Fig. 4. Detaching kinetochores from microtubules leads to Mad2 binding, and tension from a microneedle leads to Mad2 loss. (A,B) Phase contrast images of the cell in life. The chromosomes labeled with blue and yellow asterisks were detached from the spindle with a micromanipulation needle, moved to the cytoplasm, and kept detached for 10 minutes. The chromosome labeled with the yellow asterisk was then moved so that both kinetochores pointed to the lower pole. After 3 minutes, its kinetochores had attached and the chromosome was pulled away from the pole (red arrow), imposing tension; tension was greater on the left kinetochore than the other one (note that the left arm of the chromosome is more greatly stretched and is thinner). The chromosome was kept under tension for 7 minutes and then the cell was fixed and immunostained. (C) Mad2 immunostaining (green) superimposed on a phase contrast image. The kinetochores of the detached chromosome are brightly labeled (blue arrows), but the kinetochores of the chromosome under tension from the microneedle (yellow arrows) are unlabeled (left) or very lightly labeled (right). The kinetochores of unmanipulated chromosomes, which had been under tension from mitotic forces, are unlabeled (yellow arrowheads). The approximate positions of the spindle poles are labeled ‘p’. Bar, 10 µm.