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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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Identification of the AKAP450 cis-Golgi–targeting domain by serial truncations. (A and D) Schematic representation of the AKAP450-truncated mutants used in this study. (right) A summary of the subcellular localization of each fragment. (B, C, and E) GFP-AK1 (B)–, GFP-AK4 (C)–, or GFP-AK1B (E)–expressing cells were labeled for GMAP210 (B and E) or γ-tubulin (C). Merged images are shown on the right. (F) WB of RPE-1 cells expressing AKAP450-truncated mutants used in this study. Molecular masses are given in kilodaltons. (G) Box and whisker plot showing quantification of GA recruitment for each N-terminal mutant. Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Asterisks denote significant statistical differences (P < 0.001; Tukey honestly significant difference [HSD]). n = 30 cells scored per mutant from three independent experiments. AU, arbitrary unit. (H) Merged images of cells expressing flag-AK1 or flag-AK1B double labeled for flag and either GM130 or Golgin-245. Fluorescence intensity profiles corresponding to lines drawn in each image from the top gallery are shown on the bottom. PACT, pericentrin-AKAP450 centrosomal targeting domain; WT, wild type. Bars, 5 µm.
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fig1: Identification of the AKAP450 cis-Golgi–targeting domain by serial truncations. (A and D) Schematic representation of the AKAP450-truncated mutants used in this study. (right) A summary of the subcellular localization of each fragment. (B, C, and E) GFP-AK1 (B)–, GFP-AK4 (C)–, or GFP-AK1B (E)–expressing cells were labeled for GMAP210 (B and E) or γ-tubulin (C). Merged images are shown on the right. (F) WB of RPE-1 cells expressing AKAP450-truncated mutants used in this study. Molecular masses are given in kilodaltons. (G) Box and whisker plot showing quantification of GA recruitment for each N-terminal mutant. Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Asterisks denote significant statistical differences (P < 0.001; Tukey honestly significant difference [HSD]). n = 30 cells scored per mutant from three independent experiments. AU, arbitrary unit. (H) Merged images of cells expressing flag-AK1 or flag-AK1B double labeled for flag and either GM130 or Golgin-245. Fluorescence intensity profiles corresponding to lines drawn in each image from the top gallery are shown on the bottom. PACT, pericentrin-AKAP450 centrosomal targeting domain; WT, wild type. Bars, 5 µm.

Mentions: To characterize the regions of AKAP450 required for its localization to the GA, we divided the full-length protein in four large fragments (AK1–AK4), all of them containing long coiled-coil regions (Fig. 1 A). Truncated mutants were fused to different tags and transfected in RPE-1 cells. Only the AK1 fragment corresponding to the N terminus of the protein was targeted to the GA as revealed by double labeling for GMAP210. Some punctuate and fibrillar structures were also observed throughout the cytoplasm (Fig. 1 B). AK2 and AK3 fragments were cytoplasmic (Fig. S1 A), whereas the C-terminal fragment AK4 that contains the pericentrin-AKAP450 centrosomal targeting domain (Gillingham and Munro, 2000) localized to the CTR as expected (Fig. 1 C). Similar distribution was observed in MCF10A cells (Fig. S1 B).


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)

Identification of the AKAP450 cis-Golgi–targeting domain by serial truncations. (A and D) Schematic representation of the AKAP450-truncated mutants used in this study. (right) A summary of the subcellular localization of each fragment. (B, C, and E) GFP-AK1 (B)–, GFP-AK4 (C)–, or GFP-AK1B (E)–expressing cells were labeled for GMAP210 (B and E) or γ-tubulin (C). Merged images are shown on the right. (F) WB of RPE-1 cells expressing AKAP450-truncated mutants used in this study. Molecular masses are given in kilodaltons. (G) Box and whisker plot showing quantification of GA recruitment for each N-terminal mutant. Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Asterisks denote significant statistical differences (P < 0.001; Tukey honestly significant difference [HSD]). n = 30 cells scored per mutant from three independent experiments. AU, arbitrary unit. (H) Merged images of cells expressing flag-AK1 or flag-AK1B double labeled for flag and either GM130 or Golgin-245. Fluorescence intensity profiles corresponding to lines drawn in each image from the top gallery are shown on the bottom. PACT, pericentrin-AKAP450 centrosomal targeting domain; WT, wild type. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Identification of the AKAP450 cis-Golgi–targeting domain by serial truncations. (A and D) Schematic representation of the AKAP450-truncated mutants used in this study. (right) A summary of the subcellular localization of each fragment. (B, C, and E) GFP-AK1 (B)–, GFP-AK4 (C)–, or GFP-AK1B (E)–expressing cells were labeled for GMAP210 (B and E) or γ-tubulin (C). Merged images are shown on the right. (F) WB of RPE-1 cells expressing AKAP450-truncated mutants used in this study. Molecular masses are given in kilodaltons. (G) Box and whisker plot showing quantification of GA recruitment for each N-terminal mutant. Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Asterisks denote significant statistical differences (P < 0.001; Tukey honestly significant difference [HSD]). n = 30 cells scored per mutant from three independent experiments. AU, arbitrary unit. (H) Merged images of cells expressing flag-AK1 or flag-AK1B double labeled for flag and either GM130 or Golgin-245. Fluorescence intensity profiles corresponding to lines drawn in each image from the top gallery are shown on the bottom. PACT, pericentrin-AKAP450 centrosomal targeting domain; WT, wild type. Bars, 5 µm.
Mentions: To characterize the regions of AKAP450 required for its localization to the GA, we divided the full-length protein in four large fragments (AK1–AK4), all of them containing long coiled-coil regions (Fig. 1 A). Truncated mutants were fused to different tags and transfected in RPE-1 cells. Only the AK1 fragment corresponding to the N terminus of the protein was targeted to the GA as revealed by double labeling for GMAP210. Some punctuate and fibrillar structures were also observed throughout the cytoplasm (Fig. 1 B). AK2 and AK3 fragments were cytoplasmic (Fig. S1 A), whereas the C-terminal fragment AK4 that contains the pericentrin-AKAP450 centrosomal targeting domain (Gillingham and Munro, 2000) localized to the CTR as expected (Fig. 1 C). Similar distribution was observed in MCF10A cells (Fig. S1 B).

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