Effects of vimentin repositioning on ER morphology. (A and B) Representative image of a COS-7 cell stained for endogenous calnexin (ER) and vimentin, and (B) intensity profile along the indicated line. Images were collected using STED microscopy. (C–E) U2OS cells co-transfected with Vim-mCh-SspB, iLID-GFP-GCN4-ppKin14, and Halo-KDEL were first imaged for 5 min without 488-nm pulsing (−5 min, 0 min), then imaged for 25 min with whole-cell 488-nm pulsing to induce optogenetic vimentin clustering (5, 10, 25 min). (C) Spinning disc confocal images from the experiment, with black dashed boxes indicating the region of the cell enlarged in the zoom panels. (D) Plot showing ratio of perinuclear fluorescence intensity to intensity at the cell periphery. (E) Plot showing loss of total fluorescence signal over time. (F) Images of COS-7 cells expressing Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14. Transfected cells are outlined with dashed lines. After 1 h of treatment with or without rapalog, cells were fixed and stained for vimentin and calnexin. The images show the distribution of the ER network, visualized by calnexin staining, in both transfected (T) and untransfected (U) cells, in the presence and absence of rapalog. Dashed boxes show the regions that have been enlarged and shown as masks in the zoom images.