Fig. 3

Mutations predicted to be benign to TRIO-RAC1 interaction do not interfere with TRIO-9’s influence on glutamatergic synapse function. a GEF1 mutations predicted to be benign b Average AMPAR-eEPSC amplitudes (± SEM) of neurons expressing WT TRIO-9, TRIO-9 Y1394A and TRIO-9 T1394A normalized to their respective average control AMPAR-eEPSC amplitudes. Wilcoxon Rank Sum Test was used to compare related samples (* = p < 0.05). c, e In Residue Models, TRIO protein shown in grey, RAC1 shown in cyan. Mutated residues and close contacts shown in sticks, with TRIO residues in grey and mutation in green. c Interactions of T1394 amino acid residue in WT and mutant protein. d, f 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 T1394A expression increased AMPAR-eEPSC amplitude (n = 7 pairs, p < 0.05, Wilcoxon signed-rank test), e Interactions of S1403 amino acid residue in WT and mutant protein. f TRIO-9 S1403F expression increased AMPAR-eEPSC amplitude (n = 7 pairs, p < 0.05, Wilcoxon signed-rank test) g Representative immunoblots of lysates from HEK293 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. h 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 acitvity. 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, two-tailed Mann Whitney test) i Phasor plots of fluorescence 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