Stu1 inversely regulates kinetochore capture and spindle stability

Abstract

The Saccharomyces cerevisiae CLASP (CLIP-associated protein) Stu1 is essential for the establishment and maintenance of the mitotic spindle. Furthermore, Stu1 localizes to kinetochores. Here we show that, in prometaphase, Stu1 assembles in an Ndc80-dependent manner exclusively at kinetochores that are not attached to microtubules. Stu1 relocates to microtubules when a captured kinetochore reaches a spindle pole. This relocation does not depend on kinetochore biorientation, but requires a functional DASH complex. Stu1 at detached kinetochores facilitates kinetochore capturing. Furthermore, since most of the nuclear Stu1 is sequestered by one or a few detached kinetochores, the presence of detached kinetochores prevents Stu1 from localizing the spindle, and therefore from stabilizing the spindle. Thus, the sequestering of Stu1 by detached kinetochores serves as a checkpoint that keeps spindle poles in close proximity until all kinetochores are captured. This is likely to facilitate kinetochore biorientation. In agreement with the findings described above, a kinetochore mutant (okp1-52) that fails to release Stu1 from the kinetochore displays a severe spindle defect upon spindle pole body separation, and this defect can be rescued by destroying the okp1-52 kinetochore.

Figure 1.

okp1-52 and ame1-2 mutants display a severe spindle defect, although Okp1 and Ame1 do not localize to spindles. (A) ame1-2 and okp1-52 display a severe spindle defect. Spindles were quantified 180 min after the release from G1 at 37°C. n > 100. Bar, 2 μm. (B) Okp1 and Ame1 do not localize to the spindle. (C–E) Most okp1-52 and ame1-2 cells display a spindle defect at a spindle length ≤2 μm. (C) Starting 60 min after the release from G1 at 37°C (0′), spindles (GFP-Tub1) were visualized by time-lapse microscopy. Individual frames for the indicated times are shown. Bar, 2μm. (D) Quantification of cells visualized in C with a defective 2-μm (or shorter) spindle. n > 50. (E) GAL1-CDC20 cells were arrested in metaphase by Cdc20 depletion (Δcdc20) at 25°C and then shifted to 37°C. At the indicated time points after the temperature shift, spindles (Tub1-GFP) were quantified as intact (as shown for wild type) or defective (as shown for okp1-52). Only cells that had separated spindle poles 2 μm or less were included in the count. n > 100. Bar, 2 μm.

Figure 2.

okp1-52 KTs fail to release Stu1. (A–C) Stu1 localization in wild-type cells. Bar, 2 μm. (A) Stu1 and spindle were visualized at the indicated cell cycle phases. Arrows indicate the position of KTs. (B) Stu1 localizes at the KT early in mitosis. Cells were analyzed 80 min after the release from G1. The phenotype shown was observed in 24% of the cells. n > 100. (C) Stu1 localizes to the spindle in metaphase. GAL-CDC20 cells were arrested in metaphase by Cdc20 depletion. (D) Stu1 remains at okp1-52 KTs during spindle pole separation. Cells were analyzed 120 min after the release from G1 at 37°C. Bar, 2 μm. (E) In okp1-52 cells, Stu1, in contrast to Ase1, fails to locate to ipMTs stabilized by KIP3 deletion. Δkip3 cells were analyzed 150 min after the release from G1 at 37°C. Bar, 2 μm. (F) Quantification of Ase1 and Stu1 localization as observed in E. n > 100. (G) In okp1-52 cells, Stu1, in contrast to Ase1, does not localize to the midzone of Δkip3-stabilized ipMTs upon overexpression of Cdc14. Cells were released from G1 at 25°C in raffinose, the temperature was shifted to 37°C, Cdc14 overexpression was induced (2% galactose) 90 min after the release, and spindles were analyzed 210 min after the release. Bar, 2 μm. (H) Quantification of Ase1 and Stu1 localization as observed in G. n > 100. (I) Compromising okp1-52 KTs by Cse4 depletion rescues spindle formation. GAL-CSE4 cells were depleted of Cse4 during a G1 arrest. One-hundred-twenty minutes after the release at 37°C, spindle phenotypes (GFP -Tub1) and Stu1-3mCherry localization were quantified.

Figure 3.

