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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 the removal of CP110 and Cep97 from centrioles upon serum starvation. (a) TEM analysis of wild-type (WT) and centrin2- cells after 48-h serum starvation. (b) Immunofluorescence microcopy of CP110 and acetylated tubulin (Ac. tub) in asynchronous or 48-h serum-starved wild-type and centrin2- cells. Insets in b–f show enlarged images of centrioles/basal bodies. (c) Immunofluorescence microscopy of Cep97 and acetylated tubulin in 48-h serum-starved wild-type and centrin2- cells. (d) Immunofluorescence microscopy of CP110 and PCM1 in serum-starved wild-type and centrin2- cells. (e) Immunoblot analysis of the indicated proteins in asynchronous (AS) and 48-h serum-starved (SS) wild-type and centrin2- cells. (f) Immunofluorescence microscopy of CP110 and acetylated tubulin in mock-treated or CP110-depleted cells. (g) Immunoblot analysis of the indicated proteins in mock, GAPDH, and CP110-depleted WT and centrin2- cells after 24-h serum starvation after transfection. (h) Frequency of ciliated cells in mock, GAPDH, and CP110-depleted wild-type and centrin2- cells either grown asynchronously or serum starved for 24-h after transfection. Histogram shows means + SD of three independent experiments in which ≥200 cells were quantitated. ***, P < 0.001, in comparison to mock-treated control cells by unpaired t test. (i) Immunoblot analysis of the indicated proteins in wild-type and centrin2- cells after serum starvation for the indicated period. Numbers indicate the mean cyclin F band intensity per lane expressed as a percentage of the signal in wild type at 0 h (n = 3). Bars: (a) 200 nm; (b, c, and f [main images]) 10 µm; (b, c, and f [insets]) 1 µm.
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fig4: Centrin2 is required for the removal of CP110 and Cep97 from centrioles upon serum starvation. (a) TEM analysis of wild-type (WT) and centrin2- cells after 48-h serum starvation. (b) Immunofluorescence microcopy of CP110 and acetylated tubulin (Ac. tub) in asynchronous or 48-h serum-starved wild-type and centrin2- cells. Insets in b–f show enlarged images of centrioles/basal bodies. (c) Immunofluorescence microscopy of Cep97 and acetylated tubulin in 48-h serum-starved wild-type and centrin2- cells. (d) Immunofluorescence microscopy of CP110 and PCM1 in serum-starved wild-type and centrin2- cells. (e) Immunoblot analysis of the indicated proteins in asynchronous (AS) and 48-h serum-starved (SS) wild-type and centrin2- cells. (f) Immunofluorescence microscopy of CP110 and acetylated tubulin in mock-treated or CP110-depleted cells. (g) Immunoblot analysis of the indicated proteins in mock, GAPDH, and CP110-depleted WT and centrin2- cells after 24-h serum starvation after transfection. (h) Frequency of ciliated cells in mock, GAPDH, and CP110-depleted wild-type and centrin2- cells either grown asynchronously or serum starved for 24-h after transfection. Histogram shows means + SD of three independent experiments in which ≥200 cells were quantitated. ***, P < 0.001, in comparison to mock-treated control cells by unpaired t test. (i) Immunoblot analysis of the indicated proteins in wild-type and centrin2- cells after serum starvation for the indicated period. Numbers indicate the mean cyclin F band intensity per lane expressed as a percentage of the signal in wild type at 0 h (n = 3). Bars: (a) 200 nm; (b, c, and f [main images]) 10 µm; (b, c, and f [insets]) 1 µm.

Mentions: TEM confirmed the integrity of the centrioles in centrin2-deficient cells (Fig. 4 a). This analysis also demonstrated that centrioles docked successfully with ciliary vesicles, a key early step in ciliogenesis that is directed by Cep164 (Fig. 4 a; Sorokin, 1962; Schmidt et al., 2012; Tanos et al., 2013). Despite the abnormal Cep164 distribution that we saw in centrin-deficient cells, this observation indicates that the distal appendages are capable of allowing docking and that the defect in ciliation lies at a later stage in the process.


