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.

内质网是一种连续的膜细胞器，在蛋白质和脂肪合成、钙储存和信号传递、内质网相关蛋白降解以及细胞对应激的反应中发挥关键作用。内质网包含多个功能不同的结构域，然而衍射受限荧光显微镜的时空分辨率阻碍了理解纳米级内质网组织和蛋白质分布的努力。在我之前的NSERC发现资助过程中，我开发了单分子定位显微镜(SMLM)和受激发射耗尽(STED)超分辨率显微镜方法来克服这些限制。三重标记3D STED成像表征了内质网管腔和膜报告蛋白以及内源性内源ER蛋白的纳米结构。我们发现内质网形成蛋白和CLIMP-63控制着纳米结构域的组织和外周内质网小管中内质网蛋白的分布。我们现在将使用抗体标记和CRISPR/Cas敲入GFP标记的ER蛋白来分析其他内源性ER蛋白。我们将使用STED和SMLM来定义各类ER蛋白的分布、动力学和分子组织，从而定义ER纳米结构域组织。目的1：3DSTED对内质网蛋白质分布的全局分析通过固定细胞3DSTED分析，我们将多个ER蛋白定位到周期性的内质网最小值，但彼此之间最小的共定位。利用4通道3D STED，我们将确定这些与多种内质网成分(内质网整形蛋白、内质网伴侣蛋白、转位蛋白、核糖体蛋白、内质网相关降解蛋白)之间的关系，并确定内质网整形蛋白如何控制它们之间的相互作用。目的：利用CRISPR/Cas蛋白敲入标记的ER蛋白，研究外周ER小管的蛋白质动力学，利用荧光漂白后恢复法(FRAP)和活细胞2D-STED研究外周ER小管的蛋白质动力学，确定不同ER蛋白复合体与外周ER小管ERmoxGFP的动态关系，特别是与外周ER小管网状结构的动态关系。目的3.用SMLM确定ER纳米结构我们将使用电子显微镜和SMLM来确定内腔ER纳米结构域的大小，并通过SMLM网络分析来确定这些结构域的分子结构，以及CLIMP-63和网状蛋白如何影响ER小管中的管腔间距。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，以及对不同群体的研究生(1-2)和本科生(5)以及2个PDF的培训。HQP培训将通过每周与SFU的Hamarneh计算机科学小组以及与我担任主任的LSI成像设施的其他用户举行的跨学科超分辨率虚拟实验室会议来分享技术和研究进展，从而进一步促进HQP培训。