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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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Reassembly of the GA after NZ washout is perturbed at different stages in AK1- and AK1B-transfected cells. (A–C) RPE-1 cells expressing GT-mCherry (A) or coexpressing GT-mCherry and either GFP-AK1 (B) or GFP-AK1B (C) were treated with NZ for 3 h and incubated at 4°C for 1 h in the presence of the drug. After washout, cells were recorded by video microscopy. Time after NZ removal is shown. (A) Video frames illustrating two-step reassembly of the GA in control cells. Golgi elements partially fuse in the cell periphery before being translocated toward the CTR. (B) In AK1-transfected cells, Golgi elements fuse in the cell periphery at lower rates than in control cells and do not apparently move toward the cell center. (C) In AK1B-transfected cells, Golgi elements are radially translocated toward the CTR without peripheral fusion. (right) An identical experiment in fixed cells. CTR position is indicated (yellow arrows). γtub, γ-tubulin. Bars, 3 µm.
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fig6: Reassembly of the GA after NZ washout is perturbed at different stages in AK1- and AK1B-transfected cells. (A–C) RPE-1 cells expressing GT-mCherry (A) or coexpressing GT-mCherry and either GFP-AK1 (B) or GFP-AK1B (C) were treated with NZ for 3 h and incubated at 4°C for 1 h in the presence of the drug. After washout, cells were recorded by video microscopy. Time after NZ removal is shown. (A) Video frames illustrating two-step reassembly of the GA in control cells. Golgi elements partially fuse in the cell periphery before being translocated toward the CTR. (B) In AK1-transfected cells, Golgi elements fuse in the cell periphery at lower rates than in control cells and do not apparently move toward the cell center. (C) In AK1B-transfected cells, Golgi elements are radially translocated toward the CTR without peripheral fusion. (right) An identical experiment in fixed cells. CTR position is indicated (yellow arrows). γtub, γ-tubulin. Bars, 3 µm.

Mentions: To gain insights into the GA formation process, we used time-lapse microscopy to compare GA reassembly after NZ treatment in control, AK1-, or AK1B-expressing cells (Fig. 6). RPE-1 cells grown on gridded dishes were either transfected with GT-mCherry (control) or cotransfected with GT-mCherry and GFP-AK1 or GFP-AK1B constructs. After NZ removal, double-transfected cells were recorded for several hours (Fig. 6, left) and then fixed and processed by IF to determine CTR position (Fig. 6, right).


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)

Reassembly of the GA after NZ washout is perturbed at different stages in AK1- and AK1B-transfected cells. (A–C) RPE-1 cells expressing GT-mCherry (A) or coexpressing GT-mCherry and either GFP-AK1 (B) or GFP-AK1B (C) were treated with NZ for 3 h and incubated at 4°C for 1 h in the presence of the drug. After washout, cells were recorded by video microscopy. Time after NZ removal is shown. (A) Video frames illustrating two-step reassembly of the GA in control cells. Golgi elements partially fuse in the cell periphery before being translocated toward the CTR. (B) In AK1-transfected cells, Golgi elements fuse in the cell periphery at lower rates than in control cells and do not apparently move toward the cell center. (C) In AK1B-transfected cells, Golgi elements are radially translocated toward the CTR without peripheral fusion. (right) An identical experiment in fixed cells. CTR position is indicated (yellow arrows). γtub, γ-tubulin. Bars, 3 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Reassembly of the GA after NZ washout is perturbed at different stages in AK1- and AK1B-transfected cells. (A–C) RPE-1 cells expressing GT-mCherry (A) or coexpressing GT-mCherry and either GFP-AK1 (B) or GFP-AK1B (C) were treated with NZ for 3 h and incubated at 4°C for 1 h in the presence of the drug. After washout, cells were recorded by video microscopy. Time after NZ removal is shown. (A) Video frames illustrating two-step reassembly of the GA in control cells. Golgi elements partially fuse in the cell periphery before being translocated toward the CTR. (B) In AK1-transfected cells, Golgi elements fuse in the cell periphery at lower rates than in control cells and do not apparently move toward the cell center. (C) In AK1B-transfected cells, Golgi elements are radially translocated toward the CTR without peripheral fusion. (right) An identical experiment in fixed cells. CTR position is indicated (yellow arrows). γtub, γ-tubulin. Bars, 3 µm.
Mentions: To gain insights into the GA formation process, we used time-lapse microscopy to compare GA reassembly after NZ treatment in control, AK1-, or AK1B-expressing cells (Fig. 6). RPE-1 cells grown on gridded dishes were either transfected with GT-mCherry (control) or cotransfected with GT-mCherry and GFP-AK1 or GFP-AK1B constructs. After NZ removal, double-transfected cells were recorded for several hours (Fig. 6, left) and then fixed and processed by IF to determine CTR position (Fig. 6, right).

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