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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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The GA of AK1B-transfected cells exhibits diminished GT mobility, whereas it is not perturbed in AK1-transfected cells. (A) FRAP experiments were performed in RPE-1 GT-GFP–expressing cells transfected with mCherry-centrin (top row) or cotransfected with mCherry-centrin and either flag-AK1 (middle row) or flag-AK1B (bottom row). (left) Images of selected cells are shown before bleaching (prebleach) with CTR in red and the GA in green. Video frames of the same cells 0 s after bleaching (middle images) and 200 s after bleaching (right images) are shown. Bleached regions are indicated by white squares. (B) Graph showing the percentage of fluorescence recovery intensity for nontransfected, flag-transfected, flag-AK1–, and flag-AK1B–transfected cells. n = 30 for each condition from five independent experiments. (C) Bar graph shows the percentage of GT-GFP mobile fraction under each condition for the same cells (*, P < 0.001; Holm-Sidak). Error bars indicate standard deviations. Bars, 5 µm.
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fig5: The GA of AK1B-transfected cells exhibits diminished GT mobility, whereas it is not perturbed in AK1-transfected cells. (A) FRAP experiments were performed in RPE-1 GT-GFP–expressing cells transfected with mCherry-centrin (top row) or cotransfected with mCherry-centrin and either flag-AK1 (middle row) or flag-AK1B (bottom row). (left) Images of selected cells are shown before bleaching (prebleach) with CTR in red and the GA in green. Video frames of the same cells 0 s after bleaching (middle images) and 200 s after bleaching (right images) are shown. Bleached regions are indicated by white squares. (B) Graph showing the percentage of fluorescence recovery intensity for nontransfected, flag-transfected, flag-AK1–, and flag-AK1B–transfected cells. n = 30 for each condition from five independent experiments. (C) Bar graph shows the percentage of GT-GFP mobile fraction under each condition for the same cells (*, P < 0.001; Holm-Sidak). Error bars indicate standard deviations. Bars, 5 µm.

Mentions: A puzzling result was that both AK1 and AK1B fragments inhibited MT nucleation at the GA, yet their effects on GA morphology and positioning were strikingly different. GA-nucleated MTs have been proposed to be required for tangential Golgi stack linking within the Golgi ribbon. To test the continuity of the GA in cells expressing AKAP450-truncated mutants, we performed FRAP experiments in a RPE-1 cell line stably expressing the galactosyltransferase (GT) membrane fragment GT-GFP (Fig. 5). To identify transfected cells and to localize CTRs, GT-GFP cells were transiently transfected with an mCherry-centrin construct or cotransfected with mCherry-centrin and either flag-AK1 or flag-AK1B in a 1:3 proportion. Nontransfected GT-GFP cells were also analyzed as an additional control (Fig. 5, A and B). The flow of GT was determined by fluorescence recovery at 200 s after bleaching a selected area (Fig. 5 C). Notably, fluorescence was recovered at almost similar levels in controls and in AK1-expressing cells (∼50%), whereas it was incomplete in cells expressing the small AK1B fragment (∼25%). Determination of the GT mobile fraction revealed that in the presence of the AK1B fragment, the enzyme mobility between stacks was reduced by 40% (Fig. 5 C). These results indicate that the Golgi ribbon of AK1B-expressing cells is interrupted, possibly because of stacks being disconnected. On the other hand, the expression of the large AK1 fragment did not significantly impair GT mobility between stacks, suggesting the possibility of generating an intact Golgi ribbon far from the CTR.


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)

The GA of AK1B-transfected cells exhibits diminished GT mobility, whereas it is not perturbed in AK1-transfected cells. (A) FRAP experiments were performed in RPE-1 GT-GFP–expressing cells transfected with mCherry-centrin (top row) or cotransfected with mCherry-centrin and either flag-AK1 (middle row) or flag-AK1B (bottom row). (left) Images of selected cells are shown before bleaching (prebleach) with CTR in red and the GA in green. Video frames of the same cells 0 s after bleaching (middle images) and 200 s after bleaching (right images) are shown. Bleached regions are indicated by white squares. (B) Graph showing the percentage of fluorescence recovery intensity for nontransfected, flag-transfected, flag-AK1–, and flag-AK1B–transfected cells. n = 30 for each condition from five independent experiments. (C) Bar graph shows the percentage of GT-GFP mobile fraction under each condition for the same cells (*, P < 0.001; Holm-Sidak). Error bars indicate standard deviations. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: The GA of AK1B-transfected cells exhibits diminished GT mobility, whereas it is not perturbed in AK1-transfected cells. (A) FRAP experiments were performed in RPE-1 GT-GFP–expressing cells transfected with mCherry-centrin (top row) or cotransfected with mCherry-centrin and either flag-AK1 (middle row) or flag-AK1B (bottom row). (left) Images of selected cells are shown before bleaching (prebleach) with CTR in red and the GA in green. Video frames of the same cells 0 s after bleaching (middle images) and 200 s after bleaching (right images) are shown. Bleached regions are indicated by white squares. (B) Graph showing the percentage of fluorescence recovery intensity for nontransfected, flag-transfected, flag-AK1–, and flag-AK1B–transfected cells. n = 30 for each condition from five independent experiments. (C) Bar graph shows the percentage of GT-GFP mobile fraction under each condition for the same cells (*, P < 0.001; Holm-Sidak). Error bars indicate standard deviations. Bars, 5 µm.
Mentions: A puzzling result was that both AK1 and AK1B fragments inhibited MT nucleation at the GA, yet their effects on GA morphology and positioning were strikingly different. GA-nucleated MTs have been proposed to be required for tangential Golgi stack linking within the Golgi ribbon. To test the continuity of the GA in cells expressing AKAP450-truncated mutants, we performed FRAP experiments in a RPE-1 cell line stably expressing the galactosyltransferase (GT) membrane fragment GT-GFP (Fig. 5). To identify transfected cells and to localize CTRs, GT-GFP cells were transiently transfected with an mCherry-centrin construct or cotransfected with mCherry-centrin and either flag-AK1 or flag-AK1B in a 1:3 proportion. Nontransfected GT-GFP cells were also analyzed as an additional control (Fig. 5, A and B). The flow of GT was determined by fluorescence recovery at 200 s after bleaching a selected area (Fig. 5 C). Notably, fluorescence was recovered at almost similar levels in controls and in AK1-expressing cells (∼50%), whereas it was incomplete in cells expressing the small AK1B fragment (∼25%). Determination of the GT mobile fraction revealed that in the presence of the AK1B fragment, the enzyme mobility between stacks was reduced by 40% (Fig. 5 C). These results indicate that the Golgi ribbon of AK1B-expressing cells is interrupted, possibly because of stacks being disconnected. On the other hand, the expression of the large AK1 fragment did not significantly impair GT mobility between stacks, suggesting the possibility of generating an intact Golgi ribbon far from the CTR.

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