Mitotic spindle disassembly occurs via distinct subprocesses driven by the anaphase-promoting complex, Aurora B kinase, and kinesin-8.

Yeast strains

The yeast strains used in this study are derivatives of S288C and are listed in Table S1 . doc1Δ , cdh1Δ , dbf2Δ , dcc1Δ , ctf8Δ , mcm21Δ , and kip3Δ null mutants originated from the Research Genetics Collection. The Ase1-4GFP and Cin8-4GFP strains were provided by E. Schiebel (ZMBH, Heidelberg, Germany). The phosphomutant alleles kip3-4A (S74A, T172A, S402A, and S792A) and she1-5A (T14A, S165A, S269A, T280A, and S325A) were generated using QuikChange site-directed mutagenesis. Ipl1 phosphorylation sites selected for mutation matched the (R/K)X(T/S)(I/L/V/T) consensus motif ( Cheeseman et. al, 2002 ). Plasmids containing full-length kip3-4A and she1-5A genes were integrated at each gene’s endogenous locus in kip3Δ and she1Δ strains, respectively. The bim1-6A and bim1-6D strains were provided by S. Westermann (Research Institute of Molecular Pathology, Vienna, Austria), and the Kip3-GFP strain was provided by K. Bloom (University of North Carolina at Chapel Hill, Chapel Hill, NC).

Fluorescence microscopy

Live cell microscopy at room temperature was performed using a microscope (IX-71; Olympus), a 100× NA 1.4 objective, and a camera (Orca-ER; Hamamatsu Photonics). All microscopy at 37°C was performed using a microscope (IX-81; Olympus) equipped with a temperature-controlled enclosure (Precision Control Weather Station), a 100× NA 1.4 objective, and a camera (Orca-ER). Two-color images were obtained by sequential switching between RFP and GFP filter sets. For time-lapse microscopy of shrinking half-spindles, actinomyosin ring contraction, and Bim1-3GFP localization, images were collected at 10-s intervals with 300-ms exposures. Each image represents a maximum intensity projection from a z stack containing six planes 0.2 µm apart. All image processing was performed using MetaMorph software (MDS Analytical Technologies).

Synthetic lethal profile comparison

To examine the synthetic interactions for SHE1 , we used the synthetic lethal and synthetic sick dataset described previously by Costanzo et al. (2010) and Tong et al. (2004) . Although synthetic interactions between SHE1 and CDH1 and between SHE1 and DOC1 did not appear in these lists, we had previously identified such interactions through tetrad analysis. When comparing the synthetic interaction profiles of KIP3 , CDH1 , and SHE1 , we used only the raw dataset with an intermediate cutoff applied (|ε| > 0.08, P < 0.05) Costanzo et al., 2010) , plus several interactions we had identified in our laboratory.

Detection of protein phosphorylation

To detect phosphorylation-dependent mobility shifts of She1-13Myc, Kip3-13Myc, and Bim1-HA, whole cell extracts were loaded onto 6–8% SDS polyacrylamide gels containing either 20 µM or 30 µM Phos-tag acrylamide (Hiroshima University, Hiroshima, Japan) and 40 µM or 60 µM MnCl 2 , respectively. Membranes were then probed with 1:500 mouse anti-Myc (9E10; our laboratory) or 1:1,000 mouse anti-HA (12CA5; Roche).

Immunoprecipitation

Immunoprecipitation was performed essentially as described previously ( Woodruff et al., 2009 ) with a few minor modifications. Lysates were incubated with mouse anti-Myc antibody for 1 h and then passed over protein G–Sepharose beads (GE Healthcare) to purify She1-13Myc. The column was washed with lysis buffer and eluted with SDS loading buffer.

Chromatin immunoprecipitation

Chromatin immunoprecipitation was conducted essentially as described previously ( Kang et al., 2001 ) with a few minor changes. Cells were fixed for 1 h in 1% formaldehyde. Whole cell extracts were passed over IgG Sepharose (GE Healthcare) to pull down She1-TAP and Mif2-TAP. Chromosome spreads Chromosome spreads were prepared as described previously ( Cheeseman et al., 2001 ). Anti-Duo1 antibody ( Hofmann et al., 1998 ) was used at a dilution of 1:2,000 to detect Duo1, and anti-GFP (rabbit; Torrey Pines Institute) was used at a dilution of 1:2,000 to detect She1-3GFP. Online supplemental material Fig. S1 describes spindle morphology in cells compromised for Kip3 and APC Cdh1 activity. Fig. S2 depicts the pairwise comparison of previously described synthetic genetic interactions shared between CDH1 , KIP3 , and SHE1 . Fig. S3 shows She1 phosphorylation and localization to the kinetochore and localization of Cdc14 and Kip3 in an she1Δ mutant. Fig. S4 shows Ipl1-3GFP localization in dbf2Δ cells. Videos 1–3 show spindle disassembly relative to cytokinetic ring contraction in wild-type, cdh1Δ , and kip3Δ cells, respectively.

Table

S1 lists the strains used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201006028/DC1 .

Online supplemental material Fig. S1 describes spindle morphology in cells compromised for Kip3 and APC Cdh1 activity. Fig. S2 depicts the pairwise comparison of previously described synthetic genetic interactions shared between CDH1 , KIP3 , and SHE1 . Fig. S3 shows She1 phosphorylation and localization to the kinetochore and localization of Cdc14 and Kip3 in an she1Δ mutant. Fig. S4 shows Ipl1-3GFP localization in dbf2Δ cells. Videos 1–3 show spindle disassembly relative to cytokinetic ring contraction in wild-type, cdh1Δ , and kip3Δ cells, respectively.

Table

S1 lists the strains used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201006028/DC1 .