CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

<div><p>Integrity of the microtubule spindle apparatus and intact cell division checkpoints are essential to ensure the fidelity of distributing chromosomes into daughter cells. Cytoskeleton-associated protein 2, CKAP2, is a microtubule-associated protein that localizes to spindle poles and aids in microtubule stabilization, but the exact function and mechanism of action are poorly understood. In the present study, we utilized RNA interference to determine the extent to which the expression of CKAP2 plays a role in chromosome segregation. CKAP2-depleted cells showed a significant increase of multipolar mitoses and other spindle pole defects. Notably, when interrogated for microtubule nucleation capacity, CKAP2-depleted cells showed a very unusual phenotype as early as two minutes after release from mitotic block, consisting of dispersal of newly polymerized microtubule filaments through the entire chromatin region, creating a cage-like structure. Nevertheless, spindle poles were formed after one hour of mitotic release suggesting that centrosome-mediated nucleation remained dominant. Finally, we showed that suppression of CKAP2 resulted in a higher incidence of merotelic attachments, anaphase lagging, and polyploidy. Based on these results, we conclude that CKAP2 is involved in the maintenance of microtubule nucleation sites, focusing microtubule minus ends to the spindle poles in early mitosis, and is implicated in maintaining genome stability.</p></div

CKAP2 is required for anchoring of centrosome-nucleated microtubules to the spindle pole.

<p>(A) Nucleation was assessed after treating cells with 10 µg/ml nocodazole for 30 minutes and released into fresh media for 2, 30, and 60 minutes. shCTL and CKAP2-depleted cells were co-immunostained with γ-tubulin (green), Tyr-tubulin (red), and merged with DAPI (blue). (B) Two minutes post-nocodazole release, a cage-like structure was often observed in CKAP2-depleted cells. Representative images for each experimental group are shown. (C) Thirty minutes post-nocodazole release, microtubules are tethered at distinct poles, often with an increase cells with multipolar spindle poles in CKAP2-depleted cells. Representative images for each experimental group are shown. (D) Sixty minutes post nocodazole block, both the control and CKAP2-depleted cells have structured bipolar assembly. Representative images for each experimental group are shown. (E) Quantification of the shCTL cells with non-centrosomal α-tubulin staining at 2, 30, and 60 minutes is shown. Approximately 50 cells were counted per condition. (F) Measurements of the total γ-tubulin in both the centrosomes and the spindle pole area for CKAP2-depleted cells and controls. Y-axis indicates signal intensity units for γ-tubulin.</p

CKAP2-depleted cells show increased chromosome missegregation.

<p>(A) Control (shCTL) and CKAP2-depleted (shCKAP2) cells were transfected with histone H2B-Cherry constructs, selected with geneticin (G418), and analyzed with live-cell imaging. The movie shows CKAP2-depleted histone H2B-Cherry positive cells undergoing aberrant mitosis with chromosome missegregation resulting in two daughter nuclei with micronuclei. Arrows indicate lagging chromosomes and resultant micronuclei. (B) CKAP2-depleted cells were immunostained for Hec1 (green), α-tubulin (red), and merged with DAPI (blue). Cells with lagging chromosomes in anaphase and telophase were analyzed for merotelic attachments. Representative images of lagging chromosomes in anaphase and telophase are shown here. Magnified views emphasize merotelic attachments in lagging chromosomes. (C) The histogram represents the number of chromosome missegregation events for each histone H2B-Cherry positive experimental group. (D) Asynchronous shCTL and CKAP2-depleted cells were analyzed for evidence of chromosome missegregation, including micronuclei, nuclear blebs, and anaphase bridges. The results are plotted as the mean ± SD.</p

Centrosome nucleation capacity is unaffected in CKAP2-depleted cells.

<p>(A) Two minutes post-nocodazole release shCTL and CKAP2-depleted cells were co-immunostained with α-tubulin (DM1A) (green), pericentrin (red), and merged with DAPI (blue). One hundred cells with non-centrosomal tubulin staining were measured per experimental group. As already demonstrated, a cage-like structure was observed two minutes post-nocodazole release. Representative images for each experimental group are shown. (B) Nucleation capacity was determined by measuring the mean of α-tubulin fluorescence for both the control and CKAP2-depleted cells.</p

Spindle pole defects in CKAP2-depleted cells.

