Overexpression of CEP72 causes abnormal mitotic spindle assembly

of the mitotic spindle (Vicente & Wordeman 2015). To clarify the effects of enhanced microtubule plus-end assembly during mitosis caused by CEP72 overexpression, the morphology of mitotic spindles was investigated. Cells with a transient overexpression of CEP72 were synchronized in metaphase by double thymidine block and subsequent treatment with the proteasome inhibitor MG132. When cells enter mitosis in the presence of MG132 a normal spindle formation is warranted while the metaphase to anaphase transition is inhibited. Thus, cells were arrested in metaphase and analysed by immunofluorescence microscopy. Cells with a transient overexpression of CEP72 exhibited an aberrant spindle morphology (Figure 3.12 a). While control cells showed normal, ball-like spindles, cells with a CEP72 overexpression exhibited curved and distorted spindle microtubules, which appeared to be longer. The quantification revealed 4.5% curved spindles in control cells, which was increased by CEP72 overexpression to 14.5% (1µg), 15.0% (3 µg) and 14% (5µg) (Figure 3.12 b).

In addition, cells with a transient BRCA1 repression were compared to cells in which CEP72 was transiently overexpressed. To further elucidate, whether the spindle morphology aberrations are caused by increased spindle microtubule plus-end assembly, the cells were simultaneously treated with either DMSO or 0.2 nM Taxol^®. The analysis revealed that also cells with a transient knock down of BRCA1 exhibited curved spindles in 21.3%, which was comparable to 20% of curved spindles in cells overexpressing CEP72 (Figure 3.12 c). Additionally the portion of curved spindles was reduced to 9% in

69 Figure 3.12 The overexpression of CEP72 leads to spindle morphology alterations during mitosis, which is dependent on increased spindle microtubule plus-end assembly. (a) Representative images showing the mitotic spindle morphology of HCT116 cells stably transfected with either an empty vector or a plasmid for CMV promoter driven expression of CEP72. The cells were synchronized in G1/S phase via double thymidine block, released into medium for 6.5 h and arrested in mitosis by treatment with 20 µM MG132 for further 3 h. By immunofluorescence staining the spindles (α-tubulin, green), kinetochores (Crest, red) and the DNA (Hoechst33342, blue) were visualized. Scale bar: 10 µm. (b) Quantification of curved spindles in HCT116 cells after transient CEP72 overexpression at indicated concentrations. Cells were treated as described in (a).

Subsequently the spindle morphology was detected and quantified by immunofluorescence analysis (mean ± s.d.; t-test, n=300 bipolar spindles of three independent experiments). (c) Quantification of curved spindles in HCT116 cells with a transient overexpression of CEP72 or BRCA1 knock down.

The cells were treated either with DMSO or 0.2 nM Taxol^® for 24h prior to immunofluorescence analysis (mean ± s.d.; t-test, n=300 bipolar spindles of three independent experiments).

cells with a repression of BRCA1 and to 8% in CEP72 overexpressing cells by treatment with low dose Taxol^®. In control transfected cells no difference could be detected between cells treated with DMSO (8.3%) and 0.2 nM Taxol^® (8%).

RESULTS

70 Figure 3.13 Cells showing spindle morphology alterations after CEP72 overexpression exhibit an enhanced inter centrosomal distance and an increase in the average microtubule length. (a) In order to determine the pole-to-pole distance of normal and curved spindles in cells transiently overexpressing CEP72, cells were synchronized in G1/S phase via double thymidine block, released into medium for 6.5 h and arrested in mitosis by treatment with 20 µM MG132 for further 3 h. Subsequently, the distance between the centrosomes was measured on the basis of immunofluorescence images (spindles, anti-α-tubulin: green; centromeres,

-tubulin: red; chromosomes, Hoechst 33342: blue; scale bar, 10 μm). The box and whisker plot shows the range, mean and quartile of the measurements (t-test, n=61-64 cells from three independent experiments). (b) Schematic depiction for the determination of the average microtubule length from pole to pole. (c) For the measurement of the average microtubule length from pole to pole of normal and curved spindles in cells with a transient CEP72 overexpression, cells were arrested in mitosis as described in (a). Subsequently the average microtubule length form pole to pole was determined on the basis of immunofluorescence images as depicted in (b).

The box and whisker plot shows the range, mean and quartile of the measurements ( t-test, n=61-64 cells from three independent experiments).

Since the aberrant spindles appear to be longer when compared to control ones, the distance between the centrosomes was determined in proper spindles of control and CEP72 overexpressing cells and compared to the pole-to-pole distance of spindles with a curved morphology. The measurement revealed an inter-centrosomal distance of 11.1 µm for normal spindles of control transfected cells (Figure 3.13 a). Compared to this, no difference in the pole-to-pole distance of proper spindles in cells overexpressing CEP72 (11.3 µm) was detectable. However, curved spindles in control and CEP72 overexpressing cells showed a significantly increased pole-to-pole distance of 14.6 µm and 15.0 µm, respectively.

Furthermore, the length of microtubules from pole to pole was measured following the curved morphology. For this the microtubule length was determined at both outer sides as well as in the middle of the spindle as depicted in Figure 3.13 b. Subsequently, the average microtubule length from pole to pole was calculated. Whereas normal spindles showed an average microtubule length of 14.4 µm in control and 14.7 µm in CEP72

71 overexpressing cells, the microtubule length from pole to pole of curved spindles was enhanced to 17.2 µm and 18.0 µm, respectively (Figure 3.13 c)

These data indicate that a CEP72 overexpression or repression of BRCA1 lead to aberrations of the mitotic spindle morphology, which is characterized by a curved and distorted phenotype, an enhanced inter-centrosomal distance and elongated microtubules. Additionally these abnormal spindles are dependent on increased spindle microtubule plus-end assembly rates.

3.2.4 Overexpression of CEP72 causes the generation of lagging chromosomes