Fig. 2

Mutations predicted to compromise TRIO-RAC1 stability disrupt TRIO-9’s influence on glutamatergic synapse function. a GEF1 mutations predicted to disrupt domain stability. TRIO protein shown in grey, RAC1 shown in cyan. b Average AMPAR-eEPSC amplitudes (± SEM) of neurons expressing WT TRIO-9 Y1318G, TRIO-9 E1304G, TRIO-9 G1453W and TRIO-9 Y1383A normalized to respective average control AMPAR-eEPSC amplitudes. Wilcoxon signed-rank test was used to compare related samples (p < 0.05). d, f, h, j Scatterplots show AMPAR-eEPSC amplitudes for 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). c, e, g, i TRIO protein shown in grey, RAC1 shown in cyan. Mutated residue and close contacts are shown in sticks, with TRIO residues in grey, RAC1 residues in cyan, and mutations in magenta. Blue dotted lines indicate Hydrogen bonds. c Interactions of E1304 amino acid residue in WT and mutant protein. d TRIO-9 E1304G expression showed no increase in AMPAR-eEPSC amplitude (n = 6 pairs, p < 0.05, Wilcoxon signed-rank rest), e Interactions of Y1318 amino acid residue in WT and mutant protein. f TRIO-9 Y1318G expression showed no increase in AMPAR-eEPSC amplitude (n = 11 pairs, p < 0.05, Wilcoxon signed-rank test), g Interactions of G1453 amino acid residue in WT and mutant protein. h TRIO-9 G1453W expression showed no increase in AMPAR-eEPSC amplitude (n = 8 pairs, p < 0.05, Wilcoxon signed-rank test), i Interactions of Y1383 amino acid residue in WT and mutant protein. j TRIO-9 Y1383A expression showed an increase in AMPAR-eEPSC amplitude (n = 6 pairs, p < 0.05, Wilcoxon signed-rank test) k 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. l 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 = 20 per condition, 2 experimental replicates, ****p < 0.0001, two-tailed Mann Whitney test) m 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