The role of Extended-Synaptotagmins in the cortical endoplasmic reticulum of neurons

描述（由申请人提供）：内质网（ER）负责合成各种膜蛋白和脂质，这些蛋白和脂质通过囊泡运输递送至目的地;然而，ER也与质膜（PM）形成直接接触。组成性ER-PM（皮质ER）接触已被证明是至关重要的，在肌肉中传播钙信号和诱导接触在所有细胞（由刺激1-奥赖介导）负责存储操作的Ca 2+进入。最近，我们的实验室表征了由一组ER蛋白介导的新型ER-PM接触，即Extended-Synaptotagmins（Esyt 1 -3）。这些通过它们的C2结构域与PM中的PI（4，5）P2形成接触，并且另外通过胞质Ca 2+的增加来调节。E-Syts的晶体结构表明，它们含有SMP结构域，这是一种具有疏水腔的管状模块，其中含有脂质。这与越来越多的证据表明膜接触位点在ER和PM之间的脂质调节和转移中的作用一致。E-Syt可能在神经元中很重要，因为它们在大脑中高度表达。由于神经元的突起通常很长，新合成的膜脂通过分泌途径的囊泡运输是困难的。通过E-Syt接触位点直接转移新合成的蛋白质在长轴突和树突的生长过程中或在神经传递过程中可能特别重要，此时膜蛋白的快速耗尽需要从ER有效补充。事实上，神经元对脂质转移的中断特别敏感，这可能导致膜运输缺陷和受损的神经突生长。本提案的目标是检验E-Syt介导的接触在神经元维持、突触传递和神经突发育中起关键作用的假设。我将通过检查索马、轴突和树突中的皮质ER来表征E-Syt介导的神经元中的ER-PM接触。我将使用连续块面扫描电子显微镜，TIRF，旋转盘共聚焦，并在体内双光子显微镜检查这些接触在脑组织，培养的神经元，和活的动物。我将测试的假设，即E-Syt接触参与i神经传递，通过确定去极化E-Syt招聘到膜的影响。此外，我将通过评估E-Syt敲低对分支和树突棘发育的影响来确定E-Syt接触是否在神经突发育中发挥作用。

DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) is responsible for the synthesis of a variety of membrane proteins and lipids, which are delivered to their destination by vesicular transport; however the ER also forms direct contacts with the plasma membrane (PM). Constitutive ER-PM (cortical ER) contacts have been shown to be crucial in muscle for propagating calcium signals and inducible contacts in all cells (mediated by Stim1-Orai) are responsible for store-operated Ca2+ entry. Recently our laboratory characterized a new type of ER-PM contact mediated by a set of ER proteins, the Extended-Synaptotagmins (Esyt1-3). These form contacts with the PI(4,5)P2 in PM via their C2 domains and are additionally regulated by increase in cytosolic Ca2+. The crystal structure of E-Syts shows that they contain an SMP domain, a tubular module with a hydrophobic cavity that harbors lipids. This is consistent with growing evidence for a role of membrane contact sites in lipid regulation and transfer between the ER and PM. E-Syts are likely to be important in neurons as they are highly expressed in the brain. Since neuron processes are often very long, vesicular transport of newly synthesized membrane lipids via the secretory pathway is difficult. Direct transfer of newly synthesized via E-Syt contact sites could be especially important during outgrowth of long axons and dendrites or during neurotransmission when rapid depletion of membrane proteins requires efficient replenishment from the ER. Indeed, neurons are particularly sensitive to disruptions in lipid transfer, which can cause both membrane trafficking defects and impaired neurite outgrowth. The goal of this proposal is to test the hypothesis that E-Syt mediated contacts play a critical role in neuron maintenance, synaptic transmission, and neurite development. I will characterize E-Syt mediated ER-PM contacts in neurons by examining cortical ER in the soma, axons and dendrites. I will use serial blockface scanning electron microscopy, TIRF, spinning disc confocal, and in vivo two-photon microscopy to examine these contacts in brain tissue, cultured neurons, and living animals. I will test the hypothesis that E-Syt contacts are involved i neurotransmission by determining the effect of depolarization on E-Syt recruitment to the membrane. Furthermore I will determine whether E-Syt contacts play a role in neurite development by assessing the effect of E-Syt knockdown on branching and dendritic spine development.