CONICET | Buscador de Institutos y Recursos Humanos

Background: When purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilamentsstart growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane. Isolatedphagosomes are metabolically active organelles that contain enzymes and metabolites necessary for lipid interconversion.Hence, addition of ATP, lipids, and actin to the system alter the steady-state composition of the phagosomal membrane at thesame time that the actin nucleation is initiated. Our aim was to model all these processes in parallel.Results: We compiled detailed experimental data on the effects of different lipids and ATP on actin nucleation and weinvestigated experimentally lipid interconversion and ATP metabolism in phagosomes by using suitable radioactive compounds.In a first step, a complex lipid network interconnected by chemical reactions catalyzed by known enzymes was modelled inCOPASI (Complex Pathway Simulator). However, several lines of experimental evidence indicated that only thephosphatidylinositol branch of the network was active, an observation that dramatically reduced the number of parameters inthe model. The results also indicated that a lipid network-independent ATP-consuming activity should be included in the model.When this activity was introduced, the set of differential equations satisfactorily reproduced the experimental data. On the otherhand, a molecular mechanism connecting membrane lipids, ATP, and the actin nucleation process is still missing. We thereforeadopted a phenomenological (black-box) approach to represent the empirical observations. We proposed that lipids and ATPinfluence the dynamic interconversion between active and inactive actin nucleation sites. With this simple model, all theexperimental data were satisfactorily fitted with a single positive parameter per lipid and ATP.Conclusion: By establishing an active 'dialogue' between an initial complex model and experimental observations, we couldnarrow the set of differential equations and parameters required to characterize the time-dependent changes of metabolitesinfluencing actin nucleation on phagosomes. For this, the global model was dissected into three sub-models: ATP consumption,lipid interconversion, and nucleation of actin on phagosomal membranes. This scheme allowed us to describe this complexsystem with a relatively small set of differential equations and kinetic parameters that satisfactorily reproduced the experimentaldata.