Figure 5. Lte1p is not required for inappropriate mitotic exit. Deletion of LTE1 does not decrease the frequency of inappropri- ate mitotic exit in mutants affecting cytoplasmic microtubule function—bim1D, tub2-401, and cnm67D. Shown are the percent- age of cells that exit mitosis inappropriately (white columns), the percentage of cells that delay spindle movement into the neck (gray columns), and the percentage of cells that delay spindle movement into the neck and exit mitosis inappropriately (black columns). Cell numbers for each column from left to right: ts-arp1 bim1D, 259, 259, 168; ts-arp1 bim1D lte1D, 121, 121, 95; tub2-401, 142, 142, 97; tub2-401 lte1D, 80, 80, 54; cnm67D, 178, 178, 86; cnm67D lte1D, 150, 150, 119. Error bars represent SEP. checkpoint mechanism is simply unable to provide the longer delays necessary to maintain coordination with mi- totic exit. However, many cells with mispositioned spin- dles appeared to delay mitotic exit indefinitely, arguing that the checkpoint has the capacity to arrest mitotic exit as long as is necessary (Fig. 3 C). In addition, inappropri- ate mitotic exit was much more frequent in ts-arp1 bim1D cells than in ts-arp1 stt4-7 cells, despite the similar severity of spindle mispositioning in these mutants (Fig. 3, A and B). Thus, the frequency of inappropriate mitotic exit is not simply correlated with the frequency or duration of de- layed spindle movement to the neck. Inappropriate Mitotic Exit in the Absence of SPB Contact with the Bud The SPB model for the spindle position checkpoint pre- dicts that the SPB must interact with the bud to cause mi- totic exit (Bardin et al., 2000; Bloecher et al., 2000). We asked whether SPB interactions with the neck always oc- curred before inappropriate mitotic exit by examining mi- crotubule and spindle behavior in the cells that underwent inappropriate exit from mitosis. We found that the SPB did not always interact with the neck before inappropriate mitotic exit. In 15% (7/48) of the ts-arp1 bim1D cells that underwent inappropriate exit from mitosis, we did not observe any spindle pole contacts with the neck (Videos 7 and 8; Fig. 4 C). Therefore, mitotic exit does not require interaction of the SPB with the neck. Mi- totic exit in these cells was associated with transient or pro- longed loss of cytoplasmic microtubule–neck contacts: ei- ther cytoplasmic microtubules failed to grow into the bud at all, or they transiently depolymerized out of the neck (7/7 cells; Videos 7 and 8; Fig. 4 C). Inappropriate mitotic exit followed the loss of microtubules from the neck if mi- crotubules were absent from the neck for .1 min, and the spindle failed to move into the neck within z20 min of micro- tubule loss from the neck. Short-term loss of cytoplasmic microtubules from the bud is frequent in bim1D and ts-arp1 bim1D cells because of defects in microtubule dynamics and microtubule capture at the bud cortex (Adames and Cooper, 2000). In the remaining 85% (41/48) of ts-arp1 bim1D cells that underwent inappropriate exit from mitosis, the spindle first made an abortive movement to the neck, placing one SPB close to or inside the neck. The spindle then moved back out into the mother before or during mitotic exit. Mi- crotubules that extended into the bud were frequently sheared off, indicating that once mitotic exit starts it is ir- reversible (Videos 9–11; Fig. 4 D). Abortive spindle move- ment to the neck also preceded inappropriate exit from mitosis in 5/5 bim1D cells, 4/4 ts-arp1 cells, 3/3 stt4-7 cells, and 4/4 ts-arp1 stt4-7 cells. In contrast to the ts-arp1 bim1D and ts-arp1 stt4-7 mu- tants, inappropriate mitotic exit in the ts-arp1 bub2D and ts- arp1 bfa1D mutants frequently occurred without abortive spindle movements to the neck (10/24 ts-arp1 bub2D cells and 9/23 ts-arp1 bfa1D cells in which inappropriate mitotic exit occurred) and without the loss of microtubules from the neck (23/24 ts-arp1 bub2D cells and 23/23 ts-arp1 bfa1D cells in which inappropriate mitotic exit occurred; Video 12; Fig. 4 E). These results further underscore the differ- ence between the bub2D or bfa1D mutants and the bim1D or stt4-7 mutants. Although the former are essential for the spindle position checkpoint to function, the infrequent in- stances of checkpoint failure in the latter are associated with abnormal microtubule and/or spindle behaviors. Inappropriate Mitotic Exit in the Absence of Lte1p The frequent occurrence of abortive spindle movements into the neck before inappropriate exit from mitosis in ts- arp1 bim1D and other mutants is consistent with the possi- bility that interaction of Tem1p on the spindle pole with Lte1p in the bud occurred when the SPB transiently pene- trated the neck, and that this triggered mitotic exit. How- ever, inappropriate exit from mitosis occurred at the same frequency in ts-arp1 bim1D lte1D cells as in the ts-arp1 bim1D mutant (Fig. 5), indicating that Lte1p is not re- quired for inappropriate mitotic exit. As in the ts-arp1 bim1D mutants, inappropriate mitotic exit in ts-arp1 bim1D lte1D cells was preceded by abortive SPB–neck in- teractions (19/31 cells) or, in cells where the spindle did not move to the neck (12/31 cells), by the loss of cytoplas- mic microtubules from the neck (10/12 cells). Conceivably, some other Tem1p activator may be located within the bud, and the abortive spindle movement to the neck might provide access of this hypothetical activator to the Tem1p on the spindle pole. However, this type of model cannot easily explain why inappropriate exit from mitosis occurs in some cells that never move a spindle pole to the neck. Inappropriate Mitotic Exit in Mutants Defective for Cytoplasmic Microtubules The spindle positioning defects associated with inappropri- ate mitotic exit in ts-arp1 bim1D cells is likely due to changes in cytoplasmic microtubule dynamics and defects in capture of cytoplasmic microtubules at the bud cortex (Tirnauer et al., 1999; Adames and Cooper, 2000; Korinek et al., 2000; Adames et al. Yeast Spindle Position Surveillance Mechanism 165