The Role of Dynamins in the Fusion of Synaptic Vesicles and their Subsequent Recycling

Abstract

Kiss and run (KR) is a highly debated mode of synaptic vesicle (SV) recycling in neurons, and limited research has investigated the protein pathways that regulate it. This thesis demonstrates that protein kinase A (PKA) activation can specifically switch the reserve pool (RP) of SVs to KR, whilst PKA inhibition switches the readily releasable pool (RRP) of SVs to full fusion (FF) for some stimuli. This thesis also demonstrated that cytosolic Dynamin-I (Dyn-I) is not required to mediate the basal KR observed during exocytosis, but a membrane bound sub-pool of Dyn-I is. KR can only occur when actin filaments are polymerised or able to polymerise, and actin polymerisation is also required to mobilise the RP to fuse at the active zone (AZ). Activation of adenylyl cyclase (AC) can block release of the RP by lowering intracellular Ca2+ ([Ca2+]i) levels via activation of exchange-proteins activated by cyclic-AMP (EPACs), but activation of AC can also switch the RRP to a KR mode of exocytosis by increasing [Ca2+]i during certain stimulation paradigms. This thesis also validates that Serine (Ser-795) is an in vivo phosphorylation site, and a confirmed target of protein kinase C (PKCs) and protein phosphatase 1 or 2A (PP1 or PP2A). Activation of PKA significantly decreases the basal phosphorylated state of Ser-795, A conditions which increases the prevalence of KR. These results reveal significant new roles for PKA and AC in regulation of SV exocytosis, for distinct pools, and highlight the sub-pool of membrane bound Dyn-I and the vital role of actin during exocytosis. Certainly future research may reveal the overall importance of dysfunction in these processes and the roles they could play in understanding neuronal disorders and disease states as dysfunctional communication has been associated with many of these. The understanding of how distinct modes of recycling are regulated by protein pathways is vital to this research.