NuMA binds to the minus ends of laser-ablated microtubules. (A) Schematic of a TIRF microscopy assay with microtubules elongating from surface-immobilized GMPCPP-seeds being severed by laser ablation in the presence of mScarlet-NuMA^FL. (B and C) Representative TIRF microscopy time course images of a microtubule elongating from a surface-immobilized Atto647N-labeled GMPCPP-seed (magenta) in the presence of 40 nM mScarlet-NuMA^FL (green) and 10 µM Atto647N-tubulin (magenta). Upon severing of the microtubule by laser ablation (asterisks), NuMA binds selectively to the newly generated minus end (arrowheads). Timestamps refer to mm:ss. (B) Ablation performed at one (top panel) or two (bottom panel) sites close to the microtubule minus end generates short segments that diffuse away in solution, thus only the minus end of the longest fragment which stays anchored to the surface can be observed. (C) Ablation performed farther from the microtubule minus end produces longer fragments that remain attached to the surface, allowing visualization of both new plus and minus ends. Related to Video 3. (D) Frequency of NuMA localization to freshly generated plus and minus ends after laser ablation in the presence of 40 nM mScarlet-NuMA^FL and 10 µM Atto647N-tubulin (mean ± SEM, five biological replicates). Each color represents a replicate; n = 54 ablation sites. All experiments were performed in NuMA microscopy buffer.