Stage-specific differential association of Brr6.NEGFP with mitotic SPBs. (A) Mid-log phase cdc7.HA cells were processed for immunofluorescence microscopy and stained with antibodies to Brr6, 12CA5 antibodies to the HA epitope on Cdc7.HA, and DAPI. Although the Cdc7.HA staining of the new SPB recognizes the pole opposite the one recognized by the Brr6 antibodies in cell 1, the two signals coincide on the same SPB in cells 3 and 4. Note that although the HA staining of cell 2 indicates that this cell is at metaphase, there is no Brr6 staining detected at this SPB. This highlights the departure of Brr6 from SPBs after insertion into the nuclear envelope. (B and C) Live cell imaging of brr6.NEGFP (B) and brr6.NEGFP cdc25.22 (C) cells. Although there is variation in absolute timing of events in individual cells, in each case, the initially strong association of Brr6.NEGFP with the SPB diminishes as cells get to metaphase to rise once more midway through anaphase B (indicated by the thick lines under the panels). The cells in B were arrested at the G2/M boundary for 4.25 h by incubation at 36°C before the temperature was returned to 25°C to induce mitosis. The increased cell volume arising from the prolonged arrest results in an extension in the duration of anaphase B and so extends the duration between the first and second phases of Brr6.NEGFP recruitment. Bars, 5 µm.