Abstract
Calcium-stimulated secretion is a process with many well-characterized players, yet the exact mechanism by which calcium triggers the vesicle fusion process remains under investigation. Synapsins are a family of abundant synaptic phosphoproteins that dynamically associate with synaptic vesicles. Synapsins have sites for phosphorylation by multiple calcium/calmodulin kinases (CaMKs) and protein kinase A (PKA). Chi et al. used green fluorescent protein (GFP)-labeled synapsin Ia to monitor synapsin localization before, during, and after action potential stimulated secretion in hippocampal neurons. In addition, they monitored synaptic vesicle turnover using the fluorescent dye FM 4-64. Endogenous and GFP-synapsin disperses away from synaptic vesicles into the axon in response to action potential stimulation. Chi et al. were able to measure the rate of synapsin disperson and, using a series of mutants for the various CaMK sites, studied how phosphorylation controlled the kinetics of this process. Analysis of vesicle fusion and synapsin movement in cells expressing wild-type synapsin Ia and phosphorylation mutants of synapsin Ia showed a strong correlation between the rate of synapsin dispersion and the rate of vesicle fusion: The slower the synapsin dispersion, the slower the rate of vesicle turnover. This suggests that synapsin acts as a rate regulator, not an "on-off" switch for vesicle fusion. The analysis of the phosphorylation mutants expressed in wild-type or synapsin I- and II-deficient neurons led to two conclusions. First, the CaMK II phosphorylation sites (sites 2 and 3) appeared to regulate synapsin homo- and heterodimer formation. Second, the CaMK I and IV site (site 1), which is also a PKA phosphorylation site, appeared to regulate a direct interaction between synapsin and the vesicle that is independent of the synapsin-synapsin interaction. These real-time experiments provide "in synapse" verification for a model based on biochemical experiments for synapsin as an inhibitor of vesicle fusion under nonstimulated conditions and the release of synapsin upon calcium-dependent phosphorylation, allowing vesicle fusion to occur (see Murthy).P. Chi, P. Greengard, T. A. Ryan, Synapsin dispersion and reclustering during synaptic activity. Nature Neurosci. 4, 1187-1193 (2001). [Online Journal]V. N. Murthy, Spreading synapsins. Nature Neurosci. 4, 1155-1156 (2001). [Online Journal]
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