Figure 7. Inappropriate mitotic exit in cnm67D cells. Still images from Video 14. Representative cells showing two examples of inappropriate mitotic exit, one after the SPB touches the neck (*), and one without any SPB– neck interactions (#). Also shown are cells that move the spindle into the neck within the normal time for mitotic exit (x). Cells of interest are outlined. Supplemental videos are avail- able at http://www.jcb.org/cgi/ content/full/153/1/159/DC1. Bar, 5 mm. cence was weak, presumably due to inefficient incorpora- tion of GFP–tubulin, and we could not detect cytoplasmic microtubules with certainty. However, we could see the spindle and noted that spindles were misaligned (.458 from the mother–bud axis) in 4/4 of the cells that underwent mi- totic exit without spindle movement to the neck, suggesting that cytoplasmic microtubules had failed to align the spindle (presumably due to loss of contact with the bud) in these cells (Video 13; Fig. 6 D). In contrast, cells (68/164 anaphase cells) in which spindles were retained in the mother but properly aligned (,458) along the mother–bud axis never underwent inappropriate mitotic exit. Another mutant that displays defects in cytoplasmic mi- crotubules and produces binucleate and anucleate cells is cnm67D (Hoepfner et al., 2000). Cnm67p is an SPB outer plaque protein that is required for formation of the outer plaque to anchor cytoplasmic microtubules to the SPB. In cnm67D cells, cytoplasmic microtubules frequently detach from the SPB, becoming free in the cytoplasm (Hoepfner et al., 2000). Consequently, cnm67D mutants are unable to move the spindle into the neck efficiently (Hoepfner et al., 2000). We confirmed that some cnm67D cells underwent in- appropriate mitotic exit, resulting in binucleate and anucle- ate cells (Video 14; Fig. 7). In 8/12 of these cells, the spindle never touched the neck. In cnm67D cells, the free cytoplas- mic microtubules are stable and move along the cell cortex, sometimes moving through the neck. Thus, in some cases the number and movement of the free cytoplasmic micro- tubules made it difficult to determine if cytoplasmic micro- tubules were absent from the neck. As shown above for ts-arp1 bim1D cells at 378C, Lte1p was not required for inappropriate mitotic exit in cnm67D mutants at 378C and tub2-401 mutants at 208C (Fig. 5). In 14/34 cnm67D lte1D cells and 2/20 tub2-401 lte1D cells that exited mitosis inappropriately, the spindle never directly interacted with the neck. Therefore, neither Lte1p nor spindle interaction with the neck are required for inappro- priate mitotic exit in any of the mutants defective for cyto- plasmic microtubule function. Discussion Recent work has suggested a model for how exit from mi- tosis is coordinated with partitioning of the nucleus be- tween mother and bud. In the current model, Bub2p– Bfa1p is responsible for keeping Tem1p in an inactive state on the SPB while the spindle is in the mother (Bardin et al., 2000; Bloecher et al., 2000; Pereira et al., 2000). Sub- sequent spindle movement into the neck promotes exit from mitosis by allowing Lte1p in the bud access to Tem1p at the SPB (Bardin et al., 2000; Hoyt, 2000; Pereira et al., 2000). This model predicts that Lte1p and spindle pole movement into the bud are essential for mitosis. Our results here provide two conclusions that are not explained by this model. First, Lte1p is not necessary for mitotic exit (at least at high temperatures). Mitotic exit oc- curred at the normal time in all lte1D cells in which the spindle moved into the neck. Moreover, inappropriate mi- totic exit in mutants lacking Lte1p occurred with at least the same frequency as in their LTE1 counterparts, even at low temperatures. Also, in some mutants, mitotic exit oc- curred without spindle movement into the neck or interac- tion of an SPB with the neck. These cases occurred in three mutants—bim1D, tub2-401, and cnm67D—where cy- toplasmic microtubules are defective. In these mutants, 47 of 196 instances of inappropriate mitotic exit occurred without any SPB–neck interactions. The GTPase-activating protein activity of Bub2p–Bfa1p is thought to constitutively maintain Tem1p in the GDP- bound state in the current model (Bardin et al., 2000; Hoyt, 2000; Pereira et al., 2000). Lte1p activation of Tem1p in the bud then overrides inhibition by Bub2p–Bfa1p. However, the observation that loss of Bub2p–Bfa1p activity allows mitotic exit even when spindles are mispositioned in the mother supports the observation that Tem1p activation can occur without Lte1p, which resides in the bud. In fact, we found that in dynactin mutants lacking Bub2p or Bfa1p, the spindle poles often do not enter or even touch the neck, and yet the timing of mitotic exit was normal. Never- theless, there was a remarkable constancy in the timing of mitotic exit relative to spindle movement into the neck in strains with functional Bub2p–Bfa1p. Together, these re- sults suggest that Bub2p–Bfa1p is inactivated upon spindle movement into the neck, allowing the intrinsic activity of Tem1p to signal mitotic exit. In this model, the guanine– nucleotide exchange factor activity of Lte1p aids mitotic exit at low temperatures that possibly reduce the intrinsic rate of nucleotide exchange on Tem1p. Whether Bub2p–Bfa1p inhibition of Tem1p is really regulated or not remains to be determined. However, we do have some clues to how such regulation could occur. The mutants we used to study inappropriate mitotic exit Adames et al. Yeast Spindle Position Surveillance Mechanism 167