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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02757

Journal of Cell Science 119, 519-531 (2006)
Published by The Company of Biologists 2006
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Deficiency of centromere-associated protein Slk19 causes premature nuclear migration and loss of centromeric elasticity

Tao Zhang*, Hong Hwa Lim*, Chee Seng Cheng and Uttam Surana{ddagger}

Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673


Figure 1
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  		Fig. 1. Nuclear division in slk19{Delta} cells. WT (US3824) and slk19{Delta} cells (US3449) were arrested in G1 using {alpha} factor and released into YEPD at 24°C. Samples were collected every 15 minutes and analyzed for extent of budding, the state of nuclear division (a total of 150 cells were counted for each time point; 90 and 110 minute samples are shown), DNA content and the levels of Clb2 and Cdc28.

 

Figure 2
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  		Fig. 2. (A) Partial nuclear division in slk19{Delta} cells occurs prior to initiation of anaphase. cdc20{Delta} GAL-CDC20 (US3398) and cdc20{Delta} slk19{Delta} GAL-CDC20 (US3399) cells were arrested in G1 using {alpha} factor and released into YEPD at 24°C. At the end of 4 hours, the cells were collected and analyzed for the state of nuclear (DAPI) division (top panel and bottom graph; for each time point, 150 cells were counted). In a parallel experiment, G1 synchronized cdc20{Delta} (US3568) and cdc20{Delta} slk19{Delta} (US3579) cells containing SCC1-cmyc18 were released into YEPD at 24°C. Samples were analyzed for the levels of Scc1, Clb2 and tubulin, and DNA content. *Denotes cleaved Scc1 (middle panel). To check whether partial nuclear division occurs in another kinetochore-component-defective mutant, cdc20{Delta} ndc10-1 GAL-CDC20 (US4708) cells were subjected to identical experimental regime as described above (bottom panel). (B) Spindles in slk19{Delta} cells. Samples taken at 240 minutes from both US3398 and US3399 cells as described in (A) were stained using anti-Tub4 antibodies.

 

Figure 3
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  		Fig. 3. Bipolar attachment in slk19{Delta} cells. WT (US3437) and slk19{Delta} (US3502) strains carrying TetO/GFP-TetR constructs were arrested in G1 using {alpha} factor and released into YEPD at 24°C. Samples withdrawn every 15 minutes were analyzed for state of nuclear division, centromeric markers (for each time point, 150 cells were counted) and DNA content. The bottom panels show the state of GFP-marked centromeric markers at 90', 105' and 120' time points in both WT and slk19{Delta} cells.

 

Figure 4
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  		Fig. 4. Nuclear dynamics in Slk19-deficient cells. cdc20{Delta} (US4235) and cdc20{Delta} slk19{Delta} (US4260) with integrated HTA2-EGFP were arrested in G1 with {alpha} factor and released into YEPD medium. The cells were immobilized onto gelatin-coated glass slides containing 2% glucose in low immunofluorescence yeast nitrogen base with complete drop-out medium supplemented with adenine. GFP signals were sampled every 4 minutes. For each time point, seven Z-sections (0.5 µm apart) were taken and Z projections of these planes were made using Metamorph software.

 

