Cyclin-dependent kinase 11 antibody and localization. (A) Western blot showing cyclin-dependent kinase 11 (CDK11; left) protein levels in control (−) and CDK11 RNA interference (RNAi)-treated cells (+). Treatment depletes CDK11 but not the γ-tubulin loading control (right). (B) Immunolocalization of CDK11 protein kinases in HeLa cells throughout the cell cycle. In interphase and early prophase, CDK11 (red and lower panels) is sequestered into nuclei, in which it forms distinct speckles/punctae on the DNA (blue). Colocalization with γ-tubulin (green and middle panels) shows that CDK11 associates with centrosomes throughout the cell cycle. Centrosomal labelling of CDK11 increases as the bipolar spindle forms and the chromosomes congress. This signal diminishes as cells transit into anaphase. During telophase and cytokinesis, CDK11 again becomes concentrated within the reforming nuclei. Scale bar, 10 μm.

Cyclin-dependent kinase 11 RNA interference leads to monopolar and short spindles with diminished levels of centrosomal γ-tubulin. Distribution of β-tubulin (green), γ-tubulin (red and lower panels in monochrome) and DNA (blue) throughout mitosis in control (A) or CDK11 (cyclin-dependent kinase 11) RNA interference (RNAi)-treated cells (B). During mitosis, γ-tubulin is recruited to the centrosomes and reaches a maximum during prometaphase (insets). (B) The spindles in CDK11 RNAi cells are short and one or more chromosomes often lag at the spindle poles 48 h after treatment. At 72 h after treatment, most chromosomes show congression defects on spindles that are poorly defined or are monopolar. Note the hypercondensed chromosomes in these cells. Scale bar, 10 μm. (C) Relative quantification of γ-tubulin immunofluorescence signal intensity at the centrosomes in control (black) and CDK11 RNAi-treated cells (grey). In controls, centrosomal γ-tubulin levels are maximal during prometaphase, but consistently at least 5–7 times higher in mitosis relative to G2 stages. After depletion of CDK11, the centrosomal γ-tubulin signal intensity does not increase beyond G2 levels.

Cyclin-dependent kinase 11 depletion prevents efficient bipolar spindle formation, microtubule nucleation and centrosomal recruitment of Aurora A and Plk1 proteins. (A) Selected frames from time-lapse recordings of mitosis in a control (left) and a CDK11 (cyclin-dependent kinase 11) RNA interference (RNAi)-treated cell (right) expressing green fluorescent protein (GFP)-tagged α-tubulin. In control cells, the centrosomes are already partially separated by prophase (00:00) and maintain this separation or move further apart during spindle formation (00:05). By metaphase (00:20–01:10), the spindle reaches a steady-state length before elongating after anaphase onset (01:35) and cytokinesis (01:50; supplementary Movie 1 online). In this CDK11-depleted cell, the centrosomes were partially separated during prophase (00:00) but did not undergo any further movement, resulting in monopolar spindle and mitotic arrest (01:54; supplementary Movie 2 online). Scale bar, 5 μm. (B) CDK11 RNAi-treated cells have reduced microtubule nucleating potential. A microtubule regrowth assay was performed following cold-induced depolymerization in control (top) and CDK11-depleted cells (bottom) expressing GFP-tagged α-tubulin. In control cells, the centrosomes nucleate robust microtubule asters after being returned to 37°C for 1 and 2 min. Centrosomes in CDK11 RNAi cells fail to nucleate more than a few short microtubules. After 3 min, a period in which control cells start to form a bipolar spindle, only atrophied asters can be detected in CDK11 RNAi cells. Scale bar, 10 μm. (C) Localization of Aurora A in the presence (37°C) or absence of microtubules (ice) in control (left panels) or CDK11-depleted mitoses (right panels). In a control metaphase cell (left), Aurora A (red and lower panel) localizes with the centrosomes (γ-tubulin; green in the middle panels) and spindle microtubules (α-tubulin; green in the top panels). After cold-induced microtubule depolymerization, the kinase still associates with the centrosomes. In the presence of microtubules, CDK11 knockdown (left panels) does not prevent centrosome or spindle microtubule association of Aurora A. In the absence of microtubules, CDK11 RNAi cells fail to accumulate Aurora A kinase at their centrosomes. (D) Efficient recruitment of Plk1 to centrosomes and kinetochores requires CDK11. In control cells, Plk1 (red in the left-hand panels, insets at right are enlargements of the bottom-most centrosome in each corresponding panel) accumulates at the centrosomes and kinetochores as cells progress into metaphase. In CDK11 RNAi cells (right panels), the levels of Plk1 at the centrosomes and kinetochores are markedly reduced (compare control and CDK11 RNAi columns). Scale bar, 10 μm, insets are a × 5 magnification.

Green fluorescent protein (GFP)–CDK11^p58 but not GFP–CDK11^p110 is a centrosome-associated active mitotic kinase. (A) Western blots of cell extracts from HeLa cells and HeLa transgenic cell lines expressing GFP–CDK11^p58 and GFP–CDK11^p110. β-Tubulin is the loading control. (B) Immunoprecipitation (IP) was performed with GFP–CDK11^p58 (left) and GFP–CDK11^p110 (right) cell extracts during interphase (I) or mitosis (M), and the recovered proteins were subjected to a kinase assay. The upper panel shows a CDK11 western blot and the lower panel, the corresponding histone H1 kinase assay. GFP–CDK11^p58 protein kinase is more active during mitosis than interphase, whereas GFP–CDK11^p110 is more active during interphase than mitosis. Specific kinase activity determined is shown in the right panel. (C) Western blots of GFP–CDK11^p58 and GFP–CDK11^p110 cell lines treated with CDK11 short interfering RNA (siRNA). The engineering of silent mutations into each of the GFP–CDK variants makes them resistant to CDK11 RNA interference (RNAi; R), whereas the endogenous proteins are depleted relative to the controls (C). (D) Localization of GFP–CDK11^p58. Cells were fixed and stained with an anti-GFP antibody (green and lower panels), anti-pericentrin antibody (red) and co-stained for DNA (blue). GFP–CDK11^p58 can be detected in the nucleus and cytoplasm during interphase (1). Following mitotic entry (2), GFP–CDK11^p58 is found in the cytoplasm and at the centrosomes. The kinase remains associated with the centrosome during prometaphase (3) and metaphase (4), but is no longer detectable on centrosomes during cytokinesis (5). Scale bar, 10 μm. (E) GFP–CDK11^p58 but not GFP–CDK11^p110 can rescue the CDK11 RNAi phenotype. The different cell lines were treated with CDK11 RNAi (1–3) and the distribution of tubulin (green), γ-tubulin (red) and DNA (blue) was examined. GFP–CDK11^p58 cells depleted of CDK11 (1) were indistinguishable from control cells (4), and formed bipolar spindles. Treatment of GFP–CDK11^p110 cells with CDK11 siRNA (2) resulted in the same short or monopolar spindle phenotype seen in HeLa cells (3). Scale bar, 10 μm.