Nanodomain Structure of the Endoplasmic Reticulum by Super Resolution Microscopy

内质网（ER）是一个连续的膜细胞器，在蛋白质和脂质合成、钙储存和信号传导、内质网相关蛋白质降解以及细胞对应激的反应中起着关键作用。内质网包含多个功能不同的结构域，但衍射受限荧光显微镜的时空分辨率阻碍了对纳米内质网组织和蛋白质分布的理解。在我之前的NSERC发现资助的过程中，我开发了单分子定位显微镜（SMLM）和受激发射损耗（STED）超分辨率显微镜方法来克服这些限制。三重标记3D STED成像表征了内源性内质网蛋白的内源性内质网管和膜报告蛋白的纳米结构。我们发现内质网成形蛋白reticulon和clip -63控制外周内质网小管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CRISPR/Cas敲入gfp标记的内源性内质网蛋白来分析其他内源性内质网蛋白。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纳米结构域的组织。通过固定细胞3D STED分析，我们将多个内质网蛋白定位到周期性的管腔内质网最小值，但彼此之间的共定位最小。使用4通道3D STED，我们将确定这些和多种内质网成分（内质网成形蛋白、内质网伴侣、转座子成分、核糖体蛋白、内质网相关降解蛋白）之间的关系，并确定内质网成形蛋白如何控制它们的相互作用。我们将利用CRISPR/Cas蛋白敲入标记的内质网蛋白，利用光漂后荧光恢复（FRAP）和活细胞2D-STED研究外周内质网蛋白动力学，确定不同内质网蛋白复合物与管腔ERmoxGFP之间的动态关系，特别是clip -63与外周内质网蛋白之间的动态关系。目标3。我们将使用电子显微镜和SMLM来确定内质网纳米结构域的大小，并使用SMLM网络分析来确定这些结构域的分子结构以及clip -63和reticulon如何影响内质网小管的腔间距。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，以及研究生（1-2）和本科生(5)学生的不同群体的培训以及2个pdf文件。HQP培训将通过每周与SFU的Hamarneh计算机科学小组以及LSI成像设备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，进一步促进HQP培训。

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.