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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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Cell polarization and migration analysis in AK1- and AK1B-expressing cells. (A) Reorientation of the CTR and the GA after wounding in AK1 (top)- and AK1B (bottom)-transfected cells. The white lines indicate the scratch orientation. Pink or white angles indicate nontransfected or transfected cells, respectively. (B) Graph showing the percentage of control, AK1-, and AK1B- transfected cells with the CTR (circles) or the GA (squares) reoriented toward the wound at each time point. Random orientation is expected to be 25%. n ≥ 80 cells for each condition from two independent experiments. (C) Wound-healing assays in either control (top row), AK1 (middle row)-, and AK1B (bottom row)-transfected cells. Frames from time-lapse phase-contrast videos at the indicated times are shown. 13 selected cells were false colored and numbered to better visualize their movement. Tracks indicating frame by frame movement of cells are overlaid in right images. (D) Quantitative analysis of wound-healing assays by using the WimScratch software (Wimasis). Graph represents the percentage of scratch area at each time point in control, AK1-, and AK1B-expressing cells. Values are from three independent experiments. Error bars indicate standard deviations. Bars, 12.5 µm.
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fig8: Cell polarization and migration analysis in AK1- and AK1B-expressing cells. (A) Reorientation of the CTR and the GA after wounding in AK1 (top)- and AK1B (bottom)-transfected cells. The white lines indicate the scratch orientation. Pink or white angles indicate nontransfected or transfected cells, respectively. (B) Graph showing the percentage of control, AK1-, and AK1B- transfected cells with the CTR (circles) or the GA (squares) reoriented toward the wound at each time point. Random orientation is expected to be 25%. n ≥ 80 cells for each condition from two independent experiments. (C) Wound-healing assays in either control (top row), AK1 (middle row)-, and AK1B (bottom row)-transfected cells. Frames from time-lapse phase-contrast videos at the indicated times are shown. 13 selected cells were false colored and numbered to better visualize their movement. Tracks indicating frame by frame movement of cells are overlaid in right images. (D) Quantitative analysis of wound-healing assays by using the WimScratch software (Wimasis). Graph represents the percentage of scratch area at each time point in control, AK1-, and AK1B-expressing cells. Values are from three independent experiments. Error bars indicate standard deviations. Bars, 12.5 µm.

Mentions: To study the specific role of Golgi positioning and integrity in cell polarity, scratch wounding was performed on control, AK1-, and AK1B-transfected cells (Fig. 8 A). The CTR and GA were counted as oriented when most of the labeling was located within the 90° angle facing the wound (Fig. 8 A). As expected, ∼80% of control cells had already oriented both the CTR and GA at 3 h after wounding (Fig. 8 B). In AK1-expressing cells, however, the percentage of reoriented GA was only 45%, even 8 h after wounding. Interestingly, the same number of cells had reoriented the CTR at this time point. Finally, although the kinetics of GA and CTR reorientation in AK1B-expressing cells was slower, 8 h after wounding, the number of reoriented cells was only slightly reduced with respect to control cells (<10%).


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)

Cell polarization and migration analysis in AK1- and AK1B-expressing cells. (A) Reorientation of the CTR and the GA after wounding in AK1 (top)- and AK1B (bottom)-transfected cells. The white lines indicate the scratch orientation. Pink or white angles indicate nontransfected or transfected cells, respectively. (B) Graph showing the percentage of control, AK1-, and AK1B- transfected cells with the CTR (circles) or the GA (squares) reoriented toward the wound at each time point. Random orientation is expected to be 25%. n ≥ 80 cells for each condition from two independent experiments. (C) Wound-healing assays in either control (top row), AK1 (middle row)-, and AK1B (bottom row)-transfected cells. Frames from time-lapse phase-contrast videos at the indicated times are shown. 13 selected cells were false colored and numbered to better visualize their movement. Tracks indicating frame by frame movement of cells are overlaid in right images. (D) Quantitative analysis of wound-healing assays by using the WimScratch software (Wimasis). Graph represents the percentage of scratch area at each time point in control, AK1-, and AK1B-expressing cells. Values are from three independent experiments. Error bars indicate standard deviations. Bars, 12.5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: Cell polarization and migration analysis in AK1- and AK1B-expressing cells. (A) Reorientation of the CTR and the GA after wounding in AK1 (top)- and AK1B (bottom)-transfected cells. The white lines indicate the scratch orientation. Pink or white angles indicate nontransfected or transfected cells, respectively. (B) Graph showing the percentage of control, AK1-, and AK1B- transfected cells with the CTR (circles) or the GA (squares) reoriented toward the wound at each time point. Random orientation is expected to be 25%. n ≥ 80 cells for each condition from two independent experiments. (C) Wound-healing assays in either control (top row), AK1 (middle row)-, and AK1B (bottom row)-transfected cells. Frames from time-lapse phase-contrast videos at the indicated times are shown. 13 selected cells were false colored and numbered to better visualize their movement. Tracks indicating frame by frame movement of cells are overlaid in right images. (D) Quantitative analysis of wound-healing assays by using the WimScratch software (Wimasis). Graph represents the percentage of scratch area at each time point in control, AK1-, and AK1B-expressing cells. Values are from three independent experiments. Error bars indicate standard deviations. Bars, 12.5 µm.
Mentions: To study the specific role of Golgi positioning and integrity in cell polarity, scratch wounding was performed on control, AK1-, and AK1B-transfected cells (Fig. 8 A). The CTR and GA were counted as oriented when most of the labeling was located within the 90° angle facing the wound (Fig. 8 A). As expected, ∼80% of control cells had already oriented both the CTR and GA at 3 h after wounding (Fig. 8 B). In AK1-expressing cells, however, the percentage of reoriented GA was only 45%, even 8 h after wounding. Interestingly, the same number of cells had reoriented the CTR at this time point. Finally, although the kinetics of GA and CTR reorientation in AK1B-expressing cells was slower, 8 h after wounding, the number of reoriented cells was only slightly reduced with respect to control cells (<10%).

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