The endoplasmic reticulum adopts two distinct forms of tubules

Abstract Being the largest and most expansive organelle in the cell, the endoplasmic reticulum (ER) carries diverse key functions from protein and lipid synthesis, protein folding and modification, transport, calcium storage, to organelle interactions1-6. The shaping mechanism of this complex, membrane-bounded organelle is thus of fundamental significance7-21. Using super-resolution microscopy, we uncover the coexistence of two distinct, well-defined forms of ER tubules in the mammalian cell. Whereas an ultrathin form, R1, is consistently covered by the membrane curvature-promoting protein Rtn4, in the second form, R2, Rtn4 curiously appears as two parallel lines at a conserved separation of ~105 nm over long ranges. The two tubule forms together account for ~90% of the total tubule lengths, with either one being dominant in different cell types. The R1-R2 dichotomy and the final tubule geometry are both co-regulated by Rtn4 and the ER sheet-maintaining protein Climp63, which respectively define the edge curvature and lumen height of the R2 tubules to generate a ribbon-like structure of well-defined width. The R1 and R2 tubules undergo active remodeling in the cell as they differently accommodate proteins, with the former effectively excluding ER-luminal proteins and ER-membrane proteins with large intraluminal domains. We thus unveil a dynamic ER-tubule structural dichotomy with intriguing functional implications.