Figure 5
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  		Fig. 5. (A) Partial nuclear division in slk19{Delta} results from the force exerted by the spindle. G1 synchronized cdc20{Delta} GAL-CDC20 (US3398) and cdc20{Delta} slk19{Delta} GAL-CDC20 (US3399) strains were released into YEPD containing nocodazole (15 µg/ml). Panel a shows cdc20{Delta} and panel b cdc20{Delta} slk19{Delta} cells. Top row shows cells at 240 minutes after release from {alpha} factor into YEPD. Middle row shows cells at 240 minutes after release from {alpha} factor into YEPD containing nocodazole. Bottom row shows cells that were first arrested in G2-M with short spindle (240 minutes) and then treated with nocodazole for 1.5 hours. (Panel c) Co-localization of Ndc10 with Slk19 in cdc20{Delta} GAL-CDC20 cells (US4194) arrested in G2-M by Cdc20 depletion in glucose medium. (B) Spindle length distribution in cdc20{Delta} and cdc20{Delta} slk19{Delta} cells. cdc20{Delta} GAL-CDC20 (US3398) and cdc20{Delta} slk19{Delta} GAL-CDC20 (US3399) cells were arrested in G1 using {alpha} factor and released into YEPD at 24°C. At the end of 4 hours, cells were stained with anti-tubulin antibodies. The spindle length was determined using Metamorph software (a total of 270 cells were counted). (C) Greater variation in distance between SPBs in slk19{Delta} cells. WT (US3786) and slk19{Delta} (US4164) strains expressing Spc42-GFP integrated at the TRP1 locus were immobilized onto gelatin coated slides and immunofluorescent signals were observed every 5 minutes. The distance between the Spc42-GFP spots is plotted versus time.

 

Figure 6
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  		Fig. 6. (A) Centromeric cohesion loses its elasticity in the absence of Slk19. cdc20{Delta} GAL-CDC20 (US3444) and cdc20 slk19{Delta} GAL-CDC20 (US3448) strains carrying TetO/GFP-TetR constructs were arrested in G1 using {alpha} factor and then released into YEPD for 240 minutes to arrest them in G2-M. Each culture was divided into two halves; nocodazole was added to one half of the culture for 1.5 hours. Samples were collected and proportion of cells (a total of 150 cells were counted) with divided centromeric markers was determined (bottom left panel; also see Materials and Methods). The distance between the centromeric markers is graphically depicted in the bottom right panel. (B) Physical association of Slk19 and Scc1. (a) WT cells with endogenously tagged SLK19-HA6 and SCC1-cmyc18 at their respective loci (US3779) were arrested in G1 ({alpha} factor), S phase (hydroxyurea) and metaphase (nocodazole). Immunoprecipitates from the cell extracts were obtained by using rabbit anti-HA or rabbit anti-cmyc beads and analyzed by western blotting using mouse anti-cmyc or mouse anti-HA antibodies respectively. *,** Indicate the cleaved forms of Scc1 and Slk19, respectively. The extracts were analyzed by western blotting. (b) Negative control. Extracts were immunoprecipitated with goat anti-HA or rabbit anti-cmyc antibodies and analyzed by western blots using mouse anti-cmyc or mouse anti-HA antibodies, respectively. Lane 1: WT with endogenously tagged SLK19-HA6 and SCC1-cmyc18 at their respective loci (US3779); Lane 2: untagged WT (US1363); Lane 3: WT with endogenously tagged SCC1-cmyc18 and untagged Slk19 (US3335); Lane 4: WT with endogenously tagged SLK19-HA6 and untagged Scc1 (US4305). (c) Extracts were immunoprecipitated with goat anti-HA or rabbit anti-cmyc beads and immunoprecipitates were analyzed by western blotting using mouse anti-cmyc or mouse anti-HA antibodies, respectively. Lane 1: Extracts from asynchronously growing untagged WT (US1363); Lane 2: WT carrying endogenously tagged SLK19-HA6 and SCC1-cmyc18 (US3779); Lane 3: WT with endogenously tagged SCC1-cmyc12 and carrying slk19-({Delta}1-77)-HA3 on CEN plasmid (US3972). (d) The results from a MBP pull down assay. Bacterially produced MBP or MBP-Slk19 fusion proteins immobilized onto beads were incubated with various extracts and analyzed by western blotting against mouse anti-cmyc antibodies or rabbit anti-Clb2 antibodies. Lane 1: MBP-Slk19 beads with extracts containing SCC1-cmyc18 (from US3335); Lane 2: MBP beads with extracts containing SCC1-cmyc18 (from US3335); Lane 3: MBP-Slk19 beads; Lane 4: MBP beads; Lane 5: extracts containing SCC1-cmyc18 (from US3335); Lane 6: extracts from untagged WT (US1363) as a negative control.

