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Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

Singhmar P, Kumar A - PLoS ONE (2011)

Bottom Line: Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis.The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis.Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, Karnataka, India.

ABSTRACT
Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

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Abnormal cytokinesis and apoptosis in UBE3A knockdown cells.(A) Note cells undergoing cytokinesis with elongated nuclear morphology giving rise to abnormal number of nuclei. An extended midbody (arrowheads) and micronuclei (arrows) can be seen in all the panels. ASPM was found to be diffusely present at the midbody. Scale = 2 µm. (B) Quantitation of apoptosis by in vivo detection of caspase-3 activity. Note UBE3A shRNA clones showed a significant increase in apoptotic cells as compared to scrambled clones. Cells were analyzed by flow cytometry using FL-1 channel (10,000 cells were measured for each sample). Mean ± SEM values for the samples is as follows: scrambled clone P = 6.150±0.2972, scrambled clone K = 4.920±0.0986, UBE3AshRNA clone T = 7.700±0.2663, and UBE3AshRNA clone U = 14.99±0.2929. Data are representative of three independent experiments. Unpaired Student's t-test was used to determine the significance of difference between scrambled and UBE3A knockdown clones.
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pone-0020397-g008: Abnormal cytokinesis and apoptosis in UBE3A knockdown cells.(A) Note cells undergoing cytokinesis with elongated nuclear morphology giving rise to abnormal number of nuclei. An extended midbody (arrowheads) and micronuclei (arrows) can be seen in all the panels. ASPM was found to be diffusely present at the midbody. Scale = 2 µm. (B) Quantitation of apoptosis by in vivo detection of caspase-3 activity. Note UBE3A shRNA clones showed a significant increase in apoptotic cells as compared to scrambled clones. Cells were analyzed by flow cytometry using FL-1 channel (10,000 cells were measured for each sample). Mean ± SEM values for the samples is as follows: scrambled clone P = 6.150±0.2972, scrambled clone K = 4.920±0.0986, UBE3AshRNA clone T = 7.700±0.2663, and UBE3AshRNA clone U = 14.99±0.2929. Data are representative of three independent experiments. Unpaired Student's t-test was used to determine the significance of difference between scrambled and UBE3A knockdown clones.

Mentions: We next examined if chromosome missegregation/lag in UBE3A depleted cells is leading to cytokinesis failure and micronuclei formation. Cytokinesis failure is characterized by an extended nuclear morphology reminiscent of missegregated chromosomes and an elongated midbody [17]. We could notice many of these phenotypes in UBE3A depleted cells (Figure 8A). The scrambled clones P and K displayed normal cytokinesis (Figure 6E). These defects appear when the lagging chromosomes are pushed by the cleavage furrow into either one of the two daughter cells, leading to micronuclei formation and abnormal cytokinesis (Figure 8A). We were unable to score for cytokinesis abnormalities due to low percentage of these cells. This may be due to rescue of the abnormal phenotype or cell death. Malfunctioning of chromosome segregation can generate aneuploid cells and these cells are usually inviable. With this reasoning, we examined the caspase-3 activation in cells to monitor apoptosis. We noticed a significantly higher percentage of caspase-3 positive cells in UBE3A depleted cells as compared to scrambled cells (Figure 8B). For example, the percent of apoptotic cells in UBE3A depleted clone U was 3-fold higher as compared to scrambled clone K (Figure 8B).


Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

Singhmar P, Kumar A - PLoS ONE (2011)

Abnormal cytokinesis and apoptosis in UBE3A knockdown cells.(A) Note cells undergoing cytokinesis with elongated nuclear morphology giving rise to abnormal number of nuclei. An extended midbody (arrowheads) and micronuclei (arrows) can be seen in all the panels. ASPM was found to be diffusely present at the midbody. Scale = 2 µm. (B) Quantitation of apoptosis by in vivo detection of caspase-3 activity. Note UBE3A shRNA clones showed a significant increase in apoptotic cells as compared to scrambled clones. Cells were analyzed by flow cytometry using FL-1 channel (10,000 cells were measured for each sample). Mean ± SEM values for the samples is as follows: scrambled clone P = 6.150±0.2972, scrambled clone K = 4.920±0.0986, UBE3AshRNA clone T = 7.700±0.2663, and UBE3AshRNA clone U = 14.99±0.2929. Data are representative of three independent experiments. Unpaired Student's t-test was used to determine the significance of difference between scrambled and UBE3A knockdown clones.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102111&req=5

pone-0020397-g008: Abnormal cytokinesis and apoptosis in UBE3A knockdown cells.(A) Note cells undergoing cytokinesis with elongated nuclear morphology giving rise to abnormal number of nuclei. An extended midbody (arrowheads) and micronuclei (arrows) can be seen in all the panels. ASPM was found to be diffusely present at the midbody. Scale = 2 µm. (B) Quantitation of apoptosis by in vivo detection of caspase-3 activity. Note UBE3A shRNA clones showed a significant increase in apoptotic cells as compared to scrambled clones. Cells were analyzed by flow cytometry using FL-1 channel (10,000 cells were measured for each sample). Mean ± SEM values for the samples is as follows: scrambled clone P = 6.150±0.2972, scrambled clone K = 4.920±0.0986, UBE3AshRNA clone T = 7.700±0.2663, and UBE3AshRNA clone U = 14.99±0.2929. Data are representative of three independent experiments. Unpaired Student's t-test was used to determine the significance of difference between scrambled and UBE3A knockdown clones.
Mentions: We next examined if chromosome missegregation/lag in UBE3A depleted cells is leading to cytokinesis failure and micronuclei formation. Cytokinesis failure is characterized by an extended nuclear morphology reminiscent of missegregated chromosomes and an elongated midbody [17]. We could notice many of these phenotypes in UBE3A depleted cells (Figure 8A). The scrambled clones P and K displayed normal cytokinesis (Figure 6E). These defects appear when the lagging chromosomes are pushed by the cleavage furrow into either one of the two daughter cells, leading to micronuclei formation and abnormal cytokinesis (Figure 8A). We were unable to score for cytokinesis abnormalities due to low percentage of these cells. This may be due to rescue of the abnormal phenotype or cell death. Malfunctioning of chromosome segregation can generate aneuploid cells and these cells are usually inviable. With this reasoning, we examined the caspase-3 activation in cells to monitor apoptosis. We noticed a significantly higher percentage of caspase-3 positive cells in UBE3A depleted cells as compared to scrambled cells (Figure 8B). For example, the percent of apoptotic cells in UBE3A depleted clone U was 3-fold higher as compared to scrambled clone K (Figure 8B).

Bottom Line: Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis.The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis.Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, Karnataka, India.

ABSTRACT
Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

Show MeSH
Related in: MedlinePlus