Stu1 selectively interacts with detached KTs and relocalizes to the spindle when captured KTs reach a spindle pole. Relocalization depends on a functional DASH complex but not on biorientation. (A,B) Wild-type cells were analyzed 150 min after the release from a G1 arrest into nocodazole. (A) Occurrence of clearly detached KTs. Cells with a detached KT V, as shown, were quantified. n > 100. Bar, 2 μm. (B) Stu1 interacts selectively with detached KTs. Stu1 localization was quantified as indicated. n > 100. Bar, 2 μm. (C,D) Wild-type cells were released from G1 into nocodazole. One-hundred-twenty minutes after the release, nocodazole was removed. (C) Stu1 moves with the captured KTs to a spindle pole and then relocalizes to spindle MTs. Starting 50 min after the nocodazole washout, the cells were visualized by time-lapse microscopy (3 or 1 min per frame). Individual frames with the indicated times are shown for two different cells. Arrows indicate detached KTs. Bar, 2 μm. (D,E) Biorientation is not required for Stu1 relocalization to the spindle. (D) Stu1 localization in respect to spindle poles and KTs of chromosome V in 8% of the cells (n > 50) 60 min after the nocodazole washout. Bar, 2 μm. Note that Stu1 localizes to the spindle and not to the KTs, although the latter have not achieved biorientation. (E) Stu1 localizes to spindles in scc1-72 cells. Cells were analyzed 120 min after the release from G1 at 37°C. Stu1 localized to the spindle in 100% of the cells. Phenotypes for spindles with a length of ≥4 μm were quantified as shown. n > 100. Bar, 2 μm. (F) Stu1 relocalization to the spindle fails in dad2-9 cells. Cells were processed as in C, but shifted to 37°C when released from G1 and analyzed 60 min after the nocodazole washout. The phenotype shown was observed in 92% of the cells. n > 100. Bar, 2 μm.

Figure 4.

Spindle-associated Stu1 relocates to detached KTs. (A) Stu1 relocates from captured KTs to the spindle and then returns to the remaining detached KTs. Cells were processed as in C. Magnification of the boxed area is as indicated. (White arrows) Captured KTs; (red arrows) detached KTs that acquired Stu1 only in the second round. Bar, 2 μm or as shown. (B) Stu1 relocates to detached KTs in metaphase. GAL-CDC20 cells were arrested in metaphase by Cdc20 depletion, treated with nocodazole, and visualized by time-lapse microscopy. (0′) Nocodazole addition. Magnification of the boxed area is as indicated. Bar, 2 μm or as shown. (C) Stu1 relocates to KTs when the okp1-52 mutation is induced in metaphase. GAL-CDC20 cells were arrested in metaphase by Cdc20 depletion, then shifted to 37°C and analyzed at the indicated time after the temperature shift. Cells with KT-localized Stu1 were quantified (n > 100) and classified into type 1 + 2 (intact spindle) and type 3 + 4 (defective spindle). Bar, 2 μm.

Figure 5.

Most of the nuclear Stu1 associates firmly with detached KTs. (A) Stu1 polymerizes at detached KTs. Stu1 was overexpressed from a GAL promoter in a wild-type STU1 background during a G1 arrest. Cells were analyzed 240 min after the release from G1 into nocodazole. (B) Stu1 localization at detached KTs is stable. FRAP analysis of Stu1-GFP. (C) Stu1 localization at metaphase spindles is dynamic. FRAP analysis of Stu1-GFP in cells arrested in metaphase by Cdc20 depletion.

Figure 6.

Stu1 is required for KT capturing. Cells were depleted of Stu1 during a G1 arrest and otherwise processed as in Figure 3C. KT capturing was assayed by quantifying cells with a detached KT V (as shown) at the indicated time after the nocodazole washout. n > 100. Bar, 2 μm.

Figure 7.

KT localization depends on Ndc80 but not on Mad2 or Bub1. (A) Localization of Stu1 to detached KTs depends on Ndc80. GAL-NDC80 cells were depleted of Ndc80 during a G1 arrest, and Stu1 localization was quantified 105 min after the release. n > 100. Bar, 2 μm. (B) Localization of Stu1 to detached KTs does not depend on Mad2 or Bub1. Ninety minutes after the release from G1 into nocodazole, spindle poles (Spc72-3mCherry), KTs (Ame1-GFP), and Stu1-CFP were visualized, and Stu1 localization was quantified. n > 100.

Figure 8.

Stu1 domain analysis. (A) C-terminal sequences are required for KT localization of Stu1. stu1Δ1175–1513 cells were analyzed 150 min after the release from G1 into nocodazole. Bar, 2 μm. (B) stu1Δ1175–1513 cells display an increased number of detached KTs per cell. Quantification of detached KTs or KT clusters in wild-type and stu1Δ1175–1513 cells processed as in A. n > 100. (C) Stu1 polymerization at detached KTs depends on Stu1's MBD (amino acids 461–716). Stu1Δ887–1513 and Stu1Δ461–716 were overexpressed from a GAL promoter in a wild-type STU1 background during a G1 arrest. Cells were analyzed 240 min after the release from G1 into nocodazole. Note that Stu1Δ887–1513 can still copolymerize with wild-type Stu1, whereas Stu1Δ461–716 cannot. Bar, 2 μm. (D,E) Putative models illustrating the phenotypes observed in C. (F) Overexpression of Stu1Δ461–716 results in detached KTs. Stu1Δ461–716 overexpression was as in C. Cells were analyzed 120 min after the release from G1. Bar, 2 μm. (G) Summary of Stu1 domain functions. (ND) Not done. (H) Model describing the role of Stu1 during KT capturing. For details, see the text.