Figure 6. Loss of cytoplasmic microtubules and inappropriate mitotic exit in tub2-401 cells. (A) At semipermissive tempera- tures, cytoplasmic microtubule staining is greatly reduced. At 208C, many of the spindles are misaligned and lack cytoplasmic Lee et al., 2000). If so, other mutants affecting cytoplasmic microtubules should also show inappropriate mitotic exit while the spindle is in the mother. At least two mutants with cytoplasmic microtubule defects frequently give rise to binu- cleate and anucleate cells—tub2-401 and cnm67D (Sullivan and Huffaker, 1992; Hoepfner et al., 2000). We tested whether these mutants underwent inappropriate mitotic exit with or without SPB contact with the neck. We first examined the cold-sensitive tub2-401 tubulin mutant, which undergoes a preferential loss of cytoplasmic microtubules at the semipermissive temperature of 188C (Sullivan and Huffaker, 1992). We obtained the same re- sults at 208C, without adverse effects on the spindle (Fig. 6 A). In many of these cells, cytoplasmic microtubules did not appear to penetrate or touch the neck (Fig. 6, A and B). Cytoplasmic microtubules are responsible for align- ment of the spindle along the mother–bud axis before spindle movement into the neck (Sullivan and Huffaker, 1992; Miller and Rose, 1998; Adames and Cooper, 2000). Accordingly, tub2-401 cells grown at 208C showed an in- crease in misaligned spindles compared with cells grown at 308C (Fig. 6, A and B). As with ts-arp1 bim1D mutants, many tub2-401 cells that delayed spindle movement into the neck also delayed exit from mitosis (Fig. 6 C). Movie analysis of tub2-401 cells revealed that inappro- priate mitotic exit was frequent at 208C but was rarely ob- served at 308C (Fig. 6 B). When we examined the events leading up to mitotic exit at 208C, we found that some cells (4/35 cells that exited mitosis inappropriately) exited mito- sis without moving the spindle to the neck (Video 13; Fig. 6 D), whereas most (31/35 cells) exhibited abortive spindle movement into the neck before inappropriate mitotic exit (Video 13; Fig. 6 D). These events resemble those ob- served in the ts-arp1 bim1D strain. We attempted to use GFP–tubulin to visualize cytoplas- mic microtubules in the tub2-401 mutant, but the fluores- microtubules or show no interactions between short cytoplasmic microtubules and the neck (arrows). Misaligned spindles lacking cytoplasmic microtubule interactions with the neck are also present at 308C but are much less frequent. Shown are collages of representative cells. (B) Frequency of the lack of cytoplasmic mi- crotubule interactions with the neck (white columns), spindle misalignment (gray columns), and inappropriate mitotic exit (black columns) in tub2-401 cells. Cell numbers for each column from left to right: 208C, 108, 108, 142; 308C, 150, 150, 54. (C) The spindle position checkpoint is intact in the tub2-401 mutant. At 208C, the majority of cells that delay spindle movement into the neck by .40 min arrest with an anaphase spindle at the neck on the mother side of the neck (white columns). At the lower tem- perature, few cells that delay spindle movement into the neck correct the defect and move the spindle into the neck (gray col- umns). Many of these cells undergo inappropriate mitotic exit (black columns). Cell numbers: 208C, 85; 308C, 12. (D) Still im- ages from Video 13. Events preceding inappropriate mitotic exit in the tub2-401 mutant. Cells of interest are outlined for clarity. In this series, two cells exit mitosis inappropriately after an SPB touches the neck (*). One cell exits mitosis without any SPB– neck interaction (#). Note the poor alignment of the spindle with respect to the mother–bud axis in this cell. After mitotic exit, Hof1–GFP disappears or immediately relocates to the necks of the new buds (arrows). Error bars represent SEP. Supplemental videos are available at http://www.jcb.org/cgi/content/full/153/1/ 159/DC1. Bars, 5 mm. The Journal of Cell Biology, Volume 153, 2001 166