were analyzed with NIH Image 1.62 (written by Wayne Rasband at National Institutes of Health). Comparisons of statistical significance were by t test. Immunofluorescence For microtubule staining, tub2-401 cells were grown to OD600 5 0.5 and synchronized with 0.2 M hydroxyurea for 2 h to arrest the cells in G2/M. The culture was washed and released from arrest, and split into two; one culture was grown at 208C for 60 min and the other at 308C for 30 min to allow the cells to enter anaphase. Very few cells showed anaphase spin- dles in the mother at 308C. However, at 208C, many anaphase spindles were in the mother. Immunofluorescence was performed as described (Amatruda and Cooper, 1992) using a mouse a-Drosophila a-tubulin pri- mary antibody (1:10) and a rhodamine-conjugated donkey a-mouse sec- ondary antibody (1:100). Online Supplemental Material Videos of cells depicted in Figs. 1, 2, 4, 6, and 7 are available at http:// www.jcb.org/cgi/content/full/153/1/159/DC1. Supplemental Videos 1–14 correspond to Figs. 1 A, 2 C, 2 D, 4 A–E, 6 D, and 7, respectively. Results Mitotic Exit in the Absence of Lte1p The model proposed by Bardin et al. (2000) and Pereira et al. (2000) invokes a failure of Tem1p to interact with Lte1p as the basis for the delayed mitotic exit in cells with mispositioned spindles. lte1D mutants are cold sensitive (Shirayama et al., 1994) but are able to grow at higher temperatures. Whether the dynamics of mitotic exit are af- fected at higher temperatures is unknown. Using long-term (3 h) movies of cells, we analyzed pro- gression through mitosis in lte1D cells. Most experiments in this study were done at 378C for comparison with strains carrying a temperature-sensitive allele for dynactin (ts- arp1; Muhua et al., 1998). We found an excellent correla- tion between the timing of spindle disassembly (detected using GFP–Tub1p) and the timing of cytokinesis (detected using either Hof1p–GFP or Myo1p–GFP; Materials and Methods), and these events were used interchangeably to determine the timing of mitotic exit. Selected time points from movies of representative cells are shown. We found that Lte1p was not required for mitotic exit at 378C. All of the 112 anaphase lte1D cells we observed pro- ceeded through anaphase and divided (Video 1; Fig. 1 A). Furthermore, loss of Lte1p had little effect on the timing of mitotic exit compared with wild-type cells (Fig. 1 B). Thus, Tem1p can apparently be activated with near normal ki- netics without Lte1p under these conditions. Lte1p was also not necessary for mitotic exit at lower temperatures (208C), although mitotic exit appeared slow compared with LTE1 cells at the same temperature (see below). Although Lte1p was not necessary for mitotic exit, there was a remarkable consistency in the timing of mitotic exit relative to spindle movement into the neck, even in mu- tants that delay spindle movement. In ts-arp1 lte1D mu- tants, when the spindle did eventually move into the neck, the time until mitotic exit was similar to that observed in the wild-type strain or ts-arp1 single mutant (Fig. 1 B). Mi- totic exit always occurred within z20 min of spindle move- ment into the neck regardless of how long it took to move the spindle into the neck (compare the interval from spin- dle movement into the neck to mitotic exit in Figs. 1 B, 2 A, and 3 A). This observation implies the existence of a Lte1p-independent mechanism that couples spindle move- ment into the neck with exit from mitosis. Figure 1. Lte1p is not necessary for mitotic exit. (A) Still images from Video 1. Representative field showing mitosis in lte1D cells. Cells expressed GFP–tubulin to allow visualization of the spindle (bright thick bar of fluorescence) and cytoplasmic microtubules. Cells also expressed Hof1–GFP to allow visualization of mitotic exit. Hof1–GFP initially localizes to the septin ring structures at the neck (arrowhead). During mitotic exit, Hof1–GFP moves to the constricting actomyosin ring and then disappears from the neck (Lippincott and Li, 1998a). Not all cells in the population expressed Hof1–GFP to the same extent. Time (min) is indicated in the upper right. (B) Loss of Lte1p has little effect on the kinet- ics of mitotic exit. Shown are the mean time intervals from the start of anaphase (spindle elongation) to spindle movement into the neck (white columns), from spindle movement into the neck to mitotic exit (spindle break down and constriction of the acto- myosin ring; gray columns), and the total time for anaphase (black columns). The lte1D mutant was slightly slower than the wild-type strain (P , 0.01). Number of events for each column from left to right on the chart: wild type, 108, 83, 83; lte1D, 115, 112, 112; ts-arp1, 120, 116, 114; ts-arp1 lte1D, 48, 66, 48. Error bars represent SEM. Supplemental videos are available at http:// www.jcb.org/cgi/content/full/153/1/159/DC1. Bar, 5 mm. In all of the 1,082 cells we observed in which the spindle did successfully move into the neck (after variable delays), mitotic exit always occurred within 25 min of the initial in- teraction of the SPB with the neck. When spindles failed to Adames et al. Yeast Spindle Position Surveillance Mechanism 161