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Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody.

Lee M, Rhee K - Endocrinol Metab (Seoul) (2014)

Bottom Line: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.Ninein signals were significantly impaired in CPAP-depleted cells.The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis.

Methods: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.

Results: Ninein signals were significantly impaired in CPAP-depleted cells.

Conclusion: The results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

No MeSH data available.


Related in: MedlinePlus

Centrosomal ninein distribution in centrosomal P4.1-associated protein (CPAP)-depleted mitotic cells. (A) The cell cycle of CPAP-depleted cells was synchronized during the G2 phase using a double thymidine block and release. The cells were placed on ice for 90 minutes to disrupt microtubules, and coimmunostained with acetylated-tubulin (red), cyclin B1 (red), and ninein (green) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. (B) Ninein staining patterns at the spindle poles were categorized into four groups: paired ring; ring with a bar; single ring; and disrupted. More than 300 cells were analyzed across three independent experiments. The data are means±SE. aP<0.05 was considered statistically significant compared to the controls.
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Figure 4: Centrosomal ninein distribution in centrosomal P4.1-associated protein (CPAP)-depleted mitotic cells. (A) The cell cycle of CPAP-depleted cells was synchronized during the G2 phase using a double thymidine block and release. The cells were placed on ice for 90 minutes to disrupt microtubules, and coimmunostained with acetylated-tubulin (red), cyclin B1 (red), and ninein (green) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. (B) Ninein staining patterns at the spindle poles were categorized into four groups: paired ring; ring with a bar; single ring; and disrupted. More than 300 cells were analyzed across three independent experiments. The data are means±SE. aP<0.05 was considered statistically significant compared to the controls.

Mentions: CPAP is an essential component of centriole assembly, because CPAP depletion results in defects in centriole duplication [10]. We previously proposed that CPAP is also critical for the maturation of mother centrioles [6]. Therefore, we immunostained CPAP-depleted cells with the ninein antibody to determine the structure of mature mother centrioles. Ninein signals were detected in cyclinB1-positive G2 cells, which became weaker and dispersed in M phase cells [11,12]. The majority of the control cells possessed two ninein rings corresponding to two mother centrioles (Fig. 4A). In CPAP-depleted cells, however, the number of cells with two intact ninein rings was significantly reduced (Fig. 4A); more than 30% of the CPAP-depleted cells included an intact ninein ring with a dot representing an immature centriole appendage (Fig. 4B). These results support the notion that CPAP is required for maturation of the young mother centriole.


Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody.

Lee M, Rhee K - Endocrinol Metab (Seoul) (2014)

Centrosomal ninein distribution in centrosomal P4.1-associated protein (CPAP)-depleted mitotic cells. (A) The cell cycle of CPAP-depleted cells was synchronized during the G2 phase using a double thymidine block and release. The cells were placed on ice for 90 minutes to disrupt microtubules, and coimmunostained with acetylated-tubulin (red), cyclin B1 (red), and ninein (green) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. (B) Ninein staining patterns at the spindle poles were categorized into four groups: paired ring; ring with a bar; single ring; and disrupted. More than 300 cells were analyzed across three independent experiments. The data are means±SE. aP<0.05 was considered statistically significant compared to the controls.
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Related In: Results  -  Collection

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Figure 4: Centrosomal ninein distribution in centrosomal P4.1-associated protein (CPAP)-depleted mitotic cells. (A) The cell cycle of CPAP-depleted cells was synchronized during the G2 phase using a double thymidine block and release. The cells were placed on ice for 90 minutes to disrupt microtubules, and coimmunostained with acetylated-tubulin (red), cyclin B1 (red), and ninein (green) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. (B) Ninein staining patterns at the spindle poles were categorized into four groups: paired ring; ring with a bar; single ring; and disrupted. More than 300 cells were analyzed across three independent experiments. The data are means±SE. aP<0.05 was considered statistically significant compared to the controls.
Mentions: CPAP is an essential component of centriole assembly, because CPAP depletion results in defects in centriole duplication [10]. We previously proposed that CPAP is also critical for the maturation of mother centrioles [6]. Therefore, we immunostained CPAP-depleted cells with the ninein antibody to determine the structure of mature mother centrioles. Ninein signals were detected in cyclinB1-positive G2 cells, which became weaker and dispersed in M phase cells [11,12]. The majority of the control cells possessed two ninein rings corresponding to two mother centrioles (Fig. 4A). In CPAP-depleted cells, however, the number of cells with two intact ninein rings was significantly reduced (Fig. 4A); more than 30% of the CPAP-depleted cells included an intact ninein ring with a dot representing an immature centriole appendage (Fig. 4B). These results support the notion that CPAP is required for maturation of the young mother centriole.

Bottom Line: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.Ninein signals were significantly impaired in CPAP-depleted cells.The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis.

Methods: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.

Results: Ninein signals were significantly impaired in CPAP-depleted cells.

Conclusion: The results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

No MeSH data available.


Related in: MedlinePlus