Fig. 3.

Rab5:GDI activation is tuned by free Rab5:GDP abundance. (A) Rab5 cycles between the membrane and solution before and after nucleotide exchange. (Top) sCy5-Rab5 molecule counts per frame and collective CF488A-Rab5 activation. (Bottom) Snapshots of the activation reaction. sCy5- and CF488A-Rab5 are depicted in yellow and cyan, respectively. The sCy5 channel was smoothed before merging to reduce nonspecific high-frequency noise (scale bar, 10 μm). (B) Rab5 single-molecule trajectories reveal GDP- and GTP-bound proteins on the membrane. (Top) Five hundred tracks of membrane-bound sCy5-Rab5 particles before (GDP) and after (GTP) activation. (Bottom) Frequency diagram identifies two populations with distinct lifetimes. A monoexponential decay with lifetime τ^GDP and two-exponential decay with lifetimes τ₁^GTP and τ₂^GTP was fitted to grouped data from n = 5 independent experiments, respectively (n^GDP = 4829, τ^GDP = 0.58 ± 0.04 s; n^GTP = 3090, τ₁^GTP = 1.20 ± 0.35 s, τ₂^GTP = 10.6 ± 3.3 s; errors are 95% CI). Box plot with mean lifetimes, **P < 0.01, two-sided Student’s t test (n = 5). (C) Parameter phase space of the phenomenological model for Rab5 switching, depending on the basal rate of activation $\left(a_0/a_{2}K\right)$ and the strength of positive feedback $\left(a_1/a_{2}K\right)$. Switching is defined as the relative difference in steady-state Rab5 concentration on the membrane relative to the scenario with no positive feedback. (Inset) Fold activation along the red line in the diagram. Stochasticity was introduced by solving the phenomenological model within a Fokker–Planck framework. See text for parameter definitions. (D) Stoichiometric GDI excess over Rab5 affects delay of Rab5 activation in vitro. (Left) Solid lines are mean normalized intensities over time, shaded areas correspond to SD (n = 3). (Right) Corresponding activation T_i and relative maximum rates k_max. (E) Stochastic simulations of the full model for varying initial amounts of GDI excess (0 to 2,000 particle number). Shown are curves from 10 random runs per condition; the mean line from 50 runs is depicted in bold. The effective simulation time was scaled to align with experimental results. (F) PRA1 in the membrane enhances Rab5 activation at low GEF concentrations. Solid lines are mean normalized fluorescence intensities; shaded areas correspond to SD (n = 3).