Fig. 7. The kinetochores can wander some distance from the spindle as it forms. IH2035 cells were grown to mid-log phase in rich medium at 30°C and processed for anti-Sad1 immunofluorescence. In each case the top panel is a merge of the middle Nuf2.GFP signal and the lower Sad1 staining. It is shown at twice the magnification of the images of the individual channels to show the juxtaposition of the signals most clearly. During interphase a single spot of Nuf2.GFP always associated with the Sad1 SPB stain. Upon spindle formation the association of Nuf2.GFP with Sad1 staining became much more varied. In some cases (B) association was maintained, in others (C) it was not. The two lower kinetochores in B have wandered a long way from the spindle that is forming in the upper right portion of the nucleus. At later stages of spindle formation the kinetochore marker was always seen on a line that marked the shortest route between the two Sad1-reactive SPB spots (D-E) until the kinetochores reached the poles at the end of anaphase B (F).