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. 2008 Jan 28;180(2):315-24.
doi: 10.1083/jcb.200706083.

"VSports在线直播" Loss of ATRX leads to chromosome cohesion and congression defects

Kieran Ritchie  1 , Claudia Seah, Jana Moulin, Christian Isaac, Frederick Dick, Nathalie G Bérubé
Affiliations

Loss of ATRX leads to chromosome cohesion and congression defects

Kieran Ritchie et al. J Cell Biol. .

Abstract

Alpha thalassemia/mental retardation X linked (ATRX) is a switch/sucrose nonfermenting-type ATPase localized at pericentromeric heterochromatin in mouse and human cells. Human ATRX mutations give rise to mental retardation syndromes characterized by developmental delay, facial dysmorphisms, cognitive deficits, and microcephaly and the loss of ATRX in the mouse brain leads to reduced cortical size VSports手机版. We find that ATRX is required for normal mitotic progression in human cultured cells and in neuroprogenitors. Using live cell imaging, we show that the transition from prometaphase to metaphase is prolonged in ATRX-depleted cells and is accompanied by defective sister chromatid cohesion and congression at the metaphase plate. We also demonstrate that loss of ATRX in the embryonic mouse brain induces mitotic defects in neuroprogenitors in vivo with evidence of abnormal chromosome congression and segregation. These findings reveal that ATRX contributes to chromosome dynamics during mitosis and provide a possible cellular explanation for reduced cortical size and abnormal brain development associated with ATRX deficiency. .

