Fig. 6. Weak attachment can inhibit kinetochore phosphorylation and Mad2 binding. (A,B) Phase contrast images of the cell in life. One kinetochore (upper arrow in A, ‘u’ thereafter) of a metaphase chromosome was detached from the spindle and kept detached for 10 minutes, while the other kinetochore (lower arrow in A and ‘w’ thereafter) remained weakly attached in the absence of tension. (C,D) The cell after fixation and immunostaining for 3F3/2 (C, red) and Mad2 (D, green). In both C and D the unattached kinetochore (u) is bright – it is highly phosphorylated and loaded with Mad2. The weakly attached kinetochore (w) is dim, as are the kinetochores of unmanipulated chromosomes (yellow arrowheads), which had been strongly attached. Bar, 10 µm.