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Centrin2 regulates CP110 removal in primary cilium formation.

Prosser SL, Morrison CG - J. Cell Biol. (2015)

Bottom Line: Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood.Knockdown of CP110 rescued ciliation in CETN2-deficient cells.Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway, Galway, Ireland.

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Centrin2 is required for ciliation in chicken B-cells. (a and b) Quantitation of doubling time (a) and flow cytometry analysis of DNA content (b) in DT40 cells after 48-h culture in 10% normal medium or serum starvation in 0.5% FBS. Bar graph shows means + SD of three independent experiments. *, P < 0.05, compared with controls by unpaired t test. Numbers under FACS plots indicate mean percentages of the entire population in the indicated cell cycle stage (n = 4). (c) Immunofluorescence microscopy of the cilium marker, acetylated tubulin, in serum-starved DT40 cells. Ac. tub, acetylated tubulin. Bar, 5 µm. (d) Quantitation of the number of cells with acetylated tubulin staining in DT40 cells cultured for 48 h in the indicated medium. Histogram shows means + SD of three independent experiments in which ≥500 cells were quantitated. *, P < 0.05, compared with controls by unpaired t test. (e) Quantitation of the number of cells with acetylated tubulin staining in wild-type (WT) and the indicated centrin-deficient and rescued DT40 clones after 48-h serum starvation. Histogram shows means + SD of three independent experiments in which at least 500 cells were quantitated. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by unpaired t test. Black asterisks, comparison to WT cells; red asterisks, comparison to triple Cetn4−/− Cetn2−/− Cetn3− knockout cells.
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fig1: Centrin2 is required for ciliation in chicken B-cells. (a and b) Quantitation of doubling time (a) and flow cytometry analysis of DNA content (b) in DT40 cells after 48-h culture in 10% normal medium or serum starvation in 0.5% FBS. Bar graph shows means + SD of three independent experiments. *, P < 0.05, compared with controls by unpaired t test. Numbers under FACS plots indicate mean percentages of the entire population in the indicated cell cycle stage (n = 4). (c) Immunofluorescence microscopy of the cilium marker, acetylated tubulin, in serum-starved DT40 cells. Ac. tub, acetylated tubulin. Bar, 5 µm. (d) Quantitation of the number of cells with acetylated tubulin staining in DT40 cells cultured for 48 h in the indicated medium. Histogram shows means + SD of three independent experiments in which ≥500 cells were quantitated. *, P < 0.05, compared with controls by unpaired t test. (e) Quantitation of the number of cells with acetylated tubulin staining in wild-type (WT) and the indicated centrin-deficient and rescued DT40 clones after 48-h serum starvation. Histogram shows means + SD of three independent experiments in which at least 500 cells were quantitated. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by unpaired t test. Black asterisks, comparison to WT cells; red asterisks, comparison to triple Cetn4−/− Cetn2−/− Cetn3− knockout cells.

Mentions: Exit from the cell cycle into quiescence has long been established as potentiating ciliogenesis (Dingemans, 1969; Seeley and Nachury, 2010). We tested whether the chicken DT40 lymphocyte cell line could be induced to undergo primary ciliation. Serum starvation of chicken B-lymphocyte DT40 cells led to cell cycle delay (Fig. 1, a and b) and a reproducible induction of primary cilia (Fig. 1, c and d). The ability to induce cilia in DT40 cells allowed us to explore the roles of centrin in ciliogenesis. Examining a series of genetically defined centrin knockout lines (Dantas et al., 2011), we found that centrin2 deficiency led to a significant decline in ciliation capacity that was as extensive as the decline seen in cells that lacked all three chicken centrin isoforms. Although the expression of centrin3 and centrin4 could partially rescue the absence of all chicken centrins in ciliogenesis, only the expression of centrin2 led to a complete rescue of the ability of DT40 cells to make primary cilia after serum starvation (Fig. 1 e), demonstrating that centrin2 is required for ciliation in lymphocytes. Centrin2 depletion in zebrafish gave rise to several ciliopathy phenotypes (Delaval et al., 2011) and a mouse knockout also revealed marked ciliopathy that was restricted to specific tissues (Ying et al., 2014). It was concluded that the murine phenotype in affected tissues was caused by problems in ciliary trafficking, with normal ciliogenesis initiation and axoneme formation (Ying et al., 2014). Given the partial rescue of ciliation that we see with centrin3 and centrin4 in the chicken model, it is possible that the interplay between the individual members of the centrin family determines the precise roles of an individual centrin in a given tissue.


