Nanodomain Structure of the Endoplasmic Reticulum by Super Resolution Microscopy

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相关蛋白降解以及细胞对应激的反应中起关键作用。ER包含多个功能不同的域，但是衍射限制荧光显微镜的时空分辨率阻碍了理解纳米级ER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）和模拟发射耗尽（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ER内腔和膜报告分子以及内源性ER蛋白的纳米结构域组织。我们发现ER形成蛋白reticulon和CLIMP-63控制外周ER小管中的纳米结构域组织和ER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纳米结构域组织。目标1：通过固定细胞3D STED分析，我们将多种ER蛋白定位于周期性内腔ER最小值，但彼此共定位最小。使用4通道3D STED，我们将确定这些和多个ER组分（ER成形蛋白，ER伴侣，易位子组分，核糖体蛋白，ER相关降解蛋白）之间的关系，并确定ER成形蛋白如何控制它们的相互作用。目标二：外周ER小管中的蛋白质动力学使用标记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蛋白复合物之间的动态关系和腔内ERmoxGFP，特别是外周ER小管中CLIMP-63和reticulon之间的动态关系。目标3。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，并使用SMLM网络分析来确定这些结构域的分子结构以及CLIMP-63和reticulon如何影响ER小管中的内腔间距。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，以及对研究生（1-2）和本科生（5）以及2个PDF的不同群体的培训。HQP培训将通过每周跨学科超分辨率虚拟实验室会议与SFU的Hamarneh计算机科学小组以及LSI成像设施的其他用户分享技术和研究进展来进一步促进，我是该设施的主任。

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.