Fig. 10. Macropinocytic membrane traffic in tracer-exposed DCs. The model illustrates three sequential membrane traffic phases (specified at the top and dealt with in the Discussion), occurring in DCs during the first 30 minutes of tracer (orange dots) exposure. Phase 1 starts with the application of the tracers (Bds or Dex) and ends 4 minutes later, with the change of the kinetics of tracer uptake (see Fig. 2A). During this time [Ca²⁺]_i begins to rise in DCs (see Fig. 3D,E). In addition to the low-rate macropinocytosis, the main traffic event of Phase 1 is the early exocytosis of enlargeosomes (yellow membrane), initiated within 1 minute and continued thereafter (see Fig. 8G). Some specialization of the plasma membrane by sorting of specific components, anticipating the generation of macropinosomes (purple), is assumed to take place at the bending ruffles sticking out from the cell surface. The increased rate of tracer-positive macropinosome (purple membrane) generation, which seems to depend on [Ca²⁺]_i (blocked by cell preloading with BAPTA) and PI3K activity (blocked by Wort) (see Fig. 2 and supplementary material Fig. S2), is illustrated in Phase 2. Macropinosomes, progressively enlarged (probably by fusion) and packed with the tracer, acquire in sequence various markers: first Rbk-5, then EEA1 (early endosomes, blue membrane) and finally TfR (recycling endosomes, green membrane) (Fig. 2B,C; supplementary material Fig. S2). During this phase tracer uptake predominates, however macropinosomes can undergo regulated exocytosis (regurgitation, blocked by Vac1, Fig. 7A), with discharge of their segregated tracer. This process depends on [Ca²⁺]_i. In the case of µM [Ca²⁺]_i increases, such as those induced by IONO, exocytosis becomes prompt and complete (see Fig. 5). The alternative possibility, more frequent for macropinosomes positive for TfR and no longer for EEA1 (Fig. 7C), is entering in a `deep pathway', presumably leading to processing and finally to presentation of the antigens. Phase 3, in which uptake and discharge of tracer approach equilibrium, begins after 20 minutes of application (Fig. 1B and Fig. 7) and probably continues as long as the tracer is applied. During this phase, even if macropinocytosis continues at similar rate, the number of macropinosomes per DC remains approximately stable (Fig. 1A,B and Fig. 7A) so that cell overloading is prevented.