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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.007963

Journal of Cell Science 120, 2424-2434 (2007)
Published by The Company of Biologists 2007
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CDK11p58 is required for the maintenance of sister chromatid cohesion

Dongli Hu, Marcus Valentine, Vincent J. Kidd and Jill M. Lahti*

Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA


Figure 1
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  		Fig. 1. CDK11 RNAi causes G2/M cell-cycle accumulation, impaired proliferation and cell death. (A) Western blot analysis showing downregulation of the CDK11p110 and CDK11p58 proteins. HeLa cells were transfected with CDK11 RNAi-N (pSUPER/A461), CDK11 RNAi-C (pSUPER/A2184) or the control plasmid (pSUPER). After 72 hours, cell lysates were prepared and subjected to electrophoresis and immunoblot analysis. Immunoblots were probed with anti-CDK11 P1C antibody (Trembley et al., 2002Go). (B) CDK11 RNAi impaired cell proliferation. Phase-contrast photographs of HeLa cells 72 hours after transfection with CDK11 or control RNAi plasmids. Bar, 100 µm. (C) DNA content analysis 72 hours after transfection showing increased G2/M and sub-G1 fractions resulting from CDK11 RNAi. (D) Retarded cell growth caused by CDK11 RNAi. Equal numbers of cells were transfected with pSUPER (control) or pSUPER-CDK11-RNAi (pSUPER/A2184) plasmids and plated into dishes for culturing. Cells were counted 72 hours after transfection. The bars represent the mean from triplicate experiments with standard deviation.

 

Figure 2
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  		Fig. 2. CDK11 depletion causes premature separation of condensed sister chromatids. (A,B) Partial depletion of CDK11 causes chromosome misalignment and lagging chromosomes but does not block mitotic progression. (A) Immunofluorescence analysis of HeLa cells transfected with either a CDK11 siRNA A1361 oligonucleotide or control siRNA oligonucleotide and cultured for 48 hours before immunofluorescence staining with anti-{gamma}-tubulin (red) and anti-CREST (green) antibodies and DAPI (blue). Bar, 5 µm. (B) Determination of the frequency of chromosome alignment and segregation. More than 100 mitotic cells from each treatment group (si-A1361, n=138; control, n=198) were analyzed for the presence of misaligned and lagging chromosomes. In this representative experiment, 29% of the CDK11 A1361 siRNA cells had misaligned and/or lagging chromosomes whereas only 1.5% of the control cells exhibited this phenotype. (C) Giemsa-stained mitotic chromosome spreads for cells treated with either control siRNA expression vector or CDK11 siRNA expression construct 2184 analyzed 48 hours after transfection. Bar, 5 µm. (D) CDK11 depletion results in premature sister chromatid separation. Data from a representative experiment in which cells were transfected with either the control or CDK11 siRNA expression vector and 100 mitotic cells were scored for chromosome appearance. Chromosome appearance was categorized as I: partially condensed with closed arms; II: condensed and separated arms with kinetochore linked; III: both condensed arms and kinetochores separated, still paired; IV: condensed and separated sister chromatids no longer paired. (Noco: nocodazole.) The mitotic index was determined from counting ~1000 cells from each Giemsa-stained RNAi sample.

 

Figure 3
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  		Fig. 3. CDK11p58 protein partially rescues the premature sister chromatid separation phenotype caused by CDK11 depletion. HeLa cells stably expressing GFP-tagged CDK11p58 protein from the CDK11p58-GFP cell line or the RNAi-resistant form of the CDK11p58m-GFP cell line were transfected with CDK11 RNAi or control RNAi plasmids and analyzed 48 hours later by immunoblotting for expression of CDK11 proteins with P1C antibody (A) and for mitotic chromosome appearance and mitotic index (B). Giemsa-stained cells were analyzed for mitotic chromosome appearance (100 cells) and mitotic index (>1000 cells). Representative data from one experiment are shown. Chromosome appearance was categorized as in Fig. 2.

 

Figure 4
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  		Fig. 4. CDK11 depletion results in mitotic arrest. (A) Most CDK11-depleted mitotic cells retain cyclin B despite sister chromatid separation. CDK11 RNAi or control RNAi cells were stained for cyclin A (green), cyclin B (red) and DNA (DAPI) 48 hours after transfection. A portion of these cells were treated with nocodazole for six hours before harvesting. Cyclin A and cyclin B content was analyzed by immunofluorescence in 130-250 mitotic cells from each group in a representative experiment. (B) CDK11 depletion results in mitotic arrest with BubR1 accumulation to the kinetochores. HeLa cells were analyzed by indirect immunofluorescence 48 hours after transfection with CDK11 RNAi or control plasmids using antibodies to BubR1 (red) and CREST (green). DNA is stained with DAPI (blue). Bars, 5 µm.

 

Figure 5
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  		Fig. 5. CDK11 depletion leads to premature removal of centromeric cohesin complexes. HeLa cells expressing an inducible Myc-tagged human Scc1 were transfected with CDK11 RNAi or control RNAi plasmids for 48 hours with or without 6 hours of nocodazole treatment before harvesting. The cells were analyzed by immunofluorescence staining using antibodies against the Myc tag to detect Myc-human Scc1 (red) and the kinetochores CREST antigen (green). (A) Result of the analysis of 200-300 mitotic cells examined for the presence of a Myc-Scc1 signal on centromeric kinetochores in a representative experiment. (B) Representative mitotic cells. Bar, 5 µm.

 

Figure 6
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  		Fig. 6. CDK11 depletion leads to altered Sgo1 localization on the kinetochores. (A) HeLa cells were analyzed by indirect immunofluorescence 48 hours after transfection with CDK11 RNAi or control plasmids using antibodies to Sgo1 (red) and CREST (green). DNA was stained with DAPI (blue). (B) Bub1 dissociates prematurely from the kinetochores upon depletion of CDK11. HeLa cells treated with CDK11 RNAi and control RNAi were stained for Bub1 (red) and DAPI (blue) 48 hours after transfection. Bars, 5 µm.

 

Figure 7
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  		Fig. 7. Analysis of mitotic chromosomes in cells treated with Plk1, Sgo1 and CDK11 RNAi expression constructs. (A,B) Quantitation of the chromosome phenotypes from Plk1, Sgo1 and CDK11 RNAi cells in a representative experiment. HeLa cells were transfected with CDK11, Plk1, Sgo1, Plk1-CDK11, Sgo1-CDK11, Sgo1-Plk1 or control RNAi expression constructs. Mitotic spreads were prepared and Giesma stained 48 hours later. More than 100 mitotic cells were scored for chromosome appearance. (C) The appearance of characteristic mitotic chromosomes. Bar, 3 µm.

 

Figure 8
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  		Fig. 8. Roles of CDK11p58 in regulation of sister chromatid cohesion during mitosis. CDK11p58 protects sister-chromatid cohesion at both the arms and at the centromere. The dashed circles denote the hypothetical protective action of CDK11p58 on chromosome cohesion rather than localization of CDK11p58 to the chromosome. SAC represents the spindle checkpoint. Premature dissociation of Bub1 and cohesion complexes occurs in CDK11-depleted cells. *Plk1 binding to kinetochore is reduced in CDK11 RNAi cells (Petretti et al., 2006Go), which may also influence chromosome cohesion.

 

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© The Company of Biologists Ltd 2007