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）是真核细胞的普遍配置的成分。急诊室是 在形态学和功能上不同的域分为三个：ER管，ER片和核 信封（NE）。专业秘书细胞，例如胰腺β细胞和浆细胞，挤满了 核糖体涂层的ER片，证明了ER片对有效蛋白质分泌的重要性。虽然 ER表作为秘书途径的主要入口处起着至关重要的作用，尚不知道它们是如何的 生成。拟议的工作将确定专门结构和功能的基础机制 er床单。 AIM 1将表征已知的ER成型蛋白攀岩和小说ER产生的ER表 纸调节器歌词。这些蛋白质在单独表达时会产生ER表，但它们不会 共定位时共定位。该观察结果与细胞包含不同类型的ER的想法一致 床单。这些独特的ER片的结构将通过3D电子显微镜（EM）断层扫描进行比较 在纳米分辨率下定义其形状。还将在耗竭的细胞中检查ER形态 和攀登63。最后，将检查用于Climp63和抒情诗的细胞，以检查ER表的损失以测试 认为这两个调节剂通过平行的ER生物发生途径起作用。 AIM 2将解决歌词和攀登63形成隔离的ER板域。歌词的截短突变体 将生成Climp63来定义控制其生成ER片和的能力的蛋白质域和 将彼此排除在板域中。这些实验将检验其腔内域的假设 充当垫片（以攀登为63而闻名的属性），并且该间隔活动控制了它们 本土化。另外，核糖体核糖体对抒情和攀岩蛋白分布的影响将是 通过监视Climp63和抒情板对翻译抑制剂的反应进行测试。 AIM 3将定义Climp63与歌词ER表的功能相关性。分布和密度 Climp63和歌词产生的ER片上的核糖体将通过3D EM断层扫描评估，以测试是否是否 蛋白质产生粗糙的ER。此外，Climp63和Lyric的相互作用者将通过Turboid确定以识别 ER表形态和功能的潜在下游调节剂。 拟议的培训包括3D EM断层扫描方法中的全面，一对一的指导。在 此外，该计划还将提供新成像和生化技术的培训，学习如何写作 经过同行评审的论文，管理实验室并建立独立的研究计划。实验室 机构环境将提供最先进的技术资源来支持执行此项 除了为科学和专业发展提供丰富的智力环境外，工作。