Fig. 2.

Positive feedback of Rab5 activation depends on GEF recruitment. (A) Illustration of protein interactions responsible for collective Rab5 switching. Positive feedback originates from a direct interaction between Rabex5:Rabaptin5 and Rab5:GTP. (B) Fluorescence intensity traces obtained from experiments depicted in A. Solid lines are mean normalized intensities; shaded areas are SD (Rabex5:Rabaptin5, ΔRabex5:Rabaptin5 n = 4; ΔRabex5, Rabex5:Δ_RBDRabaptin5 n = 3). (C) Stochastic model simulations with and without Rabex5:Rabaptin5:Rab5:GTP complex formation (k₅, k₆ = 0) for 200 Rabex5:Rabaptin5 particles. Average curves from 50 individual runs are depicted in bold with 10 random traces per condition. The effective simulation time was scaled to align with experimental results. (D) Kinetic traces of CF488A-Rab5 and Rabex5:sCy5-Rabaptin5 activation. Solid line is mean normalized fluorescence intensity; shaded area is SD (n = 5). (Inset) T_i for CF488A-Rab5 (blue) and Rabex5:sCy5-Rabaptin5 (orange). (E) Stochastic model simulations for Rab5 and Rabex5:Rabaptin5 membrane binding for 200 Rabex5:Rabaptin5 particles. Shown are curves from 50 independent runs; the mean line is depicted bold with 10 random traces per condition. (F) Schematic of the reconstitution experiment with preactivated SLB-immobilized Rab5Q80L-His₁₀:GTP. (G) Collective switching is faster with preactivated Rab5. (Left) Rab5 switching time courses in presence of 500 nM Rab5Q80L-His₁₀ with increasing DOGS-NTA lipid concentration in the SLB. Solid line is mean normalized fluorescence intensity over time, shaded area is SD (n = 3). (Right) Corresponding time delays T_i and relative maximum rates k_max.