Force on NOMO is modulated by ER sheet spacing and cell migration. (A) Depiction of the FRET-based force sensor. (B) Rendering of the FRET sensor inserted within (TS_in) constructs. Spheres depict Ig domains of NOMO, while a helix represents the coiled-coil topology of CLIMP-63. (C) Plots of normalized mean FRET efficiency ± SD for a TS only control, CLIMP-63 with TS inserted in the LD prior to the TM domain (CLIMP-63-TS_in), WT NOMO with TS inserted in the LD prior to the TM domain (NOMO-TS_in) or between Ig 11-12 (NOMO-TS_11-TS-12), and 4-mutant NOMO (NOMO^4-Mut-TSin). (D) Normalized mean FRET efficiency for NOMO-TS_in as in C or with CLIMP-63 silenced (siCLIMP-63) or overexpressed (F-CLIMP-63). NOMO-TS_in data are the same in C and D. (E) Schematic representation of the MEF3T3 wound migration assay. Cells at the wound edge extend protrusions and migrate into the gap, while interior confluent cells remain distal to the wound site. Arrows indicate the direction of migration. (F) NOMO-TS_in in MEF3T3 cells either migrating into a wound (Edge) or in a confluent area away from the wound (Internal). For all plots, errors reflect SD; n ≥ 30 cells for each condition measured from n ≥ 3 experiments. Statistical analyses were performed using a two-tailed unpaired Mann–Whitney test (F) or ordinary one-way ANOVA (C and D); ****P < 0.0001, ***P < 0.001, *P < 0.05, and ns, not significant. Source numerical data are available in source data.