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Disconnecting the Golgi ribbon from the centrosome prevents directional cell migration and ciliogenesis.

Hurtado L, Caballero C, Gavilan MP, Cardenas J, Bornens M, Rios RM - J. Cell Biol. (2011)

Bottom Line: We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon.Both features, however, were required for ciliogenesis.We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Señalización Celular, Centro Andaluz de Biología Molecular y Medicina Regenerativa-Consejo Superior de Investigaciones Científicas, 41092-Seville, Spain.

ABSTRACT
Mammalian cells exhibit a frequent pericentrosomal Golgi ribbon organization. In this paper, we show that two AKAP450 N-terminal fragments, both containing the Golgi-binding GM130-interacting domain of AKAP450, dissociated endogenous AKAP450 from the Golgi and inhibited microtubule (MT) nucleation at the Golgi without interfering with centrosomal activity. These two fragments had, however, strikingly different effects on both Golgi apparatus (GA) integrity and positioning, whereas the short fragment induced GA circularization and ribbon fragmentation, the large construct that encompasses an additional p150glued/MT-binding domain induced separation of the Golgi ribbon from the centrosome. These distinct phenotypes arose by specific interference of each fragment with either Golgi-dependent or centrosome-dependent stages of Golgi assembly. We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon. Both features, however, were required for ciliogenesis. We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

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Primary cilium formation is severely impaired in AK1 or AK1B fragment–expressing cells. (A) Schematic of the procedure used for primary cilium induction. (B) Representative images of control (right), flag-AK1 (middle)–, and flag-AK1B (left)–transfected cells maintained in 0.25% serum for 36 h and triple labeled for polyglutamylated tubulin (pglu-tub), AKAP450, and GMAP210. Double merged images are as indicated. Boxes are enlarged on the bottom. T, transfected cell; NT, nontransfected cells. Bars, 7.5 µm. (C) Bar graphs showing the increase on the percentage of cells with primary cilium at 0, 24, and 36 h after serum deprivation. Dotted lines indicate the percentage of cells containing a cilium before starvation. n = 300 from three independent experiments (*, P < 0.001; Tukey HSD). Error bars indicate standard deviations.
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fig9: Primary cilium formation is severely impaired in AK1 or AK1B fragment–expressing cells. (A) Schematic of the procedure used for primary cilium induction. (B) Representative images of control (right), flag-AK1 (middle)–, and flag-AK1B (left)–transfected cells maintained in 0.25% serum for 36 h and triple labeled for polyglutamylated tubulin (pglu-tub), AKAP450, and GMAP210. Double merged images are as indicated. Boxes are enlarged on the bottom. T, transfected cell; NT, nontransfected cells. Bars, 7.5 µm. (C) Bar graphs showing the increase on the percentage of cells with primary cilium at 0, 24, and 36 h after serum deprivation. Dotted lines indicate the percentage of cells containing a cilium before starvation. n = 300 from three independent experiments (*, P < 0.001; Tukey HSD). Error bars indicate standard deviations.

Mentions: Primary cilium assembly involves MT organization and polarized membrane trafficking (Satir et al., 2010). We were eager to investigate whether perturbations in GA position or integrity block primary ciliogenesis induced by serum deprivation. 6 h after transfection with AK1 or AK1B constructs, cells were serum deprived for different periods of time as represented in Fig. 9 A. Cells were fixed and stained as indicated (Fig. 9 B), and the number of ciliated cells was determined for each condition by immunostaining with antipolyglutamylated (Fig. 9 B) or acetylated tubulin (not depicted) antibodies. Quantification revealed that ∼30% of cells already contained a primary cilium at the moment of starvation. In control cells, the number of ciliated cells increased from 30 to 80% after 24 h in the absence of serum. In contrast, in AK1- or AK1B-transfected cells, only 20% of transfected cells were able to develop a cilium, even after 36 h of serum deprivation. This dramatic reduction in the ability to generate a primary cilium in AK1- and AK1B-transfected cells indicates that both the Golgi integrity and pericentrosomal position are essential to form cilia in epithelial cells.


