Regulation of ER sheet biogenesis and function

内质网是真核细胞中普遍保守的成分。急诊室是 被组织成三个形态和功能截然不同的区域：内质网小管、内质网和核 信封(NE)。专业的分泌细胞，如胰腺β细胞和浆细胞，充满了 核糖体包裹的内质网薄片，表明内质网薄片对高效蛋白质分泌的重要性。虽然 内质网作为分泌途径的主要入口点起着至关重要的作用，目前还不知道它们是如何进入的 已生成。拟议的工作将确定以下机构的专门结构和职能的基础机制 急诊单。 目标1将描述已知的内质网形成蛋白CLYP63和新的内质网产生的内质网工作表 板材调整器抒情。这些蛋白质单独过度表达时会产生内质网，但它们不会 当共表达时共定位。这一观察结果与细胞含有不同类型内质网的观点是一致的 床单。这些不同的ER片的结构将通过3D电子显微镜(EM)断层扫描进行比较 以纳米分辨率定义它们的形状。内质网形态也将在耗尽抒情的细胞中进行检查 和《气候63》。最后，将检查共耗尽了CLYP63和LYRIC的细胞的内质网片层的丢失，以测试 这两个调控因子通过平行的内质网片状生物发生途径发挥作用。 目标2将解决Lyric和Climate p63如何形成隔离的ER片域。歌词的截断突变体 并将生成CLYP63，以定义控制其生成ER片断和 从图纸域中彼此排除。这些实验将检验这样的假设，即它们的光域 起到间隔物的作用--这一特性已经在《气候变化》63中为人所知--而且这种间隔性活动控制着它们之间的隔离 本地化。此外，内质网结合的核糖体对Lyric和Climate p63蛋白分布的影响将是 通过监测CLYP63和LYRIC ER薄片对翻译抑制剂的反应进行测试。 目标3将定义气候63与抒情ER表的功能相关性。云杉的分布和密度 将通过3D EM断层扫描来评估由CLINP63和Lyric产生的ER片上的核糖体，以测试是否 蛋白质产生粗略的内质网。此外，将通过TurboID来识别CLIMP63和LYRIC的交互因子以识别 内质网形态和功能的潜在下游调控因子。 拟议的培训包括全面的、一对一的3D EM断层成像方法指导。在……里面 此外，该项目将提供新的成像和生化技术方面的培训，学习如何写作 同行评审论文，管理实验室，建立独立的研究项目。实验室 机构环境将提供最先进的技术资源，以支持执行这一 工作除了为科学和专业发展提供丰富的智力环境外。

The endoplasmic reticulum (ER) is a universally conserved component of eukaryotic cells. The ER is organized into three morphologically and functionally distinct domains: ER tubules, ER sheets, and the nuclear envelope (NE). Professional secretory cells, such as pancreatic β cells and plasma cells, are packed with ribosome-coated ER sheets, demonstrating the importance of ER sheets to efficient protein secretion. Although ER sheets play a critical role as the major entry point of the secretory pathway, it is not known how they are generated. The proposed work will identify the mechanisms underlying the specialized structure and function of ER sheets. Aim 1 will characterize ER sheets generated by the known ER-shaping protein Climp63 and the novel ER sheet regulator Lyric. These proteins generate ER sheets when individually overexpressed, but they do not colocalize when coexpressed. This observation is consistent with the idea that cells contain different types of ER sheets. The structures of these distinct ER sheets will be compared by 3D electron microscopy (EM) tomography to define their shapes at nanometer resolution. ER morphology will also be examined in cells depleted for Lyric and Climp63. Finally, cells codepleted for Climp63 and Lyric will be examined for loss of ER sheets to test the notion that these two regulators act through parallel ER sheet biogenesis pathways. Aim 2 will address how Lyric and Climp63 form segregated ER sheet domains. Truncation mutants of Lyric and Climp63 will be generated to define the protein domains that control their ability to generate ER sheets and exclude one another from sheet domains. These experiments will test the hypothesis that their luminal domains act as spacers—a property already known for Climp63—and that this spacing activity controls their segregated localization. In addition, the influence of ER-bound ribosomes on Lyric and Climp63 protein distribution will be tested by monitoring the responses of Climp63 and Lyric ER sheets to translational inhibitors. Aim 3 will define the functional relevance of Climp63 versus Lyric ER sheets. The distribution and density of ribosomes on ER sheets generated by Climp63 and Lyric will be assessed by 3D EM tomography to test if the proteins generate rough ER. In addition, interactors of Climp63 and Lyric will be identified by TurboID to identify potential downstream regulators of ER sheet morphology and function. The proposed training includes comprehensive, one-on-one instruction in 3D EM tomography methods. In addition, the program will provide training in new imaging and biochemical techniques, learning how to write peer-reviewed papers, manage a laboratory, and establish an independent research program. The laboratory and institutional environments will provide state-of-the-art technical resources to support the execution of this work in addition to providing an enriching intellectual environment for scientific and professional development.