Lte1p are in the same compartment of the cell only after the spindle moves into the neck, suggesting that move- ment of the spindle into the neck signals mitotic exit (Hoyt, 2000). Consistent with this model, multinucleate and anucleate cells were formed when overexpression of Lte1p caused some Lte1p to mislocalize in the mother (Bardin et al., 2000). Bub2p and Bfa1p (Byr4p), which form a presumed GTP- ase-activating protein thought to downregulate Tem1p, are necessary for the spindle position checkpoint (Bardin et al., 2000; Bloecher et al., 2000; Pereira et al., 2000). Bub2p and Bfa1p are located at the same SPB as Tem1p, and the localization of Tem1p depends on Bub2p and Bfa1p (Pereira et al., 2000), at least until late anaphase. These results suggest that Bub2p–Bfa1p maintains Tem1p in an inactive state on the SPB while the SPB is in the mother cell (Hoyt, 2000). Thus, the model predicts that de- layed mitotic exit in cells with mispositioned spindles is due to the absence of a positive signal for mitotic exit. Here, we test this model through direct observation of mitotic exit in lte1D mutants and show that loss of Lte1p has little effect on the kinetics of mitotic exit, at least at el- evated temperatures. Also, we confirm that the spindle position checkpoint is completely abolished in bub2D and bfa1D mutants. Mitotic exit in bub2D and bfa1D mutants occurs regardless of spindle position. Hence, Bub2p– Bfa1p inhibition of Tem1p is crucial to preventing mitotic exit but is somehow relieved upon spindle movement into the neck, even in the absence of Lte1p. To further examine how the spindle position checkpoint works, we examined several other mutants in which exit from mitosis can occur despite mispositioning of the spin- dle—bim1D (BIM1 is also known as YEB1 for yeast EB1 [Muhua et al., 1998], after the first identified member of this protein family. The name Bim1, for binds to microtu- bules, denotes how the yeast gene was first identified [Schwartz et al., 1997]. The Bim nomenclature should not be confused with the Bim genes (blocked in mitosis) identified in Aspergillus [Morris, 1975].), tub2-401, and cnm67D. In these mutants, cells with mispositioned spin- dles generally delayed mitotic exit, suggesting that the spindle position checkpoint mechanism was intact. How- ever, many mutant cells with mispositioned spindles did exit mitosis inappropriately, generating binucleate and anucleate cells. These instances of failure of the check- point were associated with one of two events: transient (but ultimately abortive and ineffective) passage of an SPB into the neck, or transient loss of cytoplasmic micro- tubule interactions with the neck while both SPBs re- mained within the mother. Deletion of LTE1 did not pre- vent inappropriate mitotic exit in these cases. Thus, the observed features of the coupling between spindle posi- tion and mitotic exit are largely unaffected by Lte1p. We propose that a parallel checkpoint pathway exists in which the interaction of cytoplasmic microtubules with the neck prevents mitotic exit through activation of Bub2p–Bfa1p. Materials and Methods Yeast Strains and Plasmids With the exception of the tub2-401 mutant, all strains were isogenic with the wild-type strain YJC1687 (MATa ade2-1 ade3 lys2-801 his3-D200 leu2-3, 112 ura3-52::GFP::TUB1) as described (Adames and Cooper, 2000). Strains differed from wild type as indicated: YJC1658 (trp1-1 bim1D::HIS3), YJC1567 (stt4-7), YJC1659 (leu2-3,112::LEU2-ts-arp1 arp1D::HIS3), YJC1655 (bim1D::HIS3 leu2-3,112::LEU2-ts-arp1 arp1D:: HIS3), YJC1571 (stt4-7 leu2-3,112::LEU2-ts-arp1 arp1D::HIS3), and YJC2228 (trp1-1 cnm67D::kanMX). Hof1–GFP on pLP14 (a gift from John Lippincott and Rong Li, Harvard University, Cambridge, MA) was integrated into the above strains to make YJC2161 (ura3::URA3- HOF1-GFP), YJC2162 (bim1D ura3::URA3- HOF1-GFP), YJC1888 (ts- arp1 ura3::URA3- HOF1-GFP), YJC1889 (ts-arp1 bim1D ura3::URA3- HOF1-GFP), and YJC2329 (cnm67D ura3::URA3- HOF1-GFP). YJC1888 was used to make YJC2456 (ts-arp1 lte1D::kanMX HOF1- GFP), YJC2327 (ts-arp1 mad2D::kanMX HOF1-GFP), YJC2288 (ts- arp1 bub2D:: kanMX HOF1-GFP), YJC2290 (ts-arp1 bfa1D::kanMX HOF1-GFP), and YJC 2327 (ts-arp1 mad2D::kanMX HOF1-GFP). YJC2161 