<p>(A) Mitosis in an asynchronous population of control (shCTL) and CKAP2-depleted (shCKAP2) cells were co-immunostained with γ-tubulin (green), α-tubulin (red), and merged with DAPI (blue). The dispersal of γ-tubulin away from the centrosome and dislocation of the centrosome from the spindle pole was analyzed in 200 cells per experimental group in two independent experiments. Representative images for each experimental group and the mitotic defect are shown (Scale bar: 2 µm). Representative images for each experimental group are shown. (B) Quantification of the cells with dispersed γ-tubulin was presented as mean ± SD. P-values were determined using the Student’s t-test. (C) Quantification of the cells with the centrosome dislocated from the spindle pole was presented as mean ± SD. P-values were determined using the Student’s t-test (D) Spindle length measured in 50 mitotic cells with bipolar spindles in both controls and CKAP2-depleted cells is shown. P-value was determined by Student’s t-test. (E) Analysis of the number of misaligned chromosomes in bipolar metaphases shows statistical significant difference between control and CKAP2-depleted cells. More than 200 cells were counted per condition. P-value was determined by Student’s t-test.</p

NuMA expression and localization is not affected by CKAP2-depletion.

<p>(A) shCTL and shCKAP2 transfected cells were co-immunostained with NuMA (green) and α-tubulin (red), and merged with DAPI (blue). The expression of NuMA was confined to the spindle pole. Representative metaphase images show that the localization of NuMA remains intact. (B) Immunoblot analysis with antibodies specific for NuMA and GAPDH showed that the amount of NuMA protein was maintained despite the silencing of CKAP2. (C) shCTL and CKAP2-depleted cells were co-immunostained with NuMA (green), CKAP2 (red), and merged with DAPI (blue) showing only partial overlay between the two protein although they both are located at the spindle pole. (D) shCTL and CKAP2-depleted cells were synchronized with nocodazole, and after two minutes post release cells were co-immunostained with NuMA (green) and α-tubulin (red). Co-localization of NuMA and α-tubulin is shown in the cage-like structures in CKAP2-depleted cells, but not in control cells.</p

Localization of CKAP2 at the spindle pole in human colorectal cancer cell line DLD-1.

<p>(A) DLD-1 cells co-immunostained with CKAP2 (green), α-tubulin (red), and DAPI (blue) depicting localization of CKAP2 to the spindle pole (Scale bar: 2 µm) (B) DLD-1 cells co-immunostained with CKAP2 (red), γ-tubulin (green) and DAPI (blue) demonstrating CKAP2 does not localize within the centrosome (Scale bar: 2 µm). (C) Mitotic cells were enriched by mitotic shake-off, lysed in hypotonic buffer, and the lysate fractionated by centrifugation. The pellet contains DNA, microtubules, and microtubule-associated proteins, whereas the supernatant contains the remaining proteins. These fractions were analyzed by immunoblot with antibodies specific to CKAP2, α/γ-tubulin, and γ-tubulin. The presence of CKAP2 in the pellet in both wild-type and nocodazole treated cells suggests that CKAP2 is indirectly associated with the mitotic spindle. (D) DLD-1 cells were transfected with shRNA, selected with puromycin, and single-cell separated by FACS based on GFP-positivity. Separated cells were synchronized overnight with nocodazole and harvested for immunoblot analysis with antibodies specific for CKAP2 and GAPDH. (E) To measure the affect of CKAP2 reduction on cell proliferation, populations of control (shCTL) and CKAP2-depleted cells (shCKAP2) were counted for six days and plotted. No significant differences in growth activity are observed.</p

CKAP2 plays a role in maintaining chromosome stability.

<p>(A–C) Mitotic cells were treated with colcemid in order to obtain metaphase spreads. Chromosome content was determined by counting the individual chromosomes in at least 100 metaphases. The results are presented as radial plots, where the concentric circle represents the relative ploidy and each symbol represents an individual cell. In parallel, here indicated are karyotypes analyzed by SKY showing the increased level of aneuploidy and chromosome instability in CKAP2-depleted cells.</p

Telomerase Variant A279T Induces Telomere Dysfunction and Inhibits Non-Canonical Telomerase Activity in Esophageal Carcinomas

<div><p>Background</p><p>Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas.</p><p>Methods</p><p>Sequencing techniques were used to evaluate mutational status of <i>telomerase reverse transcriptase (TERT)</i> and <i>telomerase RNA component (TERC)</i> in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability.</p><p>Results</p><p>Sequencing analysis revealed one deletion involving <i>TERC (TERC del 341-360)</i>, and two non-synonymous <i>TERT</i> variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage.</p><p>Conclusions</p><p>A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.</p></div

Gene expression levels of A279T-transduced cells normalized to wtTERT-transduced cells.

<p>*ND: Not detected.</p