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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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AK1 and AK1B fragments differentially affect GA positioning and morphology. (A) RPE-1 cells expressing the flag-AK1 fragment were triple labeled for flag, AKAP450, and GMAP210. Single labelings and the merge are shown. White arrows and arrowheads indicate the GA and the CTR, respectively. In B, a nontransfected cell is shown for comparison. N, nucleus. (C) Merged image of a flag-AK1–transfected cell stained for flag, α-tubulin, and GMAP210 to visualize the MT network. (D) High magnification images of a flag-AK1–transfected cell triple labeled as in C. Single labelings in black and white and the merge are shown. Yellow arrows indicate the alignment of AK1B-containing structures with MTs. (E and F) Merged images of flag-AK1B–expressing cells triple labeled as in A or C, respectively. (G) Low magnification images of flag-AK1 (top)– and flag-AK1B (bottom)–transfected cells triple labeled for flag, AKAP450, and GMAP210. Single labelings for flag are shown on the left to identify transfected cells (T). (right) Schematic of the measurement of CTR–GA distance and GA diameter over the corresponding AKAP450 and GMAP210 merged images. A straight line connecting the center of the circumferences circumscribing the GA and the CTR was measured to calculate the CTR–GA distance. GA diameter was used as an index of GA circularity. NT, not transfected. (H) Graphs showing quantification of the CTR–GA distance (top) and Golgi circularity index (bottom; n = 160 for each condition from three independent experiments). Data are presented in the same box and whisker format as in Fig 1 with the median marked as a solid line (*, P < 0.001; Tukey HSD). Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. Bars, 2.5 µm.
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fig2: AK1 and AK1B fragments differentially affect GA positioning and morphology. (A) RPE-1 cells expressing the flag-AK1 fragment were triple labeled for flag, AKAP450, and GMAP210. Single labelings and the merge are shown. White arrows and arrowheads indicate the GA and the CTR, respectively. In B, a nontransfected cell is shown for comparison. N, nucleus. (C) Merged image of a flag-AK1–transfected cell stained for flag, α-tubulin, and GMAP210 to visualize the MT network. (D) High magnification images of a flag-AK1–transfected cell triple labeled as in C. Single labelings in black and white and the merge are shown. Yellow arrows indicate the alignment of AK1B-containing structures with MTs. (E and F) Merged images of flag-AK1B–expressing cells triple labeled as in A or C, respectively. (G) Low magnification images of flag-AK1 (top)– and flag-AK1B (bottom)–transfected cells triple labeled for flag, AKAP450, and GMAP210. Single labelings for flag are shown on the left to identify transfected cells (T). (right) Schematic of the measurement of CTR–GA distance and GA diameter over the corresponding AKAP450 and GMAP210 merged images. A straight line connecting the center of the circumferences circumscribing the GA and the CTR was measured to calculate the CTR–GA distance. GA diameter was used as an index of GA circularity. NT, not transfected. (H) Graphs showing quantification of the CTR–GA distance (top) and Golgi circularity index (bottom; n = 160 for each condition from three independent experiments). Data are presented in the same box and whisker format as in Fig 1 with the median marked as a solid line (*, P < 0.001; Tukey HSD). Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. Bars, 2.5 µm.

Mentions: The expression of both AK1 and AK1B domains completely disassembled the GA-associated AKAP450 network without affecting the centrosomal fraction (Fig. 2, A and E). The large N-terminal AK1 mutant induced a dramatic change in the position of the GA relative to the CTR (Fig. 2 A). The GA appeared either as a single structure or fragmented in two or three large elements that remained together and connected by membrane tubules away from the CTR. Fig. 2 B shows the GA surrounding the CTR in a nontransfected cell. No significant perturbation of the CTR-nucleated MT network was observed in transfected cells (Fig. 2 C). AK1-containing cytoplasmic punctate or fibrillar structures aligned with MTs were also observed (Fig. 2 D). These structures became randomly distributed when MTs were depolymerized (see Fig. 4 and Fig. S2), suggesting that the AKAP450 N-terminal domain associates with MTs. In contrast, AK1B expression resulted in the collapse of the GA around the CTR (Fig. 2 E); the GA changed from an extended ribbon to a circular morphology around the CTR (compare Fig. 2, E and B). MT aster also appeared to be more focused (Fig. 2 F). These effects on GA were reminiscent of that induced by AKAP450 siRNA (Fig. S4 B), suggesting that this fragment behaves as a dominant-negative mutant. It should be noted that AK2, AK3, and AK4 fragment expression, all of them consisting of long coiled-coil domains, did not affect GA morphology or positioning regardless of their expression levels (Fig. S1), thus confirming the specificity of AK1- and AK1B-induced effects on GA.


