SYNAPTOTAGMIN 1

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Synaptotagmin 1是一种突触囊泡蛋白，已被证明作为触发快速神经递质释放的Ca2+传感器。synaptotagmin 1的大部分细胞质区是由两个c2结构域构成的，它们被称为c2a结构域和c2b结构域。我们之前已经解决了两个突触塔蛋白1 c2结构域的三维结构，并表明它们通过β -三明治尖端的环结合多个Ca2+离子。这些环也介导Ca2+依赖于带负电荷的磷脂的结合，这种活性很可能是synaptotagmin 1触发释放功能的基础。这些结果导致了c2a结构域和c2b结构域的顶部环与膜接触并在磷脂结合中合作的模型。含有synaptotagmin 1 c2结构域的片段（c2ab片段）在蛋白浓度接近1mm的Ca2+缺失或存在的溶液中是单体的。在这些研究过程中，动态光散射实验表明，c2ab片段在Ca2+依赖结合时诱导囊泡聚集，并且这种聚集可以通过添加EGTA来逆转。我们的第一个目标是利用低温电镜观察突触塔蛋白的囊泡聚集活性。最近有报道称，c2ab片段在Ca2+依赖性磷脂结合后形成七聚体低聚物。通过电子显微镜（EM）对沉积在EM网格上的脂质单层结合的c2ab片段进行阴性染色观察这些低聚物。然而，我们无法通过化学交联或荧光共振能量转移实验观察到这种低聚物。该项目的第二个目的是观察synaptotagmin是否在磷脂膜上是单体的，或者是否在单层的电子显微镜下形成七聚体环状低聚物。我们还想测量两个聚集的囊泡之间的距离，以计算囊泡之间的空间是否足够大，以容纳直径为11nm的synaptotagmin低聚物。该系统提供了研究神经功能、神经递质释放和膜融合等方面的机会。

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.