 

Figure 7
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  		Fig. 7. (A) N-terminus 77 amino acids are important in augmenting centromeric cohesion. cdc20{Delta} slk19{Delta} GAL-CDC20 strains carrying TetO/GFP-TetR constructs transformed with a CEN plasmid expressing full-length Slk19, Slk19-R77E or Slk19-({Delta}1-77) (US3986, US4024, US4025) were arrested in G2-M by growth in YEPD to allow spindle formation and transient separation of centromeres. The culture was split into two halves: nocodazole was added to one half for 1.5 hours to destroy spindles, while the other half continued without Nocodazole. Samples were collected and cells (150 cells were counted) with separated centromeres were quantitated. (B) Coiled-coil domains in C-terminus of Slk19 are important for physical association with Scc1. Extracts prepared from asynchronously growing WT strains with endogenously tagged SCC1-HA6 and carrying either SLK19-cmyc12, slk19-({Delta}327-400)-cmyc12, slk19-({Delta}419-498)-cmyc12, slk19-({Delta}511-538)-cmyc12 or slk19-({Delta}543-577)-cmyc12 on CEN plasmid (US4532, US4022, US4020, US4021, US4023) were immunoprecipitated with anti-HA or anti-cmyc beads. Immunoprecipitates were analyzed by western blotting using anti-cmyc or anti-HA antibodies respectively. Extracts from untagged WT (US1363) and WT cells with endogenously tagged SCC1-HA6 and carrying SLK19-cmyc12 on a plasmid (US4532) were used as negative and positive controls. Table: Slk19 constructs carrying deletions in the coiled coil domains are unable to restore centromeric elasticity. cdc20{Delta} slk19{Delta} cells carrying TetO/GFP-TetR constructs and transformed with various deletions in the coiled coil domains of Slk19 (US4067, US4070 and US4071) were arrested in G2. Samples were collected and proportion of cells (150 cells counted) with divided centromeres was determined.

 

Figure 8
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  		Fig. 8. (A) Onset of Scc1 cleavage remains unaffected in the absence of Slk19. cdc20{Delta} GAL-CDC20 (US3910) and cdc20 slk19{Delta} GAL-CDC20 (US3579) strains were arrested in G1 at 24°C using {alpha} factor and then released into raffinose medium to deplete Cdc20. After 240 minutes, cells were filtered and resuspended in galactose medium pre-equilibrated at 16°C to induce Cdc20 synthesis. Scc1 cleavage was monitored by western blot analysis and DNA content was measured by FACS in samples collected at various time points. (B) Chromatin Immunoprecipitation. Cross linked chromatin from WT SCC1-cmyc18 SLK19-HA6 (US3779, left panel) and slk19{Delta} SCC1-cmyc18 (US3516, right panel) was divided into two portions. One portion was used as input DNA. The other half was immunoprecipitated with anti-HA or anti-cmyc beads. The input DNA and coimmunoprecipitated samples were analyzed with PCR (24 cycles) using pairs of primers (see Materials and methods) corresponding to the following loci; CEN3, CEN16 and MET2.

 

Figure 9
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  		Fig. 9. (A) Slk19-GFP is localized to the kinetochores in the absence of Scc1 function. Scc1-73 (US4077) and wild-type (US2337) cells containing SLK19-GFP were arrested in G1 using {alpha} factor and released at 37°C. Samples were taken every 15 minutes and analyzed for state of nuclear division and GFP signal. (B) Reduced cleavage of Slk19-HA6 in the absence of Scc1 function. Scc1-73 SLK19-HA6 cells (US4530) were arrested G1 using {alpha} factor and released into either 24°C or 37°C medium. Samples were taken every 15 minutes and analyzed by western blotting.

 

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© The Company of Biologists Ltd 2006