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)

AK1 and AK1B fragments differentially affect GA positioning and morphology. (A) RPE-1 cells expressing the flag-AK1 fragment were triple labeled for flag, AKAP450, and GMAP210. Single labelings and the merge are shown. White arrows and arrowheads indicate the GA and the CTR, respectively. In B, a nontransfected cell is shown for comparison. N, nucleus. (C) Merged image of a flag-AK1–transfected cell stained for flag, α-tubulin, and GMAP210 to visualize the MT network. (D) High magnification images of a flag-AK1–transfected cell triple labeled as in C. Single labelings in black and white and the merge are shown. Yellow arrows indicate the alignment of AK1B-containing structures with MTs. (E and F) Merged images of flag-AK1B–expressing cells triple labeled as in A or C, respectively. (G) Low magnification images of flag-AK1 (top)– and flag-AK1B (bottom)–transfected cells triple labeled for flag, AKAP450, and GMAP210. Single labelings for flag are shown on the left to identify transfected cells (T). (right) Schematic of the measurement of CTR–GA distance and GA diameter over the corresponding AKAP450 and GMAP210 merged images. A straight line connecting the center of the circumferences circumscribing the GA and the CTR was measured to calculate the CTR–GA distance. GA diameter was used as an index of GA circularity. NT, not transfected. (H) Graphs showing quantification of the CTR–GA distance (top) and Golgi circularity index (bottom; n = 160 for each condition from three independent experiments). Data are presented in the same box and whisker format as in Fig 1 with the median marked as a solid line (*, P < 0.001; Tukey HSD). Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. Bars, 2.5 µm.
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fig2: AK1 and AK1B fragments differentially affect GA positioning and morphology. (A) RPE-1 cells expressing the flag-AK1 fragment were triple labeled for flag, AKAP450, and GMAP210. Single labelings and the merge are shown. White arrows and arrowheads indicate the GA and the CTR, respectively. In B, a nontransfected cell is shown for comparison. N, nucleus. (C) Merged image of a flag-AK1–transfected cell stained for flag, α-tubulin, and GMAP210 to visualize the MT network. (D) High magnification images of a flag-AK1–transfected cell triple labeled as in C. Single labelings in black and white and the merge are shown. Yellow arrows indicate the alignment of AK1B-containing structures with MTs. (E and F) Merged images of flag-AK1B–expressing cells triple labeled as in A or C, respectively. (G) Low magnification images of flag-AK1 (top)– and flag-AK1B (bottom)–transfected cells triple labeled for flag, AKAP450, and GMAP210. Single labelings for flag are shown on the left to identify transfected cells (T). (right) Schematic of the measurement of CTR–GA distance and GA diameter over the corresponding AKAP450 and GMAP210 merged images. A straight line connecting the center of the circumferences circumscribing the GA and the CTR was measured to calculate the CTR–GA distance. GA diameter was used as an index of GA circularity. NT, not transfected. (H) Graphs showing quantification of the CTR–GA distance (top) and Golgi circularity index (bottom; n = 160 for each condition from three independent experiments). Data are presented in the same box and whisker format as in Fig 1 with the median marked as a solid line (*, P < 0.001; Tukey HSD). Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. Bars, 2.5 µm.
Mentions: The expression of both AK1 and AK1B domains completely disassembled the GA-associated AKAP450 network without affecting the centrosomal fraction (Fig. 2, A and E). The large N-terminal AK1 mutant induced a dramatic change in the position of the GA relative to the CTR (Fig. 2 A). The GA appeared either as a single structure or fragmented in two or three large elements that remained together and connected by membrane tubules away from the CTR. Fig. 2 B shows the GA surrounding the CTR in a nontransfected cell. No significant perturbation of the CTR-nucleated MT network was observed in transfected cells (Fig. 2 C). AK1-containing cytoplasmic punctate or fibrillar structures aligned with MTs were also observed (Fig. 2 D). These structures became randomly distributed when MTs were depolymerized (see Fig. 4 and Fig. S2), suggesting that the AKAP450 N-terminal domain associates with MTs. In contrast, AK1B expression resulted in the collapse of the GA around the CTR (Fig. 2 E); the GA changed from an extended ribbon to a circular morphology around the CTR (compare Fig. 2, E and B). MT aster also appeared to be more focused (Fig. 2 F). These effects on GA were reminiscent of that induced by AKAP450 siRNA (Fig. S4 B), suggesting that this fragment behaves as a dominant-negative mutant. It should be noted that AK2, AK3, and AK4 fragment expression, all of them consisting of long coiled-coil domains, did not affect GA morphology or positioning regardless of their expression levels (Fig. S1), thus confirming the specificity of AK1- and AK1B-induced effects on GA.

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