SYNAPTOTAGMIN 1

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Synaptotagmin 1 is a synaptic vesicle protein that has been shown to act as the Ca2+ sensor that triggers fast neurotransmitter release. Most of the cytoplasmic region of synaptotagmin 1 is formed by two C2-domains that are referred to as C2A-domain and C2B-domain. We have previously solved the three-dimensional structures of both synaptotagmin 1 C2-domains and shown that they bind multiple Ca2+ ions through loops at the tip of a beta-sandwich. These loops also mediate Ca2+-dependent binding to negatively charged phospholipids, the activity that most likely underlies the function of synaptotagmin 1 in triggering release. These results have led to a model whereby the top loops of the C2A-domain and C2B-domain are in contact with the membrane and cooperate in phospholipid binding. A fragment containing both synaptotagmin 1 C2-domains (C2AB-fragment) is monomeric in solution in the absence or presence of Ca2+ at protein concentrations close to 1 mM. In the course of these studies, dynamic light scattering experiments suggested that the C2AB-fragment induces vesicle clustering upon Ca2+-dependent binding, and that such clustering can be reversed by addition of EGTA. Our first aim is to visualize this vesicle clustering activity of synaptotagmin using cryo-EM. It was recently reported that the C2AB-fragment forms heptameric oligomers upon Ca2+-dependent phospholipid binding. These oligomers were observed by electron microscopy (EM) performed with negative staining on samples of C2AB-fragment bound to lipid monolayers deposited on EM grids. However, we have not been able to observe such oligomers by chemical cross-linking or fluorescence resonance energy transfer experiments. The second aim of this project is to see whether synaptotagmin is monomeric on the phospholipid membranes or if it forms heptameric ring-like oligomers as observed in electron microscopy of monolayers. We would also like to measure the distance between two clustered vesicles to calculate whether the space between the vesicles is big enough to accommodate synaptotagmin oligomers of 11 nm diameter. This system afford the opportunity to study aspects of neural functioning, neurotransmitter release and membrane fusion in general.