Disconnecting the Golgi ribbon from the centrosome prevents directional cell migration and ciliogenesis.

Hurtado L, Caballero C, Gavilan MP, Cardenas J, Bornens M, Rios RM - J. Cell Biol. (2011)

Primary cilium formation is severely impaired in AK1 or AK1B fragment–expressing cells. (A) Schematic of the procedure used for primary cilium induction. (B) Representative images of control (right), flag-AK1 (middle)–, and flag-AK1B (left)–transfected cells maintained in 0.25% serum for 36 h and triple labeled for polyglutamylated tubulin (pglu-tub), AKAP450, and GMAP210. Double merged images are as indicated. Boxes are enlarged on the bottom. T, transfected cell; NT, nontransfected cells. Bars, 7.5 µm. (C) Bar graphs showing the increase on the percentage of cells with primary cilium at 0, 24, and 36 h after serum deprivation. Dotted lines indicate the percentage of cells containing a cilium before starvation. n = 300 from three independent experiments (*, P < 0.001; Tukey HSD). Error bars indicate standard deviations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105543&req=5

fig9: Primary cilium formation is severely impaired in AK1 or AK1B fragment–expressing cells. (A) Schematic of the procedure used for primary cilium induction. (B) Representative images of control (right), flag-AK1 (middle)–, and flag-AK1B (left)–transfected cells maintained in 0.25% serum for 36 h and triple labeled for polyglutamylated tubulin (pglu-tub), AKAP450, and GMAP210. Double merged images are as indicated. Boxes are enlarged on the bottom. T, transfected cell; NT, nontransfected cells. Bars, 7.5 µm. (C) Bar graphs showing the increase on the percentage of cells with primary cilium at 0, 24, and 36 h after serum deprivation. Dotted lines indicate the percentage of cells containing a cilium before starvation. n = 300 from three independent experiments (*, P < 0.001; Tukey HSD). Error bars indicate standard deviations.
Mentions: Primary cilium assembly involves MT organization and polarized membrane trafficking (Satir et al., 2010). We were eager to investigate whether perturbations in GA position or integrity block primary ciliogenesis induced by serum deprivation. 6 h after transfection with AK1 or AK1B constructs, cells were serum deprived for different periods of time as represented in Fig. 9 A. Cells were fixed and stained as indicated (Fig. 9 B), and the number of ciliated cells was determined for each condition by immunostaining with antipolyglutamylated (Fig. 9 B) or acetylated tubulin (not depicted) antibodies. Quantification revealed that ∼30% of cells already contained a primary cilium at the moment of starvation. In control cells, the number of ciliated cells increased from 30 to 80% after 24 h in the absence of serum. In contrast, in AK1- or AK1B-transfected cells, only 20% of transfected cells were able to develop a cilium, even after 36 h of serum deprivation. This dramatic reduction in the ability to generate a primary cilium in AK1- and AK1B-transfected cells indicates that both the Golgi integrity and pericentrosomal position are essential to form cilia in epithelial cells.

Bottom Line: We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon.Both features, however, were required for ciliogenesis.We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Señalización Celular, Centro Andaluz de Biología Molecular y Medicina Regenerativa-Consejo Superior de Investigaciones Científicas, 41092-Seville, Spain.

ABSTRACT
Mammalian cells exhibit a frequent pericentrosomal Golgi ribbon organization. In this paper, we show that two AKAP450 N-terminal fragments, both containing the Golgi-binding GM130-interacting domain of AKAP450, dissociated endogenous AKAP450 from the Golgi and inhibited microtubule (MT) nucleation at the Golgi without interfering with centrosomal activity. These two fragments had, however, strikingly different effects on both Golgi apparatus (GA) integrity and positioning, whereas the short fragment induced GA circularization and ribbon fragmentation, the large construct that encompasses an additional p150glued/MT-binding domain induced separation of the Golgi ribbon from the centrosome. These distinct phenotypes arose by specific interference of each fragment with either Golgi-dependent or centrosome-dependent stages of Golgi assembly. We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon. Both features, however, were required for ciliogenesis. We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

Show MeSH
Related in: MedlinePlus