Figure 3. Inappropriate mitotic exit in bim1D and stt4-7 mutants. (A) Exit from mitosis was delayed in the ts-arp1 bim1D and ts-arp1 stt4-7 mutants. Shown are the mean time intervals from the start of anaphase to spindle movement into the neck (white columns), from spindle movement into the neck to mitotic exit (gray col- umns), and the total time for anaphase (black columns). Notice the near lack of variability in the time interval from spindle movement into the neck to cytokinesis. Number of events for each column from left to right on the chart: wild type, 108, 83, 83; ts-arp1, 120, 116, 114; bim1D, 129, 170, 150; stt4-7, 49, 49, 55; ts-arp1 bim1D, 147, 135, 127; ts-arp1 stt4-7, 27, 33, 29; ts-arp1 bub2D, 68, 66, 92. Error bars represent SEM. (B) In the ts-arp1 bim1D mutant, a large fraction of cells that delayed spindle movement into the neck ex- ited mitosis inappropriately. However, inappropriate mitotic exit did not occur in all ts-arp1 bim1D cells that delayed spindle move- ment into the neck. Comparison of the percentage of cells that underwent inappropriate mitotic exit (white columns) with the percentage of cells that delayed spindle movement into the neck by .20 min (gray columns) is shown. The frequency of inappro- priate mitotic exit in cells that delayed spindle movement is also should show incomplete penetrance in their checkpoint phe- notype and undergo inappropriate mitotic exit without mov- ing the spindle into the neck only under certain conditions. We have previously reported that ts-arp1 mutants carrying additional mutations in BIM1 or STT4 sometimes undergo a checkpoint failure in which mitotic exit occurs while the spin- dle is confined to the mother cell, yielding binucleate and anucleate progeny (Muhua et al., 1998). More recently, it was shown that BUB2 and BFA1 (BYR4) are also required to prevent such inappropriate mitotic exit, whereas MAD2 is not (Bloecher et al., 2000; Pereira et al., 2000). We examined spindle position and mitotic exit in these various mutants to determine the penetrance of their checkpoint phenotypes. In wild-type cells, mitotic exit occurred within 20 min of anaphase start (spindle elongation) in nearly all cells (Fig. 2 A). Consistent with previous reports, most ts-arp1 and ts- arp1 mad2D cells that delayed spindle movement into the neck also delayed mitotic exit (Video 2; Fig. 2, A–C). Only a small fraction of these cells exited mitosis without moving the spindle into the neck (Fig. 2 B). In contrast, ts-arp1 bub2D and ts-arp1 bfa1D cells that delayed spindle move- ment into the neck did not delay exit from mitosis, which al- most always occurred within 20 min of anaphase onset (Fig. 2 A). In these strains, cells exited mitosis inappropriately when spindle movement into the neck was delayed (Video 3; Fig. 2, B and D), indicating that the bub2D and bfa1D mu- tants are completely defective for checkpoint function. Next we examined bim1D and stt4-7 single mutants and ts-arp1 bim1D and ts-arp1 stt4-7 double mutants. As ex- pected, many ts-arp1 bim1D and ts-arp1 stt4-7 cells failed to move the spindle into the neck by 20 min (Fig. 3, A and B). However, in contrast to bub2D and bfa1D, inappropri- ate mitotic exit occurred in only a fraction of cells that de- layed spindle movement (Figs. 3 B and 4 A; Video 4). Some cells that delayed spindle movement into the neck eventually succeeded in moving the spindle into the neck and divided (Figs. 3 C and 4, A and B; Videos 4–6). Other cells failed to move the spindle into the neck and remained arrested in anaphase (Fig. 3 C and 4 A; Video 4). Because many cells delayed spindle movement into the neck with- out undergoing mitotic exit, the ts-arp1 bim1D and ts-arp1 stt4-7 mutants showed, on average, a delay in mitotic exit, as did the ts-arp1 control cells (Fig. 3 A; P , 0.001). These results suggest that the spindle position checkpoint retains at least partial function in bim1D and stt4-7 mutants. What accounts for the occasional failure of the spindle position checkpoint in these mutants? Even ts-arp1 single shown (black columns). Cell numbers: wild type, 217; ts-arp1, 287; bim1D, 290; stt4-7, 72; ts-arp1 bim1D, 259; ts-arp1 stt4-7, 100; ts-arp1 bub2D, 93. (C) Fate of cells that delay spindle movement into the neck. In the ts-arp1 mutant, most cells that delay spindle movement into the neck fail to move the spindle into the neck during the course of observation (white column). In the ts-arp1 stt4-7 mutant, many cells delay spindle movement into the neck but then correct the spindle position and exit mitosis (gray col- umn). In the ts-arp1 bim1D mutant, many cells exit mitosis in- appropriately (black column) instead of correcting the spindle position. Cell numbers: wild type, 0; ts-arp1, 92; bim1D, 39; stt4-7, 29; ts-arp1 bim1D, 168; ts-arp1 stt4-7, 81; ts-arp1 bub2D, 24. Error bars represent SEP. Adames et al. Yeast Spindle Position Surveillance Mechanism 163