Analisi quantitativa dell'interazione tra proteine coinvolte nel traffico di membrana

Rab5 and Rab11 are small GTPase proteins involved in membrane trafficking, that cycle between an active and an inactive state. In particular, Rab5 and Rab11 are detected on the membrane of compartments implicated in intracellular transport such as endosomes. Live-cell fluorescence imaging provides strong evidence that Rab5 and Rab11 are simultaneously present on the endosomal membrane at the early stages of the recycling pathway. Videos composed by interlaced frames with fluorescently marked Rab5 and Rab11 are analyzed in order to infer quantitative information on the two proteins. Analysis suggests that active Rab5 and total Rab11 accumulate on mutually exclusive domains, as well as active Rab11 and total Rab5. This suggest that the two molecules are connected by a reciprocal inhibitory relation. Moreover, biochemical experiments are performed to gain further insight into the Rab5-Rab11 quantitative correlation: cells are trasfected with active and inactive forms of both proteins and subsequent levels of both active Rab forms are observed. These experiments not only support image analysis results showing an active Rab5-active Rab11 mutual exclusivity, but they also add further important information: a self-activation mechanism of both Rab5 and Rab11 emerges. Taking all observations into account we can hypothesize a relational scheme between Rab5 and Rab11. Our hypothesis involves a Rab5-Rab11 common effector that contributes to their recruitment to the membrane. In such a scenario the two molecules compete for a common effector pool: when all effector molecules are sequestered by one Rab, its activation is enhanced while the other Rab's activation is inhibited. With this scheme both the positive self-loops and the mutual inhibition emerge. Steady-state responses arising from this model are computed as functions of parameters. The behaviour of active Rab5 and active Rab11 is observed by varying parameter values in a range compatible with already known reaction rates. A qualitative feature robust to parameter variation emerges: the system always occupies two main regions corresponding either to high active Rab5-low active Rab11 concentration or low active Rab5-high active Rab11 concentration, thus qualitatively reproducing experimental observations. The transition between the two regimes is quick with respect to parameter variation: the system is ultrasensitive to fluctuations in the parameter values. Ultransensitivity is found in a number of biological phenomena such as similar protein-protein interactions or mRNA-target interaction. In order to perform a proper quantitative comparison with experimental data, simulations with the Gillespie algorithm are included in this study's future perspectives.