Fig. 1

Mutations predicted to compromise TRIO-RAC1 binding disrupt TRIO-9’s influence on glutamatergic synapse function. a GEF1 mutations predicted to disrupt Rac1 binding. TRIO-9 is shown in grey, RAC1 is shown in cyan. b Electrophysiological recording setup. c Average AMPAR eEPSC amplitudes (± SEM) of neurons expressing wild-type (WT) TRIO-9, TRIO-9 E1299W, TRIO-9 C1387W, TRIO-9 T1430W and TRIO-9 A1464W normalized to their respective average control AMPAR-eEPSC amplitudes. Wilcoxon signed-rank was used to compare related samples (* = p < 0.05). d, f, h, j, l Scatterplots show AMPAR-eEPSC amplitudes for single pairs of control and transfected neurons (open circles). Filled circles show mean ± SEM. (Insets) Current traces from control (black) and transfected (various colors) neurons (Scale bars: 20 ms, 20 pA). d TRIO-9 expression increased AMPAR-eEPSC amplitude (n = 8 pairs, p < 0.05, Wilcoxon signed-rank test). e, g, i, k TRIO protein is shown in grey, RAC1 is shown in cyan. Mutated residue and close contacts are shown in sticks, with TRIO residues in grey, RAC1 residues in cyan, and mutation in orange. Hydrogen bonds are shown as blue dotted lines. e Interactions of E1299 amino acid residue in WT and mutant protein. f TRIO-9 E1299W expression showed a significant reduction in AMPAR-eEPSC amplitude (n = 7 pairs, p < 0.05, Wilcoxon signed-rank test), g Interactions of C1387 amino acid residue in WT and mutant protein. h TRIO-9 C1387W expression showed no increase in AMPAR-eEPSC amplitude (n = 9 pairs, p < 0.05, Wilcoxon signed-rank test), i Interactions of T1430 amino acid residue in WT and mutant protein j TRIO-9 T1430W expression showed no increase in AMPAR-eEPSC amplitude (n = 7 pairs, p < 0.05, Wilcoxon signed-rank test), k Interactions of A1464 amino acid residue in WT and mutant protein. l TRIO-9 A1464W expression showed an increase in AMPAR-eEPSC amplitude (n = 7 pairs, p < 0.05, Wilcoxon signed-rank test), m Representative immunoblots of lysates from HEK293T cells expressing GFP-TRIO-9 WT and GFP-TRIO-9 binding mutants each co-immunoprecipitated with FLAG-RAC1, probed with anti-GFP, anti-FLAG and anti-GAPDH, IN denotes input fractions, IP denotes immunoprecipitated fraction. n Mean Fluorescence Lifetime (τ) (± SEM) from exponential decay fit of field of ~20 cells per condition. Lower values indicate higher FRET and increase in RAC1 activity. Two-tailed Mann Whitney test was used to compare TRIO WT against sensor alone and each mutant against TRIO WT (n = 10 per condition, 2 technical replicates, ***p < 0.0001) o Phasor plots of flurorescence lifetime data for sensor alone, TRIO-9 WT and binding mutants. The x- and y- axes respectively display g = cosθ and s = sinθ transforms of the decay lifetime curve per pixel. The red circle was positioned around the ‘sensor alone’ signal and maintained in this position for all phasor plots. Extension to the right indicates an increase in FRET above the ‘sensor alone’ condition corresponding to a decrease in donor decay lifetime