Extended Data Fig. 1: PTEN sensor linker optimization.
From: Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain
a Schematic design of the different FRET/FLIM-based mEGFP-PTEN-sREACh sensor linker versions; combinations of extended linkers on either side of mEGFP-PTEN-sREACh (EL1, EL2 respectively) and truncated linkers on either side of mEGFP-PTEN-sREACh (TL1, TL2 respectively). b Quantification of fluorescence lifetime of the different mEGFP-PTEN-sREACh linker versions. EL1 + EL2 (2.30 ± 0.002 ns, n = 310), EL1 + EL2 4 A (2.52 ± 0.002 ns, n = 223), TL1 + EL2 (2.39 ± 0.009 ns, n = 320), TL1 + EL2 4 A (2.58 ± 0.002 ns, n = 310), TL2 + EL1 (2.33 ± 0.004, n = 320), TL2 + EL1 4 A (2.56 ± 0.002 ns, n = 400), TL1 + TL2 (2.19 ± 0.004 ns, n = 288) or TL1 + TL2 4 A (2.51 ± 0.002 ns, n = 281). Comparisons between WT and 4 A mutation of each variant were all p < 0.0001. (c) Quantification of change in mean fluorescent lifetime (Δ) of the different mEGFP-PTEN-sREACh linker version 4 A mutations from WT; EL1 + EL2 4 A (0.22 ± 0.002 ns, n = 223), TL1 + EL2 4 A (0.19 ± 0.002 ns, n = 310), TL2 + EL1 4 A (0.23 ± 0.002 ns, n = 400), TL1 + TL2 4 A (0.32 ns ±0.002 ns, n = 281). ns p = 0.2106. ‘n’ denotes number of cells, error bars represent s.e.m. statistical differences were measured using one-way ANOVA followed by post-hoc Tukey’s multiple comparison test. Significant differences in (c) produced p < 0.0001, unless otherwise stated. **** p < 0.0001.
