Nanodomain Structure of the Endoplasmic Reticulum by Super Resolution Microscopy

通过超分辨率显微镜观察内质网的纳米域结构

• 批准号: RGPIN-2019-05179
• 负责人: Nabi, Ivan
• 金额: $ 4.23万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738107
• 关键词: Nanodomain; Structure; Endoplasmic; Reticulum; Super

The endoplasmic reticulum (ER) is a continuous membrane organelle that plays critical roles in protein and lipid synthesis, calcium storage and signaling, ER-associated protein degradation, and the cellular response to stress. The ER contains multiple functionally distinct domains, however spatiotemporal resolution of diffraction-limited fluorescent microscopy has hindered efforts to understand nanoscale ER organization and protein distribution. Over the course of my previous NSERC Discovery grant, I developed Single-Molecule Localization Microscopy (SMLM) and STimulated Emission Depletion (STED) super-resolution microscopy approaches to overcome these limitations. Triple labeling 3D STED imaging characterized the nanodomain organization of ER lumenal and membrane reporters and of endogenous ER proteins. We showed that the ER-shaping proteins reticulon and CLIMP-63 control nanodomain organization and ER protein distribution in peripheral ER tubules. We will now analyze other endogenous ER proteins using both antibody labeling and CRISPR/Cas knock-in of GFP-tagged ER proteins. We will use STED and SMLM to define the distribution, dynamics and molecular organization of various classes of ER proteins and thus define ER nanodomain organization. Aim 1: Global analysis of protein distribution in the ER by 3D STED By fixed cell 3D STED analysis, we localized multiple ER proteins to periodic lumenal ER minima but with minimal colocalization with each other. Using 4-channel 3D STED we will determine the relationship between these and multiple ER components (ER-shaping proteins, ER chaperones, translocon components, ribosomal proteins, ER-associated degradation proteins) and determine how ER-shaping proteins control their interaction. Aim 2: Protein dynamics in peripheral ER tubules Using CRISPR/Cas protein knock-in of tagged ER proteins, we will study protein dynamics in peripheral ER tubules using fluorescence recovery after photobleaching (FRAP) and live cell 2D-STED and determine the dynamic relationship between different ER protein complexes and lumenal ERmoxGFP and specifically the dynamic relationship between CLIMP-63 and reticulon in peripheral ER tubules. Aim 3. Defining ER nanodomain structure by SMLM We will use electron microscopy and SMLM to determine the size of lumenal ER nanodomains and SMLM network analysis to determine the molecular architecture of these domains and how CLIMP-63 and reticulon impact lumenal spacing in ER tubules. My research program will lead to novel insights into ER nanostructure, technological advances in super-resolution microscopy and training of a diverse group of graduate (1-2) and undergraduate (5) students as well as 2 PDFs. HQP training will be further promoted through sharing of technological and research advances via weekly interdisciplinary super-resolution virtual lab meetings with the Hamarneh computer science group at SFU and with other users of the LSI Imaging Facility, of which I am Director.

内质网（ER）是一种连续的膜细胞器，在蛋白质和脂质合成，钙储存和信号传​​导，与ER相关的蛋白质降解以及对胁迫的细胞反应中起着关键作用。 ER包含多个功能上不同的域，但是衍射限制荧光显微镜的时空分辨率阻碍了了解纳米级ER组织和蛋白质分布的努力。在我以前的NSERC发现赠款的过程中，我开发了单分子定位显微镜（SMLM）和刺激发射消耗（STED）超分辨率显微镜方法来克服这些限制。三型标记3D STED成像表征了ER腔内和膜记者的纳米域组织以及内源性ER蛋白的组织。我们表明，ER成形蛋白网状蛋白和攀爬-63控制纳米域组织和ER蛋白在外围ER小管中的分布。现在，我们将使用GFP标记的ER蛋白的抗体标记和CRISPR/CAS敲击来分析其他内源性ER蛋白。我们将使用STED和SMLM来定义各种ER蛋白的分布，动力学和分子组织，从而定义ER纳米域组织。 AIM 1：通过固定细胞3D Sted分析稳定的ER中蛋白质分布的全局分析，我们将多个ER蛋白定位在周期性的Lumenal ER minima中，但彼此之间的共定位最少。使用4通道3D Sted，我们将确定这些与多个ER成分之间的关​​系（ER成形蛋白，ER伴侣伴侣，转运成分，核糖体蛋白，与ER相关的降解蛋白），并确定ER成型蛋白如何控制其相互作用。目的2：使用CRISPR/CAS蛋白在周围ER蛋白质中敲入周围ER蛋白质的蛋白质动力学，我们将使用光漂白后的荧光恢复（FRAP）（frap）（frap）和Live 2d sted中的荧光恢复在周围ER小管中研究蛋白质动力学，并确定不同的ER蛋白质复合物与繁殖的动态关系之间的动态关系，并确定彼此之间的动态关系 - ER小管。 AIM 3。通过SMLM定义ER纳米域结构，我们将使用电子显微镜和SMLM来确定Lumenal ER纳米域和SMLM网络分析的尺寸，以确定这些域的分子结构以及如何攀爬63和Ericulon撞击Er tubules中的Lumenal间距。我的研究计划将导致对ER纳米结构的新见解，超分辨率显微镜的技术进步以及多样化的研究生（1-2）和本科生（5）学生以及2个PDF的培训。 HQP培训将通过与SFU的Hamarneh计算机科学集团以及与我是LSI Imaging设施的其他用户一起，通过每周跨学科的超级分辨率虚拟实验室会议来进一步促进HQP培训。