Dynamic Ca2+-Dependent Activity of Membrane-Anchored Synaptotagmin 1 Observed at the Content Mixing Level

Abstract

Synaptotagmin 1 (Syt1) is thought to be the main Ca 2þ switch for the presynaptic vesicle fusion. Although in vitro fusion assays importantly contributed to understanding the molecular mechanism of Syt1, the results was largely restricted to truncated Syt1 that retained only soluble C2AB domains. Using the single-vesicle fluorescence assay, we have recently shown the strong fusogenic activity of membrane-anchored Syt1 at physiological Ca 2þ levels (Science 328, 760 ( 2010)). Moreover, Syt1 shows a biphasic activity that Syt1 activity is observed to diminish at extraordinarily high Ca 2þ concentrations. By developing ability to detect content mixing in single vesicle fusion events, we here show that such dynamic Ca 2þ -dependent Syt1 activity is also observed at the content mixing level. In addition, we report point mutations in the linker, which was previously thought as a dull unstructured region, critically modulates the Syt1 activity. Therefore, Syt1 seems to become a far versatile fusion regulator when it in a form of membrane protein.