NuMA’s coiled-coil regulates its C terminus’ chromatin binding over the cell cycle. (A) LOVTRAP light-induced dissociation system: in the dark, LOV2 (phototropin) binds to Zdk1, while blue light induces a conformational change in LOV2 that prevents binding to Zdk1. In the Opto-NuMA engineered protein NuMA-N(1–705)-PhusionRed-LOV2 + EGFP-Zdk1-NuMA-C, both NuMA ends are linked through LOVTRAP, and blue light dissociates them from each other. (B) Representative time-lapse of an uninduced NuMA KO RPE1 cell stably expressing Opto-NuMA in the nucleus before, during (0:00-0:25), and after illumination with blue (488 nm) light, showing NuMA-N but not NuMA-C becoming diffuse upon dissociation. EGFP-Zdk1-NuMA-C cannot be imagined without dissociating Opto-NuMA. Red circle corresponds to the area chosen for intensity analysis in C. Time in minutes:seconds. Scale bar, 5 µm. See also Video 4. (C) NuMA-N(1–705)-PhusionRed-LOV2 intensity in the nucleus before, during (blue box), and after illumination with blue light. Red trace = mean; black = SEM. n = 9 cells. Two-sided Mann–Whitney test: ****, P < 0.0001. (D) Top: The schematics represent the NuMA constructs used in each gel, with amino acid numbers indicated. Bottom: EMSA using 60 ng H1-H3 spacer DNA fragment and increasing concentrations of αβtubulin, NuMA-N-SNAP, NuMA-C-SNAP, NuMA-Bonsai-SNAP, and NuMA-NC-SNAP. DNA was labeled with Sybr-Gold. Arrows indicate the minimum protein concentration for each protein with a noticeable DNA band shift. (E) Representative time-lapse images of RPE1 cells transiently expressing NuMA-FL-EGFP and stably expressing NuMA-Bonsai-EGFP, EGFP-Zdk1-NuMA-C, or NuMA-NC-EGFP and labeled with SiR-Hoechst (DNA), followed from metaphase through cytokinesis. NuMA-C and NuMA-NC localize to chromosomes at mitosis, while NuMA-Bonsai does not. Time in minutes:seconds; 0:00 corresponds to anaphase onset (AO, vertical dashed line). Arrows indicate when we detect different NuMA truncations on chromosomes. Scale bar, 5 µm. See also Video 5.