Abstract
BackgroundWhen purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilaments start growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane. Isolated phagosomes 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 the same time that the actin nucleation is initiated. Our aim was to model all these processes in parallel.ResultsWe compiled detailed experimental data on the effects of different lipids and ATP on actin nucleation and we investigated 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 in COPASI (Complex Pathway Simulator). However, several lines of experimental evidence indicated that only the phosphatidylinositol branch of the network was active, an observation that dramatically reduced the number of parameters in the 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 other hand, a molecular mechanism connecting membrane lipids, ATP, and the actin nucleation process is still missing. We therefore adopted a phenomenological (black-box) approach to represent the empirical observations. We proposed that lipids and ATP influence the dynamic interconversion between active and inactive actin nucleation sites. With this simple model, all the experimental data were satisfactorily fitted with a single positive parameter per lipid and ATP.ConclusionBy establishing an active 'dialogue' between an initial complex model and experimental observations, we could narrow the set of differential equations and parameters required to characterize the time-dependent changes of metabolites influencing 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 complex system with a relatively small set of differential equations and kinetic parameters that satisfactorily reproduced the experimental data.
Highlights
When purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilaments start growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane
By establishing an active 'dialogue' between an initial complex model and experimental observations, we could narrow the set of differential equations and parameters required to characterize the time-dependent changes of metabolites influencing actin nucleation on phagosomes
The global model was dissected into three sub-models: ATP consumption, lipid interconversion, and nucleation of actin on phagosomal membranes
Summary
When purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilaments start growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane. LBP represent the simplest imaginable membrane system, a single bilayer around a 1 μm latex bead These organelles are of amazing complexity, being composed of at least 1000 so far identified proteins (Desjardins, personal communication) as well as hundreds, if not thousands of lipids (Brouwers et al, personal communication). Among these are functional units of proteins and lipids that sense incoming signals, regulate fusion, and interact with the cytoskeleton [1]. Since all these processes have been reconstituted in vitro [2,3,4,5], the intricate membrane machinery required for their operation must be functionally intact on these isolated membrane organelle
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