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Figures

Figure 1.
Figure 1.
Transient ATRX depletion in HeLa cells induces abnormal nuclear morphology. (A) Immunofluorescence detection of ATRX (green) and the kinetochore marker CREST (red) on prometaphase and metaphase chromosomes. (B) Western blot analysis of HeLa cells transiently transfected with siATRX 19-mer duplexes demonstrate ATRX protein depletion starting at 48 h and up to 96 h after transfection. ATRX protein levels remained constant upon mock transfection and in cells transfected with a control siATRX scrambled sequence duplex (left). Both ATRX isoforms were effectively silenced by 48 h after transfection (right). α-Tubulin protein expression (αTub) was used as a control in all experiments. Numbers on top of the blot indicate hours after transfection and numbers in parenthesis indicate the molecular mass in kD. (C) Costaining of kinetochores and ATRX with the CREST and 39f antibodies, respectively, reveals loss of ATRX protein at PCH in siRNA-treated cells. (D) Cells stained with DAPI show abnormal nuclear morphology in ATRX-depleted cells compared with control cells. Common defects include lobulated nuclei and intranuclear DNA bridges (right). (E) The number of nuclei displaying abnormalities was quantified at 48, 72, and 96 h after siRNA treatment (n > 1,000 nuclei at each time point). Bars: (A and C) 5 μm; (D) 20 μm.
Figure 2.
Figure 2.
Stable and transient depletion of ATRX extends the transition to metaphase and induces congression defects. (A) HeLa-HG cells that stably express pSuper-shATRX1, pSuper-shATRX2, or pSuper (empty vector control) were generated and the level of ATRX depletion was measured by Q-RT-PCR (top) and Western blot analysis (bottom). Q-RT-PCR results were normalized to GAPDH expression and protein loading on the Western blot was controlled with α-tubulin. Error bars represent the standard deviation from triplicate samples. (B) Mitotic cells were followed in real time by videomicroscopy and the duration of prometaphase was measured for HG control and HG-shATRX1 stably depleted cells (n = 50 each) and also in transient transfections with siATRX1 (n > 120). Horizontal lines indicate the mean value of each dataset. (C) The fraction of metaphasic cells with misaligned chromosomes (inset) was evaluated in live cell cultures of HeLa-HG, HG-pSuper, HG-shATRX1, and HG-shATRX2 stable clones (n = 100 for each). (D) Live mitotic cells were followed over a 10 h period and scored for congression defects in stable (n = 50) and transient experiments (n > 120). (E) Selected panels from live cell videomicroscopy experiments of control HeLa-HG cells and ATRX-depleted cells from nuclear envelope breakdown (NEBD) to anaphase onset (AO). ATRX-depleted mitotic cells often displayed misaligned chromosomes (arrowheads) that resolved before the onset of anaphase (middle), whereas, in some cases, anaphase was initiated despite the presence of misaligned chromosomes (bottom). A subset of metaphase chromosomes underwent cycles of general decondensation and recondensation (the asterisk indicates a decondensed metaphase plate). Numbers indicate minutes after NEBD. (F) Morphological assessment of chromosome decondensation in control and transiently depleted cells. Graph depicts the percentage of metaphase spreads that display a more decondensed appearance (far right). Bars: (C) 5 μm; (E) 16 μm, (F) 20 μm.
Figure 3.
Figure 3.
Increased interkinetochore distances and reduced cohesion in ATRX-depleted cells. (A) ATRX-depleted metaphase cells were stained for the kinetochore proteins CENP-F or CENP-E (green) in combination with CREST (red) and imaged at different z planes. Images represent an extended focus rendering of deconvoluted z stacks. (B) Control and ATRX-depleted cells were stained with anti-CREST antibody to stain kinetochores. Distances between paired kinetochores (n = 100) were measured at individual z planes and were significantly increased in shATRX1 and shATRX2 cells compared with controls (P < 0.05 by ANOVA). (C) Mitotic HeLa cells were transiently transfected with either siATRX-Scr control or siATRX1 and siATRX2 duplexes. After mitotic shake-off to remove any cells in mitosis, cells were treated with colcemid and chromosome spreads were stained with DAPI and scored for the percentage of chromatids displaying cohesion defects (n = 50 per treatment). Chromosomes from ATRX-depleted cells showed reduced cohesion compared with control-treated cells. (D) Microscopic images of representative chromosome spreads scored as normal (<10% cohesion defect), moderate (10–90% cohesion defect), or severe (>90% cohesion defect). Insets show representative chromosomes from each spread with normal (left), moderate (middle), and severe (right) cohesion defects. Bars: (A and B) 5 μm; (C) 20 μm.
Figure 4.
Figure 4.
Spindle checkpoint activation and aberrant chromosome segregation in ATRX-depleted cells. (A) The spindle checkpoint protein BubR1 was detected in ATRX-depleted metaphase cells at both aligned and misaligned chromosomes (top). Another checkpoint protein, Bub1, was also found to be adjacent to the centromeres (CREST staining) in ATRX-depleted metaphase cells. (B) Mitotic index of control and ATRX-depleted cells after 16 h of nocodazole treatment. Control and ATRX-depleted cells were cultured with or without nocodazole for 16 h and live cells were photographed and scored for the percentage of mitotic rounded cells by phase imaging (n > 1,000). (C) Live cell imaging of control and ATRX-depleted mitotic cells (n = 100 for each cell line) revealed an increased number of cells that undergo repeated failed attempts at initiating anaphase, an increased number of cells that initiate anaphase without full chromosome alignment, and an increased incidence of segregation defects as indicated by the formation of chromosome DNA bridges and micronuclei. (D) The number of internuclear bridges and micronuclei indicative of chromosome missegregation were quantified at 2–4 d after transfection of the siATRX1 duplex and the fold difference between mock and depleted cells was calculated. (E) Fixed mitotic cells stained with DAPI (blue) and a phosphohistone H3S10 antibody (red) showing intranuclear DNA bridges and micronuclei in transiently depleted cells. Bars, 5 μm.
Figure 5.
Figure 5.
ATRX association with mitotic chromosomes and evidence of mitotic defects in ATRX-deficient neuroprogenitors in vivo. (A) ATRX staining of the cortex at E13.5. ATRX is highly expressed in all cells but is highest in the cortical hem (CH), which gives rise to hippocampal structures. ATRX is also expressed at increased levels in the differentiated neurons of developing cortical plate (CP). Mitotic cells that line the lateral ventricle are highly enriched for ATRX protein (arrows). Higher magnification of the cortical hem (right) demonstrates ATRX staining of mitotic chromosomes in cells that line the lateral ventricle (arrow). Punctate nuclear staining of ATRX in cycling cells of the ventricular zone (VZ) is characteristic of ATRX localization at PCH. (B) Cryosections obtained from control and ATRX null telencephalon were stained with DAPI to visualize mitotic chromosomes lining the lateral ventricle (LV) at E13.5. An increased incidence of micronuclei or dispersed chromosomes was detected in the vicinity of the mitotic layer (arrows) in the ATRX null embryonic brain. (C) The mean number of micronuclei or dispersed chromosomes were scored in the mitotic layer from point 1 to 2 as indicated by the white arrows (top) and are represented in the graph below (n = 4 for each control and ATRX null brain; P < 0.0001 by nonpaired t test). Error bars represent the standard deviation of counts from a total of 24 cortical hemisphere from a total of four brains. Cn, cortical neuroepithelium; Hh, hippocampal hem; Hp, hippocampal primordium. Bars: (A) 100 μm; (B) 40 μm; (C) 200 μm.
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