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、ER 相关蛋白质降解以及细胞对应激的反应中发挥着关键作用。内质网包含多个功能不同的结构域，然而衍射极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。在我之前获得 NSERC 发现资助的过程中，我开发了单分子定位显微镜 (SMLM) 和 STimulated Emission Depletion (STED) 超分辨率显微镜方法来克服这些限制。三标记 3D STED 成像表征了 ER 腔和膜报告基因以及内源性 ER 蛋白的纳米结构域组织。我们表明，内质网塑造蛋白网状蛋白和 CLIMP-63 控制外周内质网小管中的纳米结构域组织和内质网蛋白分布。我们现在将使用抗体标记和 GFP 标记的 ER 蛋白的 CRISPR/Cas 敲入来分析其他内源性 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 纳米域组织。目标 1：通过 3D STED 对 ER 中的蛋白质分布进行全局分析 通过固定细胞 3D STED 分析，我们将多个 ER 蛋白质定位到周期性的腔内 ER 最小值，但彼此之间的共定位最小。使用 4 通道 3D STED，我们将确定这些与多个 ER 成分（ER 塑造蛋白、ER 伴侣、易位子成分、核糖体蛋白、ER 相关降解蛋白）之间的关系，并确定 ER 塑造蛋白如何控制它们的相互作用。目标 2：外周 ER 小管中的蛋白质动力学 利用标记 ER 蛋白的 CRISPR/Cas 蛋白敲入，我们将使用光漂白后荧光恢复 (FRAP) 和活细胞 2D-STED 来研究外周 ER 小管中的蛋白质动力学，并确定不同 ER 蛋白复合物和腔内 ERmoxGFP 之间的动态关系，特别是 CLIMP-63 和外周网状蛋白之间的动态关系 ER 小管。目标 3. 通过 SMLM 定义 ER 纳米域结构 我们将使用电子显微镜和 SMLM 来确定腔内 ER 纳米域的大小，并使用 SMLM 网络分析来确定这些域的分子结构以及 CLIMP-63 和 reticulon 如何影响 ER 小管中的腔间距。我的研究项目将对 ER 纳米结构、超分辨率显微镜技术进步以及对不同群体的研究生 (1-2) 和本科生 (5) 学生的培训以及 2 个 PDF 产生新的见解。 HQP 培训将通过每周与 SFU 的 Hamarneh 计算机科学小组以及我担任主任的 LSI 成像设施的其他用户举行跨学科超分辨率虚拟实验室会议，分享技术和研究进展，从而进一步促进 HQP 培训。