was used to make YJC2443 (lte1D::kanMX). YJC1889 was used to make YJC2444 (ts-arp1 bim1D lte1D::kanMX). YJC2228 was used to make YJC2446 (cnm67D lte1D::TRP1). To make the tub2-401 mutant with GFP–tubulin and Hof1–GFP (YJC2318), we integrated pAFS92 (a gift from Aaron Straight, Harvard University, Cambridge, MA) and pLP14 into CUY66 (MATa ade2-101 ura3-52 tub2-401; a gift from Tim Huffaker, Cornell University, Ithaca, NY). YJC2318 was used to make YJC2445 (tub2-401 lte1D::kanMX). All deletion strains were analyzed by PCR to confirm replacement of the open reading frame with the appropriate selection marker. Deletion strains showed the ap- propriate phenotypes, including cold-sensitive growth in lte1D mutants. Strains expressing Myo1–GFP were also made but are not shown. There were no differences in the timing of spindle breakdown, cytokinesis, and cell separation between cells expressing Myo1–GFP or Hof1–GFP or cells expressing neither (data not shown). Media, genetic manipulations, and lithium acetate transformation were as described (Kaiser et al., 1994). Assay for Mitotic Exit Strains expressed GFP–tubulin to allow visualization of mitotic spindles (Straight et al., 1997). Some strains also expressed Myo1–GFP (not shown) or Hof1–GFP to visualize the process of cytokinesis (Bi et al., 1998; Lippincott and Li, 1998a,b; Vallen et al., 2000). The use of Myo1–GFP or Hof1–GFP to monitor cell division provides a more accurate determination of when cell division occurs than does cell separation. Both proteins constrict with the actomyosin ring during cyto- kinesis and disappear from the neck thereafter. Cells within the population expressed these GFP fusion proteins to different extents, and strong uni- form expression was seen only in slowly growing cells in dense cultures, in cells that delay spindle movement into the neck, or at low temperatures. Using GFP–tubulin allowed us to visualize spindle breakdown directly. Spindle disassembly is dependent on the same proteolytic events involved in the degradation of mitotic cyclins (Juang et al., 1997). Spindle break- down and constriction of the actomyosin ring always coincided, regardless of whether the spindle was in the neck or entirely in the mother (59/59 wild-type cells, 46/46 bim1D cells, 20/20 ts-arp1 cells, and 80/80 ts-arp1 bim1D cells). Spindles were completely disassembled 1.6 6 0.2 min after the start of actomyosin ring constriction in the wild-type strain (58 cells), 2.7 6 0.4 min after constriction in ts-arp1 cells (19 cells), and 1.7 6 0.3 min after constriction in ts-arp1 bim1D cells (65 cells). In the strains examined, cell separation occurred at the same time interval after spindle breakdown or actomyosin ring constriction (24.3 6 1.6 min, 29 cells, for wild type; 23.8 6 2.5 min, 30 cells, for bim1D; 29.3 6 7.5 min, 6 cells, for stt4-7; 29.7 6 2.6 min, 50 cells, for ts-arp1; 26.7 6 2.8 min, 79 cells, for ts-arp1 bim1D). In some experiments, cells expressed only GFP–Tub1p, and cytokinesis was presumed to occur at the time of spindle breakdown. Scoring of SPB in- teractions with the neck in cells that exited mitosis inappropriately was confirmed in blind tests. Movies and Analysis GFP–tubulin expression was induced as described (Adames and Cooper, 2000). For low-temperature experiments, the ambient temperature was maintained ,148C. An Air Therm heater (World Precision Instruments) warmed the system to the appropriate temperature. At the semipermissive temperature of 188C used by Sullivan and Huffaker (1992), spindle micro- tubules were affected, as indicated by abnormal spindle elongation, abnor- mal spindle breakdown, and the failure of some cells to enter anaphase, suggestive of spindle assembly checkpoint activity (data not shown). Therefore, tub2-401 experiments were done at 208C or the permissive tem- perature of 308C. All other experiments were at 378C. Cells were incu- bated at 378C for 1 h to inactivate Arp1p before movie collection. Movies were collected as described (Waddle et al., 1996). Z-series of 10 focal planes 0.5 mm apart were collected and projected onto a single two- dimensional image. The timing of nuclear movements and cell cycle events The Journal of Cell Biology, Volume 153, 2001 160