Using STED super-resolution microscopy, researchers at the University of British Columbia have shed new light on the compartmentalization in peripheral ER tubules. Using the lumenal ER reporter ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP), the team led by Dr. Ivan Nabi performed high-speed (40 ms/frame) live-cell STED imaging and characterized the behavior of the ER-shaping proteins. The results show that RTN and CLIMP-63 regulate the localization and dynamics of nanodomains along the tubules in very distinct ways. RTN segregates away from and restricts lumenal blob length, while CLIMP-63 associates with and increases lumenal blob length. ERmoxGFP defines local lumenal filling of nanodomains that are segregated from membrane-associated ER proteins along peripheral ER tubules. RTN and CLIMP-63 constitute key players for establishing ER nanodomain heterogeneity, interaction with ER proteins, and dynamics in ER tubules.