Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

Neuronal exocytosis is mediated by Ca2+-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca2+-sensing protein proposed to regulate fusion pore dynamics via Ca2+-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca2+-triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Here, we show that Ca2+ does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca2+-C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are approximate to11 nm long and approximate to11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca2+ first drives C2AC2B-membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca2+-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.