Regulation of ER sheet biogenesis and function

ER 表生物合成和功能的调节

• 批准号: 10321889
• 负责人: Eric M Sawyer
• 金额: $ 2.98万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2022-07-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321889
• 关键词: 3-Dimensional; Address; Affect; Behavior; Binding; Biochemical; Biogenesis; Caliber; Cells; Cellular Structures; Cellular biology; DNA; Data; Development; Dimensions; Disease; Electron Microscopy; Endoplasmic Reticulum; Environment; Eukaryotic Cell; Exclusion; Exhibits; Fluorescence Microscopy; Freezing; Functional disorder; Generations; Goals; Human; Image; Individual; Instruction; Label; Laboratories; Learning; Mass Spectrum Analysis; Measures; Mechanics; Mediating; Membrane; Membrane Proteins; Methods; Modeling; Monitor; Morphology; Nuclear Envelope; Organelles; Paper; Pathway interactions; Peer Review; Peripheral; Plasma Cells; Play; Process; Property; Protein Secretion; Proteins; Regulation; Research; Resolution; Resources; Ribosomes; Role; Rough endoplasmic reticulum; Secretory Cell; Shapes; Small Interfering RNA; Specific qualifier value; Structure; Structure of beta Cell of islet; Techniques; Tertiary Protein Structure; Testing; Training; Translations; Work; Writing; density; design; experimental study; inhibitor; insight; knock-down; mutant; nanometer resolution; novel; overexpression; pressure; programs; protein distribution; response; segregation; tomography

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.

内质网（ER）是真核细胞普遍保守的组成部分。急诊室是 组织成三个形态和功能不同的域：内质网小管、内质网片层和核 信封（NE）。专业分泌细胞，如胰腺β细胞和浆细胞，富含 核糖体包被的内质网片层，证明了内质网片层对于高效蛋白质分泌的重要性。虽然 ER 表作为分泌途径的主要入口点发挥着关键作用，但目前尚不清楚它们是如何发挥作用的 生成的。拟议的工作将确定专业结构和功能背后的机制 急诊室表。 目标 1 将表征由已知的 ER 塑造蛋白 Climp63 和新型 ER 生成的 ER 表 抒情调节表。这些蛋白质在单独过表达时会产生 ER 表层，但它们不会 共表达时共定位。这一观察结果与细胞含有不同类型 ER 的观点一致 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。还将在 Lyric 耗尽的细胞中检查 ER 形态 和克林普63。最后，将检查 Climp63 和 Lyric 的代码缺失细胞是否有 ER 表丢失，以测试 这两个调节因子通过平行的 ER 表生物发生途径发挥作用。 目标 2 将解决 Lyric 和 Climp63 如何形成隔离的 ER 表域。 Lyric 的截断突变体 将生成 Climp63 来定义控制其生成 ER 表的能力的蛋白质结构域 将彼此排除在工作表域之外。这些实验将检验他们的管腔域的假设 作为间隔物（Climp63 已知的属性），并且这种间隔活动控制它们的隔离 本土化。此外，ER结合核糖体对Lyric和Climp63蛋白分布的影响将是 通过监测 Climp63 和 Lyric ER 表对翻译抑制剂的反应进行测试。 目标 3 将定义 Climp63 与 Lyric ER 表的功能相关性。分布及密度 Climp63 和 Lyric 生成的 ER 表上的核糖体将通过 3D EM 断层扫描进行评估，以测试是否 蛋白质产生粗糙的内质网。此外，Climp63和Lyric的交互者将被TurboID识别，以识别 ER片形态和功能的潜在下游调节剂。 拟议的培训包括 3D EM 断层扫描方法的全面、一对一的指导。在 此外，该计划将提供新成像和生化技术的培训，学习如何写作 同行评审论文、管理实验室并建立独立的研究计划。实验室 和机构环境将提供最先进的技术资源来支持这一计划的执行 除了为科学和专业发展提供丰富的智力环境之外，我们还致力于工作。