Characterization of Synaptotagmin Function In Calcium Dependent Neuronal Exocytosis

Abstract

Synaptotagmin 1 is a synaptic vesicle protein believed to be the calcium sensor at the neuronal synapse. The protein consists of two calcium dependent membrane binding domains known as the C2 domains. Previous studies have shown that deletion of Synaptotagmin 1 results in a loss of calcium dependent secretion at the synaptic terminal. More recently, much work has focused on unravelling the molecular mechanism of Synaptotagmin 1 function. However, due to its highly dynamic and fast action, the biochemical assays required to elucidate its precise function have remained somewhat limited. Conventional methods, such as GST pulldowns and immunoprecipitation lack sufficient resolution to capture the protein ¡§in action¡¨. Currently, it is hypothesised that upon calcium influx into the presynaptic terminal, Synaptotagmin rapidly binds to lipid membrane resulting in the fusion of the synaptic vesicle with the plasma membrane. Recent evidence has also suggested that Synaptotagmin binds to the SNARE complex. The SNARE proteins are a superfamily of proteins that drive membrane fusion. There are two classes of SNARE proteins, the Q-SNARE and the R-SNARE, which forms a four helical bundle, required for the merger of opposing membranes. However, the role and mechanism of the SNARE complex interaction with Synaptotagmin remains unclear. Another SNARE-interacting protein that has been implicated in calcium dependent exocytosis is Complexin. The exact interplay between Complexin and Synaptotagmin is largely unknown although Complexin has been suggested to regulate the interaction of Synaptotagmin with the SNARE complex. Given as such, the major aim of this project was to develop biochemical tools to enable a more precise characterisation of the molecular mechanism of Synaptotagmin action. Due to the inherent functional complexity of the protein, assays that could better define the distinct interactions of Synaptotagmin with its binding partners were developed. The first section of this dissertation focuses on the calcium binding to the C2 domains. This aspect was explored using a method known as isothermal titration calorimetry (ITC). ITC allows the direct determination of parameters such as affinity (K), enthalpy ( ´H), entropy ( ´S) and Gibbs free energy ( ´G) using a single titration experiment. By employing this method, calcium titration into Synaptotagmin revealed that the two C2 domains of Synaptotagmin (namely C2A and C2B) binds calcium with divergent mechanisms and are independent of each other. Furthermore, no additional weak calcium binding sites were evident when calcium was titrated to the C2 domains rendered inactive by site-directed mutagenesis. The second section of this thesis details the use a fluorescence resonance energy transfer (FRET)-based method used to study the binding of Synaptotagmin to liposome membranes. This approach was employed to examine the potential effects of lipid composition and phosphatidylserine (PS) density on Synaptotagmin binding. Interestingly, increasing the amount of PS in the liposome membrane appeared to correspond with an increase the affinity of Synaptotagmin to PS hinting that microdomains might play a role in Synaptotagmin function. The final section of the thesis attempts to further clarify the interaction of Synaptotagmin with the membrane fusion machinery, i.e. SNARE complex. This was achieved using complementary gel-based and fluorescence-based assays to probe the function of the Synaptotagmin ¡V SNARE interaction. Based on the results presented herein, the binding of Synaptotagmin to SNAREs was exclusively observed for both the binary and ternary SNARE complex. In contrast to previous reports, binding of Synaptotagmin to individual Q-SNARE proteins was not detected under the assay conditions outlined in this study. It has also been suggested that Synaptotagmin might play a role in assisting SNARE complex assembly. However based on the findings presented in this study it appears highly unlikely given that the addition of Synaptotagmin both in the absence and presence of calcium does not significantly affect the kinetics of SNARE complex assembly. Finally, the potential role of Complexin in the interplay between Synaptotagmin and the SNARE complex was studied using a FRET-based assay. Synaptotagmin was able to bind to the SNARE complex in the presence of Complexin, but only in the presence of calcium. In the absence of calcium, the interaction between Synaptotagmin and the SNARE complex was abolished. Collectively, the findings of this thesis provide important new insights into the function of Synaptotagmin. However, future work remains to elucidate the exact molecular details of Synaptotagmin action in calcium-dependent exocytosis.