STED super resolution microscopy reveals CLIMP63-dependent nanodomain segregation in peripheral ER tubules Guang Gao, Chengjia Zhu, and Ivan R. Nabi* Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada *Corresponding Author: firstname.lastname@example.org
The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that exhibits nanodomain heterogeneity and communicates with other organelles at discrete membrane contact sites. ER-mitochondria contacts are linked to mitochondrial homeostasis, autophagosome formation, and mtDNA distribution. However, spatiotemporal limitations of diffraction limited fluorescent microscopy have hindered efforts to study nanodomain organization of the ER. 2D and 3D STED (stimulated emission depletion) super-resolution microscopy has revealed fenestrated sheet-like structures and periodic ER tubules. Peripheral ER tubules labeled with lumenal (ERmoxGFP) and membrane (Sec61β-GFP) ER reporters are enriched at different nanodomains and present distinct periodicity patterns of blobs and constrictions. ER resident proteins derlin-1 and calnexin were enriched in periodic constrictions of peripheral tubules labeled for ERmoxGFP but not membrane-associated Sec61β-GFP. Moreover, the lumenal ER spacer CLIMP63 shows increased association with lumenal ERmoxGFP relative to membrane-associated calnexin, derlin-1 or Sec61β-GFP and CLIMP63 knockdown disrupts derlin-1 and calnexin enrichment in ERmoxGFP constrictions. This suggests that the ER spacer CLIMP63 is a critical organizer of ER tubule nanodomain structure, defining and segregating lumenal ER protein nanodomains from ER membrane protein complexes. STED analysis has further defined the distribution of mitochondrial/ER markers at the ER-mitochondria interface.
Funding: supported by the CIHR (PJT-148698), NSERC and CFI/BCKDF. GG is the recipient of a UBC Four Year Doctoral Fellowship and CZ the recipient of an NSERC undergraduate student research award.
Credits: None available.
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