Centrin2 regulates CP110 removal in primary cilium formation.

Prosser SL, Morrison CG - J. Cell Biol. (2015)

Centrin2 is required for ciliation in chicken B-cells. (a and b) Quantitation of doubling time (a) and flow cytometry analysis of DNA content (b) in DT40 cells after 48-h culture in 10% normal medium or serum starvation in 0.5% FBS. Bar graph shows means + SD of three independent experiments. *, P < 0.05, compared with controls by unpaired t test. Numbers under FACS plots indicate mean percentages of the entire population in the indicated cell cycle stage (n = 4). (c) Immunofluorescence microscopy of the cilium marker, acetylated tubulin, in serum-starved DT40 cells. Ac. tub, acetylated tubulin. Bar, 5 µm. (d) Quantitation of the number of cells with acetylated tubulin staining in DT40 cells cultured for 48 h in the indicated medium. Histogram shows means + SD of three independent experiments in which ≥500 cells were quantitated. *, P < 0.05, compared with controls by unpaired t test. (e) Quantitation of the number of cells with acetylated tubulin staining in wild-type (WT) and the indicated centrin-deficient and rescued DT40 clones after 48-h serum starvation. Histogram shows means + SD of three independent experiments in which at least 500 cells were quantitated. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by unpaired t test. Black asterisks, comparison to WT cells; red asterisks, comparison to triple Cetn4−/− Cetn2−/− Cetn3− knockout cells.
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fig1: Centrin2 is required for ciliation in chicken B-cells. (a and b) Quantitation of doubling time (a) and flow cytometry analysis of DNA content (b) in DT40 cells after 48-h culture in 10% normal medium or serum starvation in 0.5% FBS. Bar graph shows means + SD of three independent experiments. *, P < 0.05, compared with controls by unpaired t test. Numbers under FACS plots indicate mean percentages of the entire population in the indicated cell cycle stage (n = 4). (c) Immunofluorescence microscopy of the cilium marker, acetylated tubulin, in serum-starved DT40 cells. Ac. tub, acetylated tubulin. Bar, 5 µm. (d) Quantitation of the number of cells with acetylated tubulin staining in DT40 cells cultured for 48 h in the indicated medium. Histogram shows means + SD of three independent experiments in which ≥500 cells were quantitated. *, P < 0.05, compared with controls by unpaired t test. (e) Quantitation of the number of cells with acetylated tubulin staining in wild-type (WT) and the indicated centrin-deficient and rescued DT40 clones after 48-h serum starvation. Histogram shows means + SD of three independent experiments in which at least 500 cells were quantitated. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by unpaired t test. Black asterisks, comparison to WT cells; red asterisks, comparison to triple Cetn4−/− Cetn2−/− Cetn3− knockout cells.
Mentions: Exit from the cell cycle into quiescence has long been established as potentiating ciliogenesis (Dingemans, 1969; Seeley and Nachury, 2010). We tested whether the chicken DT40 lymphocyte cell line could be induced to undergo primary ciliation. Serum starvation of chicken B-lymphocyte DT40 cells led to cell cycle delay (Fig. 1, a and b) and a reproducible induction of primary cilia (Fig. 1, c and d). The ability to induce cilia in DT40 cells allowed us to explore the roles of centrin in ciliogenesis. Examining a series of genetically defined centrin knockout lines (Dantas et al., 2011), we found that centrin2 deficiency led to a significant decline in ciliation capacity that was as extensive as the decline seen in cells that lacked all three chicken centrin isoforms. Although the expression of centrin3 and centrin4 could partially rescue the absence of all chicken centrins in ciliogenesis, only the expression of centrin2 led to a complete rescue of the ability of DT40 cells to make primary cilia after serum starvation (Fig. 1 e), demonstrating that centrin2 is required for ciliation in lymphocytes. Centrin2 depletion in zebrafish gave rise to several ciliopathy phenotypes (Delaval et al., 2011) and a mouse knockout also revealed marked ciliopathy that was restricted to specific tissues (Ying et al., 2014). It was concluded that the murine phenotype in affected tissues was caused by problems in ciliary trafficking, with normal ciliogenesis initiation and axoneme formation (Ying et al., 2014). Given the partial rescue of ciliation that we see with centrin3 and centrin4 in the chicken model, it is possible that the interplay between the individual members of the centrin family determines the precise roles of an individual centrin in a given tissue.

Bottom Line: Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood.Knockdown of CP110 rescued ciliation in CETN2-deficient cells.Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway, Galway, Ireland.

Show MeSH
Related in: MedlinePlus