SSA - The in situ molecular structure of active calcium ion channels

背景：钙通道的结构机制是多种神经功能的基础，包括感觉和记忆的获得。我们最近发表的研究表明，Ca2+信号不是通过单通道复合物传播的。相反，高阶的大分子超复合体协同作用，协调细胞内的局部结构重塑，包括通过肌动蛋白聚合扩大突触和向突触膜募集平滑内质网——这些活跃细胞过程的结构基础目前尚不清楚。我们开发了低温相关光电子显微镜、低温电子断层扫描和亚断层扫描平均研究突触原位分子结构的方法。因此，我们的目标是了解突触膜上Ca+2依赖性信号转导的结构基础和分子机制。新颖性和及时性：低温电子显微镜（cryoEM）是结构生物学中发展最快的学科，仪器和计算图像分析的进步正在改变我们对分子机制的理解。随着对cryoEM的投资，利兹处于这场革命的最前沿。我们期望通过将小鼠遗传学与冷冻电镜相结合来确定细胞和组织内的原位分子结构将代表这一领域的进一步重大进展。实验方法：我们结合小鼠遗传学、药理学荧光标签、相关光电子显微镜和低温电子断层扫描来定位组织和细胞样本中的离子通道复合物，以确定原位分子结构。细胞和组织内活性依赖的结构变化机制将使用荧光钙指示剂来定位活性膜以确定结构。

Background:Structural mechanisms involving calcium channels underpin various neurological functions including sensation and the acquisition of memory. Our recent published research indicates that Ca2+ signals are not propagated by single channel complexes. Instead, higher order macromolecular supercomplexes act in concert to coordinate local structural remodeling within the cell, includingsynapse enlargement by actin polymerization and the recruitment of smooth ER to the synaptic membrane - the structural basis for these active cellular processes is currently unknown. We have developed methods for investigating the in situ molecular structure of synapses by cryogenic correlated light-electron microscopy, cryo-electron tomography and sub-tomogram averaging. Thus, our goal is to understand the structural basis and molecular mechanism of Ca+2 - dependent signal transduction at the synaptic membrane.Novelty and timeliness:Cryo-electron microscopy (cryoEM) is the fastest growing discipline in structural biology, where advancements in instrumentation and computational image analysis are transforming our understanding of molecular mechanisms. With its investment in cryoEM Leeds is at the forefront of this revolution. We expect that by combining mouse genetics with cryoEM to determine in situmolecular structures within cells and tissues will represent a further significant advancement in this field. Experimental approach:We are combining mouse genetics, pharmacological fluorescent tags, correlated light-electron microscopy, and cryo-electron tomography to locate ion channel complexes within tissue and cell samples to determine in situ molecular structure. The mechanism of activity-dependent structural change within cells and tissues will be investigated using fluorescent calcium indicators to locateactive membranes for structure determination.