Endocrinol Metab.  2015 Mar;30(1):53-57. 10.3803/EnM.2015.30.1.53.

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

• Lee M
• Rhee K

• KMID: 2407104
• DOI: http://doi.org/10.3803/EnM.2015.30.1.53

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.

Keyword

Centrosome; Centrioles; Centrosomal P4.1-associated protein; Ninein; Cell cycle; Microcephaly

MeSH Terms

Brain
Cell Cycle
Centrioles*
Centrosome
Humans
Microcephaly
Mitosis
Mothers*
Spindle Poles

Figure

• 

  Fig. 1 Generation of a ninein polyclonal antibody. (A) Ninein is a 2096-chain amino acid protein. The 381-689 fragment of ninein (solid bar) was used to generate the ninein antibody. (B) HeLa lysates were subjected to immunoblot analysis using the affinity-purified ninein antibody. The estimated size of ninein is approximately 240 kDa.

• 

  Fig. 2 Cell cycle stage-specific localization of ninein at the centrosome. HeLa cells were synchronized with a double thymidine block and release. Cells representative of each stage were coimmunostained with ninein (green) and γ-tubulin (red) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. Insets are magnified views of the centrosomes.

• 

  Fig. 3 Ninein distribution within the centriole. Asynchronous HeLa cells were coimmunostained with centrin-2 (green) and ninein (red) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. Insets are magnified views of the centrosomes. The staining patterns of ninein on centrioles were taken from the right side.

• 

  Fig. 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.

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