Figure 5. Expression of the D933N and K1355E variants of CENP-E results in mitotic spindle abnormalities and polar chromosomes.

(a). DLD-1 cells engineered by Flp-In recombination for the conditional (doxycycline-induced) expression of siRNA-resistant CENPE and CENPE patient variants, were plated and 24hrs later subjected to CENPE siRNA targeting endogenous CENP-E only. After 40hrs approx. the cells were treated with doxycycline (Dox; 1µg/ml) for 8hrs to induce expression of the siRNA-resistant CENPE variants. Cells were then fixed and processed for immunofluorescence.

(b). Expression levels of the various CENP-E variants including a construct with both variants engineered in-cis (Double mut; double mutant) were compared to that of wild-type (WT) CENP-E following 8hrs in doxycycline.

(c). Immunofluoresence images following 8hrs treatment with doxycycline for wild-type (WT), D933N alone, K1355E alone and the double mutant (Double mut) with both D933N and K1355E in-cis co-stained with anti-CENP-E and anti-α-tubulin. Polar chromosomes not aligned properly stain strongly for CENP-E under these conditions as seen in D933N alone, K1355E alone and the double mutant. (Scale bar; 5µm).

(d). The frequency of mitotic cells with >5 unaligned polar chromosomes is elevated following expression of D933N alone, K1355E alone or following expression of both variants in-cis (Double mut). Between 70–90 mitotic cells were evaluated in total for each construct.

(e). Expression of D933N alone, K1355E alone or both variants in-cis (Double mut) resulted in an increased frequency of mitotic cells with abnormal spindle structure (monopolar plus multipolar spindles) compared to expression of wild-type (WT) CENP-E in this system (n= the number of mitotic cells scored).