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）是真核细胞中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领域：ER小管，ER片，和核 信封（NE）。专职分泌细胞，如胰腺β细胞和浆细胞， 核糖体包被的ER片层，证明ER片层对有效蛋白分泌的重要性。虽然 ER片层作为分泌途径的主要入口点起着关键作用，但目前尚不清楚它们是如何作用的。 生成的.拟议的工作将确定作为专门结构和职能基础的机制， 急诊室床单。 目的1将表征由已知的ER成形蛋白Climp 63和新的ER产生的ER片层 床单调节器抒情。这些蛋白质在单独过表达时产生ER片层，但它们不 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 薄膜.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耗竭的细胞中检查ER形态 Climp63。最后，将检查Climp 63和Lyric编码的细胞的ER片的损失，以测试细胞的细胞周期。 这两种调节因子通过平行的ER片生物发生途径发挥作用的观点。 目的2将解决Lyric和Climp 63如何形成分离的ER片层结构域。Lyric截短突变体 和Climp 63将被产生以定义控制它们产生ER片层的能力的蛋白质结构域， 从表域中排除彼此。这些实验将验证他们的管腔域 作为间隔物-Climp 63已知的属性-并且这种间隔活动控制它们的分离 本地化此外，ER结合的核糖体对Lyric和Climp 63蛋白分布的影响将被研究。 通过监测Climp 63和Lyric ER片层对翻译抑制剂的反应进行测试。 目标3将定义Climp 63与Lyric ER片材的功能相关性。的分布和密度 由Climp 63和Lyric产生的ER片层上的核糖体将通过3D EM断层扫描进行评估，以测试 蛋白质产生粗糙的ER。此外，TurboID将识别Climp 63和Lyric的交互作用者，以确定 ER片形态和功能的潜在下游调节剂。 拟议的培训包括全面的，一对一的三维电磁层析成像方法的指导。在 此外，该计划将提供新的成像和生物化学技术的培训，学习如何写 同行评审的论文，管理实验室，并建立一个独立的研究计划。实验室 和机构环境将提供最先进的技术资源，以支持执行这一目标， 除了为科学和专业发展提供丰富的知识环境外，还可以开展工作。