Centrin2 regulates CP110 removal in primary cilium formation.

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

Centrin2 is required for the removal of CP110 and Cep97 from centrioles upon serum starvation. (a) TEM analysis of wild-type (WT) and centrin2- cells after 48-h serum starvation. (b) Immunofluorescence microcopy of CP110 and acetylated tubulin (Ac. tub) in asynchronous or 48-h serum-starved wild-type and centrin2- cells. Insets in b–f show enlarged images of centrioles/basal bodies. (c) Immunofluorescence microscopy of Cep97 and acetylated tubulin in 48-h serum-starved wild-type and centrin2- cells. (d) Immunofluorescence microscopy of CP110 and PCM1 in serum-starved wild-type and centrin2- cells. (e) Immunoblot analysis of the indicated proteins in asynchronous (AS) and 48-h serum-starved (SS) wild-type and centrin2- cells. (f) Immunofluorescence microscopy of CP110 and acetylated tubulin in mock-treated or CP110-depleted cells. (g) Immunoblot analysis of the indicated proteins in mock, GAPDH, and CP110-depleted WT and centrin2- cells after 24-h serum starvation after transfection. (h) Frequency of ciliated cells in mock, GAPDH, and CP110-depleted wild-type and centrin2- cells either grown asynchronously or serum starved for 24-h after transfection. Histogram shows means + SD of three independent experiments in which ≥200 cells were quantitated. ***, P < 0.001, in comparison to mock-treated control cells by unpaired t test. (i) Immunoblot analysis of the indicated proteins in wild-type and centrin2- cells after serum starvation for the indicated period. Numbers indicate the mean cyclin F band intensity per lane expressed as a percentage of the signal in wild type at 0 h (n = 3). Bars: (a) 200 nm; (b, c, and f [main images]) 10 µm; (b, c, and f [insets]) 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: Centrin2 is required for the removal of CP110 and Cep97 from centrioles upon serum starvation. (a) TEM analysis of wild-type (WT) and centrin2- cells after 48-h serum starvation. (b) Immunofluorescence microcopy of CP110 and acetylated tubulin (Ac. tub) in asynchronous or 48-h serum-starved wild-type and centrin2- cells. Insets in b–f show enlarged images of centrioles/basal bodies. (c) Immunofluorescence microscopy of Cep97 and acetylated tubulin in 48-h serum-starved wild-type and centrin2- cells. (d) Immunofluorescence microscopy of CP110 and PCM1 in serum-starved wild-type and centrin2- cells. (e) Immunoblot analysis of the indicated proteins in asynchronous (AS) and 48-h serum-starved (SS) wild-type and centrin2- cells. (f) Immunofluorescence microscopy of CP110 and acetylated tubulin in mock-treated or CP110-depleted cells. (g) Immunoblot analysis of the indicated proteins in mock, GAPDH, and CP110-depleted WT and centrin2- cells after 24-h serum starvation after transfection. (h) Frequency of ciliated cells in mock, GAPDH, and CP110-depleted wild-type and centrin2- cells either grown asynchronously or serum starved for 24-h after transfection. Histogram shows means + SD of three independent experiments in which ≥200 cells were quantitated. ***, P < 0.001, in comparison to mock-treated control cells by unpaired t test. (i) Immunoblot analysis of the indicated proteins in wild-type and centrin2- cells after serum starvation for the indicated period. Numbers indicate the mean cyclin F band intensity per lane expressed as a percentage of the signal in wild type at 0 h (n = 3). Bars: (a) 200 nm; (b, c, and f [main images]) 10 µm; (b, c, and f [insets]) 1 µm.
Mentions: TEM confirmed the integrity of the centrioles in centrin2-deficient cells (Fig. 4 a). This analysis also demonstrated that centrioles docked successfully with ciliary vesicles, a key early step in ciliogenesis that is directed by Cep164 (Fig. 4 a; Sorokin, 1962; Schmidt et al., 2012; Tanos et al., 2013). Despite the abnormal Cep164 distribution that we saw in centrin-deficient cells, this observation indicates that the distal appendages are capable of allowing docking and that the defect in ciliation lies at a later stage in the process.

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