Figure 2. Phosphorylation mimicking mutations at S365-S366-S367 result in the PS1 conformational shift.
(A) FLIM analysis of the PS1 conformation in 7 W cells transfected with WT or phosphorylation-mimicking mutant G-PS1-R. The FRET efficiency between GFP and RFP in phospho-mutants is normalized to the average FRET efficiency of the WT G-PS1-R expressing cells (n = 42–53 cells). Mean ± SEM, ***p<0.001, one-way factorial ANOVA. (B) Spectral FRET analysis shows RFP/GFP (R/G) ratio in 7 W cells transfected with WT or single phosphorylation-mimicking mutant G-PS1-R (n = 50–69 cells). Mean ± SEM, ***p<0.001, one-way factorial ANOVA.
Figure 2—figure supplement 1. Calcium imaging and validation of the phosphorylation at the three domains-driven PS1 conformational change by the G-PS1-R FRET reporter probe.
This figure is related to the data displayed in Table 1. (A) Indo-1/Ca2+ imaging shows changes in intracellular Ca2+ levels of the 7 W cells transfected with FLAG-WT PS1, following 15 min treatment with vehicle control or with 5 µM A23187 (n = 250–297 cells). Mean ± SEM, ***p<0.001, Student’s t-test. (B) FLIM analysis of the PS1 conformation in the 7 W cells transfected with WT or domain 1, 2 or 3 phosphorylation-inhibited mutants PS1, following 15 min treatment with vehicle control or with 5 µM A23187. The FRET efficiency in each group is normalized to the average FRET efficiency of vehicle-treated cells expressing WT G-PS1-R (n = 22–55 cells). Mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, one-way factorial ANOVA.
Figure 2—figure supplement 2. Schematic image of the three domains in PS1 involved in Ca2+-triggered pathogenic ‘closed’ conformation.
This figure is related to the data displayed in Table 1. Schematic image of PS1 molecule and the three domains involved in Ca2+-triggered PS1 pathogenic ‘closed’ conformation (blue squares). Serine/threonine residues known to undergo phosphorylation are shown by red circles.