Nanodomain Structure of the Endoplasmic Reticulum by Super Resolution Microscopy

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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.