green; TPR2A, teal; TPR2B, dark blue; JD, orange. Positions of amyotrophic lateral sclerosis (ALS)-associated mutations are shown as purple spheres. (D) Rescue of DnaJC7 KO in tau biosensor cells with ALS-associated mutants of DnaJC7 and WT control, sorted by domain location. Rescue with the WT DnaJC7 construct is shown in all domains as a gray, dashed line. Error bars represent SEM of three technical replicates. *=p < 0.05, **=p < 0.01, ***=p < 0.001, ****=p < 0.0001.

Figure 5.

Figure 5—figure supplement 1. Stability and expression of DnaJC7 mutants in tau biosensors.

Figure 5—figure supplement 1.

(A) Schematic showing tau biosensor cells with or without DnaJC7 knockout (KO) transiently overexpressing different Ruby-tagged gRNA-resistant DnaJC7 constructs. Cells were allowed to express the constructs for 2 days before being plated for the seeding assay. (B) Immunoblotting for DnaJC7 confirmed expression of the Ruby-WT DnaJC7 and Ruby-DnaJC7 (HPQ) mutant constructs in tau biosensor cells without (Overexpression) and with endogenous DnaJC7 knocked out (Rescue). Ruby fusion constructs are highlighted by a red arrow. (C) Immunoblotting for DnaJC7 confirmed expression of the Ruby-WT DnaJC7 and Ruby-DnaJC7 amyotrophic lateral sclerosis (ALS) mutant constructs in tau biosensor cells with endogenous DnaJC7 knocked out. Ruby fusion constructs are highlighted by a red arrow. (D) Positive and negative controls utilized in the rescue of DnaJC7 KO in tau biosensor cells with ALS-associated mutants of DnaJC7, colored as follows: Vehicle Control, purple; Ruby Control, orange; WT DnaJC7, gray dashed; HPQ mutant, dark blue. Error bars represent SEM of three technical replicates. (E) Rosetta-calculated mean Gibbs free energy shift (ddG) of the ALS-associated mutants of DnaJC7 vs. their rescue seeding with 33nM of tau fibrils. Gray dashed line denotes a mean ddG total score of 0. Mutants are colored according to their domain localization: TPR1, green; TPR2A, teal; TPR2B, dark blue; JD, orange. (F) Quantification of the western blot signal for the different ALS-associated mutants and controls in (C). All constructs were normalized to the band intensity of the WT construct. Domains are colored as in (C). (G) Rosetta-calculated mean Gibbs free energy shift (ddG) of the ALS-associated mutants of DnaJC7 vs. their normalized western blot band intensity. Gray dashed line denotes a mean ddG total score of 0. Mutants are colored as in (C).All error bars represent SEM of three technical seeding replicates. *=p < 0.05, **=p < 0.01, ***=p < 0.001, ****=p < 0.0001. Source data for this figure are provided in Figure 5—figure supplement 1—source data 1.
Figure 5—figure supplement 1—source data 1. This source data file contains the original uncropped images for the western blots shown in Figure 5—figure supplement 1B and C.