Loss of centrioles causes chromosomal instability in vertebrate somatic cells

Abstract

Most animal cells contain a centrosome, which comprises a pair of centrioles surrounded by an ordered pericentriolar matrix (PCM). Although the role of this organelle in organizing the mitotic spindle poles is well established, its precise contribution to cell division and cell survival remains a subject of debate. By genetically ablating key components of centriole biogenesis in chicken DT40 B cells, we generated multiple cell lines that lack centrioles. PCM components accumulated in acentriolar microtubule (MT)-organizing centers but failed to adopt a higher-order structure, as shown by three-dimensional structured illumination microscopy. Cells without centrioles exhibited both a delay in bipolar spindle assembly and a high rate of chromosomal instability. Collectively, our results expose a vital role for centrosomes in establishing a mitotic spindle geometry that facilitates correct kinetochore-MT attachments. We propose that centrosomes are essential in organisms in which rapid segregation of a large number of chromosomes needs to be attained with fidelity.

Figure 1.

CEP152-KO and STIL-KO DT40 cells lack intact centrioles. (A–G) TEM of WT, CEP152-KO, and STIL-KO cells. Arrowheads indicate centriolar satellites. Bars, 100 nm. (A) A centriole pair is illustrated in WT. (B and C) CEP152-KO cells contain electron-dense structures reminiscent of partial centriole walls. Insets in A and C show a cross section of triplet MTs at high magnification. (D–F) STIL-KO cells lack centriolar structures and contain only aMTOCs. (F) Mitotic spindle pole in STIL-KO. (G) Table depicts quantification of the major phenotypes observed by TEM. Asterisks indicate electron-dense structures that can potentially correspond to centrioles.

Figure 2.

aMTOCs in KO cells are disordered and unable to overduplicate. (A) CEP152-KO and STIL-KO cells contain cytoplasmic foci enriched in γ-tubulin and CDK5RAP2. Triangles indicate mitotic cells. Asterisks and arrowheads indicate cells with undetectable or weak PCM signal, respectively. DNA is shown in blue. Bars, 5 µm. (B) 3D-SIM images of interphase and mitotic centrosomes stained with antibodies against γ-tubulin and CDK5RAP2 are shown. Bars, 0.5 µm. (C) Centrosome amplification during aphidicolin (APH)-induced arrest. Cells were treated with DMSO or aphidicolin for 24 h. Graphs show the percentages of cells without centrosome (0), one to two centrosomes (1–2), or more than two centrosomes (>2). Centrosomes are defined as γ-tubulin–positive foci. Although ∼10% CEP152-KO cells show centrosome amplification, unlike WT, these contained up to three foci. Error bars are means ± SEM. (D) Representative fields of aphidicolin-treated cells corresponding to C. Cells were stained with antibodies against γ-tubulin and the spindle pole protein TACC3. DNA is shown in blue. Bars, 10 µm.

Figure 3.

Cells lacking intact centrioles proliferate slowly but are proficient in DNA repair. (A) CEP152-KO and STIL-KO cells display growth impairment. (B) KO cells show increased apoptosis compared with WT. n = 4. Error bars indicate SEMs, analysis of variance, Tukey’s test. (C) Quantification of cell cycle profiles by FACS analysis with propidium Iodide (PI) and anti-BrdU antibodies, with >15,000 events per genotype. Percentages of cells per each cell cycle stage are indicated. (D) Exponential one phase decay graph illustrates clonogenic survival of WT, CEP152-KO, and STIL-KO cells after genotoxic treatments. The percentage of colony formation was normalized against untreated controls of the same genotype. Cells were treated with indicated doses of cisplatin for 1.5 h or irradiation (IR). Error bars indicate SDs.

Figure 4.

Bipolar spindle formation and anaphase onset are delayed in cells lacking intact centrioles. (A) CEP152-KO and STIL-KO cells contain disorganized mitotic spindles. Cells were stained with antibodies against CDK5RAP2 and α-tubulin. DNA is shown in blue. Asterisks mark cells with disorganized spindles. (B) Unfocused MTs contact kinetochores in STIL-KO cells. Cells were stained with antibodies against the kinetochore protein, CENP-O, and α-tubulin. DNA is shown in blue. A single focal plane is shown along with higher magnification of framed area. (C) Graphs show the percentage of mitotic cells with disorganized spindles in CEP152-KO and STIL-KO (n = 3, >165 cells per genotype per experiment; means ± SD). (D) Still frames from time-lapse experiments show GFP-tubulin–expressing WT, CEP152-KO, and STIL-KO cells (Videos 1, 2, and 3). NEBD marks the first frame after NEBD. Times are given in hours, minutes, and seconds. (E) Graph shows distribution of time intervals from NEBD to anaphase onset. Medians and interquartile ranges are indicated, from Kruskal–Wallis and Dunn’s test. (F) MT regrowth in mitotic cells after depolymerization. Note the absence of MTs at 0 min and enrichment of MTs near chromatin in STIL-KO cells after 3 min of regrowth. Cells were stained with antibodies against CDK5RAP2 and α-tubulin. Bars, 5 µm.

Figure 5.

CEP152-KO and STIL-KO cells exhibit chromosome instability and aneuploidy. (A) Still frames from time-lapse experiments show H2B-GFP–expressing WT, CEP152-KO, and STIL-KO cells (Videos 4, 5, and 6). Lagging chromatids (red arrowheads) or larger chromatin masses (yellow arrowheads) are evident during anaphase in KO cells. Pictures with blue asterisks are shown with increased gain setting in the framed area. Bars, 2 µm. (B) Table summarizes chromosome missegregation phenotypes from time-lapse experiments. Overlays of H2B-GFP and bright-field images were analyzed. (C) Lagging chromatids in anaphase cells show merotelic attachments. Cells were stained with antibodies against CENP-O and α-tubulin. DNA is shown in blue. Framed areas are shown at higher magnification. Bars, 5 µm. (D) Chromosome (chr) spreads of WT and CEP152-KO cells. WT cells show normal ploidy. CEP152-KO image on the left illustrates CA (trisomy of chromosome 3 but normal copy number of chromosomes 1, 2, 4, and Z) and, on the right, CIN (trisomy of chromosome 4 but normal copy number of chromosomes 1, 2, 3, and Z). Pie charts on the bottom depict the percentage of cells with indicated ploidies. Numbers of chromosomes 1–4 and Z were scored. Bar, 8 µm.