A New Platform to Study the Molecular Mechanisms of Exocytosis

Abstract

The exocytotic process in neurons and neuroendocrine cells consists of a sequence of reactions between well defined proteins. In the present study, we have created for the first time a comprehensive kinetic model that demonstrates the dynamics of interactions between key synaptic proteins that are associated with exocytosis. The interactions between the synaptic proteins were transformed into differential rate equations that, after their integration over time, reconstructed the experimental signal. The model can perfectly reconstruct the kinetics of exocytosis, the calcium-dependent priming and fusion processes, and the effects of genetic manipulation of synaptic proteins. The model suggests that fusion occurs from two parallel pathways and assigns precise, non-identical synaptic protein complexes to the two pathways. In addition, it provides a unique opportunity to study the dynamics of intermediate protein complexes during the fusion process, a possibility that is hidden in most experimental systems. We thus developed a novel approach that allows detailed characterization of the temporal relationship between synaptic protein complexes. This model provides an excellent platform for prediction and quantification of the effects of protein manipulations on exocytosis and opens new avenues for experimental investigation of exocytosis.

Vesicle Priming and Recruitment by ubMunc13-2 Are Differentially Regulated by Calcium and Calmodulin

“…5A) were accurately reconstructed by the model using the very same set of previously published parameters without any readjustments (Mezer et al, 2004(Mezer et al, , 2006. The reconstruction of the fusion dynamics recorded Figure 5.…”

“…The reaction mechanism is basically linear, in which the product of one step is the reactant for the next. The model in this manuscript is an extension of a formal model presented previously (Mezer et al, 2004(Mezer et al, , 2006. A detailed description of the logic underlying the model was published previously (Mezer et al, 2004).…”

“…The model in this manuscript is an extension of a formal model presented previously (Mezer et al, 2004(Mezer et al, , 2006. A detailed description of the logic underlying the model was published previously (Mezer et al, 2004). Briefly, the modeling of the exocytotic process was started with the reaction between SNAP-25 and syntaxin I to form the binary SNARE complex.…”

“…Finally, the cascade drives vesicle fusion from RCI and RCII with the cell membrane during Ca 2ϩ binding. The steps beyond the ubMunc13-2 pathway were kept constant according to the previously described parameters (Mezer et al, 2004(Mezer et al, , 2006.…”

“…Because ubMunc13-2 causes an apparent change in the kinetics of the SRP and has an S-shaped time course that cannot be fitted with the three-exponential fitting procedure, we decided to analyze the nature of these changes using a detailed kinetics analysis based on a previously published protein-protein interaction simulation (Mezer et al, 2004(Mezer et al, , 2006. We implemented a set of new reactions in the model that represent the binding of Ca 2ϩ /CaM to ubMunc13-2, performed a detailed kinetics search, and reconstructed the experimental data (Fig.…”