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Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication.

Zou C, Li J, Bai Y, Gunning WT, Wazer DE, Band V, Gao Q - J. Cell Biol. (2005)

Bottom Line: In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole.The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication.Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Cancer Biology, Evanston Northwestern Healthcare Research Institute, Northwestern University Feinberg School of Medicine, Evanston, IL 60201, USA.

ABSTRACT
In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

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Centrobin expression, amino acid sequence, and structure. (A and B) A tissue blot with 2 μg of polyA mRNA per lane (A) and a blot with 20 μg of total mRNA from the indicated breast cell lines (B) were probed with a 32P-labeled centrobin probe followed by autoradiography. Hybridization with the 36B4 probe was used as a loading control. (C) The predicted amino acid sequence of centrobin-α. (D) The extra 22 residues presented only in centrobin-β. (E) Coiled–coil regions of centrobin, ninein, and pericentrin as predicted by DNASTAR. Boxes indicate the coiled–coil region; lines indicate noncoiled regions. Centrobin-C, the COOH-terminal fragment of centrobin that was isolated from the yeast two-hybrid screen. (F) Centrobin protein expression. Cell lysate from the indicated cell lines or 293T cells transfected with pCR3.1 vector, Myc-centrobin, or GFP-centrobin constructs were fractionated by a 6% SDS-PAGE and blotted with affinity-purified anti-centrobin or anti-Myc antibodies.
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fig1: Centrobin expression, amino acid sequence, and structure. (A and B) A tissue blot with 2 μg of polyA mRNA per lane (A) and a blot with 20 μg of total mRNA from the indicated breast cell lines (B) were probed with a 32P-labeled centrobin probe followed by autoradiography. Hybridization with the 36B4 probe was used as a loading control. (C) The predicted amino acid sequence of centrobin-α. (D) The extra 22 residues presented only in centrobin-β. (E) Coiled–coil regions of centrobin, ninein, and pericentrin as predicted by DNASTAR. Boxes indicate the coiled–coil region; lines indicate noncoiled regions. Centrobin-C, the COOH-terminal fragment of centrobin that was isolated from the yeast two-hybrid screen. (F) Centrobin protein expression. Cell lysate from the indicated cell lines or 293T cells transfected with pCR3.1 vector, Myc-centrobin, or GFP-centrobin constructs were fractionated by a 6% SDS-PAGE and blotted with affinity-purified anti-centrobin or anti-Myc antibodies.

Mentions: We identified centrobin in a yeast two-hybrid screening with the conserved COOH-terminal 1,026 residues of BRCA2 as bait. The yeast two-hybrid screening was performed as described previously (Gao et al., 1999, 2000). One set of 11 positive clones encoded the COOH-terminal 539 aa and the 3′-untranslated region of centrobin (Fig. 1, C and E). Northern blotting revealed a single centrobin mRNA transcript of ∼3.8 kb expressed in most human tissues and all of the cell lines tested, although the levels varied (Fig. 1, A and B). The basis and significance of the interaction of centrobin with BRCA2 is currently under investigation and is not discussed here.


Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication.

Zou C, Li J, Bai Y, Gunning WT, Wazer DE, Band V, Gao Q - J. Cell Biol. (2005)

Centrobin expression, amino acid sequence, and structure. (A and B) A tissue blot with 2 μg of polyA mRNA per lane (A) and a blot with 20 μg of total mRNA from the indicated breast cell lines (B) were probed with a 32P-labeled centrobin probe followed by autoradiography. Hybridization with the 36B4 probe was used as a loading control. (C) The predicted amino acid sequence of centrobin-α. (D) The extra 22 residues presented only in centrobin-β. (E) Coiled–coil regions of centrobin, ninein, and pericentrin as predicted by DNASTAR. Boxes indicate the coiled–coil region; lines indicate noncoiled regions. Centrobin-C, the COOH-terminal fragment of centrobin that was isolated from the yeast two-hybrid screen. (F) Centrobin protein expression. Cell lysate from the indicated cell lines or 293T cells transfected with pCR3.1 vector, Myc-centrobin, or GFP-centrobin constructs were fractionated by a 6% SDS-PAGE and blotted with affinity-purified anti-centrobin or anti-Myc antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171251&req=5

fig1: Centrobin expression, amino acid sequence, and structure. (A and B) A tissue blot with 2 μg of polyA mRNA per lane (A) and a blot with 20 μg of total mRNA from the indicated breast cell lines (B) were probed with a 32P-labeled centrobin probe followed by autoradiography. Hybridization with the 36B4 probe was used as a loading control. (C) The predicted amino acid sequence of centrobin-α. (D) The extra 22 residues presented only in centrobin-β. (E) Coiled–coil regions of centrobin, ninein, and pericentrin as predicted by DNASTAR. Boxes indicate the coiled–coil region; lines indicate noncoiled regions. Centrobin-C, the COOH-terminal fragment of centrobin that was isolated from the yeast two-hybrid screen. (F) Centrobin protein expression. Cell lysate from the indicated cell lines or 293T cells transfected with pCR3.1 vector, Myc-centrobin, or GFP-centrobin constructs were fractionated by a 6% SDS-PAGE and blotted with affinity-purified anti-centrobin or anti-Myc antibodies.
Mentions: We identified centrobin in a yeast two-hybrid screening with the conserved COOH-terminal 1,026 residues of BRCA2 as bait. The yeast two-hybrid screening was performed as described previously (Gao et al., 1999, 2000). One set of 11 positive clones encoded the COOH-terminal 539 aa and the 3′-untranslated region of centrobin (Fig. 1, C and E). Northern blotting revealed a single centrobin mRNA transcript of ∼3.8 kb expressed in most human tissues and all of the cell lines tested, although the levels varied (Fig. 1, A and B). The basis and significance of the interaction of centrobin with BRCA2 is currently under investigation and is not discussed here.

Bottom Line: In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole.The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication.Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Cancer Biology, Evanston Northwestern Healthcare Research Institute, Northwestern University Feinberg School of Medicine, Evanston, IL 60201, USA.

ABSTRACT
In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

Show MeSH
Related in: MedlinePlus