Figure 1 raw data

Raw fluorescence intensities and stochastic simulation outputs, which were used to generate data, presented in Figure 1.

Figure 1. Rab5:GDI activation on phospholipid membranes is ultrasensitive and stochastic. (A) Schematic of Rab5 activation reconstitution assay on a supported lipid bilayer (SLB). (B) Top panel: addition of Rabex5:Rabaptin5 triggers nucleotide exchange by CF488A-Rab5, which can be followed by an increase of fluorescence intensity on the membrane surface. Solid line is mean normalized intensity, shaded area corresponds to SD (N = 4). Bottom: micrographs of CF488A-Rab5 binding to the SLB after addition of 200 nM GEF complex and corresponding kymograph (below) taken along the yellow line. Scale bar = 5 µm. (C) Rab5 intensity traces obtained at increasing Rabex5:Rabaptin5 concentrations. (D) Rab5:GDI activation response diagram with Rabex5:Rabaptin5. The fold change was calculated by dividing the fluorescence intensity at steady state with the average signal 10 min before GEF addition. (E) Activation delay Ti decreases with higher Rabex5:Rabaptin5 concentration. Where no detectable activation was observed within 150 min, the Tis are denoted as >150 min and shown in orange. Error bars are SD. (F) Relative maximum rates kmax against the GEF complex concentration reveal cooperativity of Rab5 activation. Without detectable activation within 150 min, the activation rate was determined to be 0 and the corresponding points are depicted in orange. Error bars are means ± SD. (G) Schematic representation of modeled molecular interactions. We constructed a model of the minimal Rab5 activation network based on the known literature (9–11, 21, 53). The ODEs were derived from mass action kinetics. (H) Stochastic model simulations of Rab5 activation at increasing Rabex5:Rabaptin5 particle numbers. Shown are average curves from 50 individual runs in bold and 10 random traces per condition. The effective simulation time was scaled to align with experimental results. (I-K) Signal fold change, temporal delays and relative maximum rates from the stochastic simulations in H. We ran 50 individual stochastic simulations per condition.