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Centrin2 regulates CP110 removal in primary cilium formation.

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

Bottom Line: We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110.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.
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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: We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110.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