Chemically induced NOD dimers exhibit directional motility, and a NOD mutant lacking one of the DNA binding domains is motile and tip tracks. (A) Schematic of the rapamycin-based approach used to dimerize NOD485 molecules. (B) TIRF image of a cell expressing NOD485-FKBP-EGFP and NOD485-FRB treated with rapamycin. (C) Select frames from the region in the white box in B showing a motile NOD puncta (marked by arrows) walking toward the cell periphery (right). (D) Kymograph of dimerized NOD485 walking on a MT toward the cell periphery (right) within the white box in B. (E) TIRF assay of taxol-stabilized MTs (blue) plus lysate containing ATP prepared from cells expressing NOD485-FKBP-EGFP (green) and NOD485-FRB in the absence of rapamycin. (F) TIRF of the same cell lysate in E but in the presence of 100 nM rapamycin. A motile NOD puncta is highlighted in the kymograph. (G) Schematic of rapamycin-induced dimerization of NOD485-FKBP-EGFP and NOD485-FRB-mCherry. (H) Representative images of cells expressing NOD485-FKBP-EGFP (green) and NOD485-FRB-mCherry (red) in the absence (top) and presence (bottom) of rapamycin in which the localization pattern was the same as NOD485 and NOD485CC, respectively. (I) Schematic of NODΔHMG in which one of the chromosome-associating domains is deleted. (J) Kymograph of a NODΔHMG cluster (red) walking along MTs (green) in a cell. (K) Example of NODΔHMG cluster in a cell exhibiting two activities on the same MT (with plus and minus-ends indicated in the tubulin kymograph): tip tracking (closed arrows) and plus end–directed motility (open arrows). (L) Histogram of the velocities of NODΔHMG puncta, with a mean of 7.63 ± 2.03 µm/min; n = 37. Horizontal bars: 10 µm (B); 1 µm (C–F, J, and K); 5 µm (H). Vertical bars: 10 s (B–F, H, and J); 1 min (K). Mean ± SD.