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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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Related in: MedlinePlus

Both AK1 and AK1B contain the GA-binding GM130-interacting domain, but only AK1 binds MTs. (A) GFP-AK1–, GFP-AK1B–, and GFP-AK2–expressing cell extracts were immunoprecipitated with the anti-GM130 antibody, and blots were revealed for GM130 and GFP. Ctl, control; SP, supernatant. (B) Co-IP from flag-AK1B and YFP-GM130 double-transfected cell extracts using an anti-flag antibody. Blots were revealed for flag and GM130. (C) Control (left) or GM130-depleted (right) cells were transfected with the flag-AK1B construct and then NZ treated to fragment the GA into stacks. Labelings were for flag, GM130, and GMAP210 as indicated. Merged images are shown. Enlarged boxes on the right show single labelings or merges corresponding to outlined areas. T, transfected; D, depleted. Arrows indicate GA ministacks. Bars, 5 µm. (D) Cells coexpressing GFP- and flag-tagged versions of AK1 (left) or AK1B (right) were immunoprecipitated with an anti-GFP antibody, and blots were revealed for GFP and flag. (E) GFP-AK1–transfected cell extracts were immunoprecipitated either with anti-p150glued or anti-GFP antibodies (left). IPs were then tested for the presence of GFP-AK1 or p150glued. (right) A similar anti-GFP IP from GFP-AK1B–expressing cells is shown. Black lines separate parts from different gels. (F) MTs were polymerized from RPE-1 cell lysates expressing GFP-AK1 or GFP-AK1B in the presence of exogenous tubulin and taxol. After centrifugation through a sucrose cushion, supernatants (SP) and pellets (MT) were analyzed by WB for GFP, α-tubulin, and p150glued as a positive control. (G) A summary of properties of AKAP450 and the N-terminal AK1 and AK1B fragments. αtub, α-tubulin; γTuSC, γ-tubulin small complex.
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fig3: Both AK1 and AK1B contain the GA-binding GM130-interacting domain, but only AK1 binds MTs. (A) GFP-AK1–, GFP-AK1B–, and GFP-AK2–expressing cell extracts were immunoprecipitated with the anti-GM130 antibody, and blots were revealed for GM130 and GFP. Ctl, control; SP, supernatant. (B) Co-IP from flag-AK1B and YFP-GM130 double-transfected cell extracts using an anti-flag antibody. Blots were revealed for flag and GM130. (C) Control (left) or GM130-depleted (right) cells were transfected with the flag-AK1B construct and then NZ treated to fragment the GA into stacks. Labelings were for flag, GM130, and GMAP210 as indicated. Merged images are shown. Enlarged boxes on the right show single labelings or merges corresponding to outlined areas. T, transfected; D, depleted. Arrows indicate GA ministacks. Bars, 5 µm. (D) Cells coexpressing GFP- and flag-tagged versions of AK1 (left) or AK1B (right) were immunoprecipitated with an anti-GFP antibody, and blots were revealed for GFP and flag. (E) GFP-AK1–transfected cell extracts were immunoprecipitated either with anti-p150glued or anti-GFP antibodies (left). IPs were then tested for the presence of GFP-AK1 or p150glued. (right) A similar anti-GFP IP from GFP-AK1B–expressing cells is shown. Black lines separate parts from different gels. (F) MTs were polymerized from RPE-1 cell lysates expressing GFP-AK1 or GFP-AK1B in the presence of exogenous tubulin and taxol. After centrifugation through a sucrose cushion, supernatants (SP) and pellets (MT) were analyzed by WB for GFP, α-tubulin, and p150glued as a positive control. (G) A summary of properties of AKAP450 and the N-terminal AK1 and AK1B fragments. αtub, α-tubulin; γTuSC, γ-tubulin small complex.

Mentions: Endogenous GM130 was able to pull down both GFP-AK1 and GFP-AK1B (Fig. 3 A). A reciprocal coimmunoprecipitation (IP; co-IP) experiment showed an interaction between GFP-AK1B and YFP-GM130 in double-transfected cells (Fig. 3 B). No interaction was detected with the other partial constructs that do not target the GA, namely GFP-AK2 (Fig. 3 A), GFP-AK3, or GFP-AK4 fragments (Fig. S4 A). These experiments demonstrate a specific interaction of both AK1 and AK1B with GM130.


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)

Both AK1 and AK1B contain the GA-binding GM130-interacting domain, but only AK1 binds MTs. (A) GFP-AK1–, GFP-AK1B–, and GFP-AK2–expressing cell extracts were immunoprecipitated with the anti-GM130 antibody, and blots were revealed for GM130 and GFP. Ctl, control; SP, supernatant. (B) Co-IP from flag-AK1B and YFP-GM130 double-transfected cell extracts using an anti-flag antibody. Blots were revealed for flag and GM130. (C) Control (left) or GM130-depleted (right) cells were transfected with the flag-AK1B construct and then NZ treated to fragment the GA into stacks. Labelings were for flag, GM130, and GMAP210 as indicated. Merged images are shown. Enlarged boxes on the right show single labelings or merges corresponding to outlined areas. T, transfected; D, depleted. Arrows indicate GA ministacks. Bars, 5 µm. (D) Cells coexpressing GFP- and flag-tagged versions of AK1 (left) or AK1B (right) were immunoprecipitated with an anti-GFP antibody, and blots were revealed for GFP and flag. (E) GFP-AK1–transfected cell extracts were immunoprecipitated either with anti-p150glued or anti-GFP antibodies (left). IPs were then tested for the presence of GFP-AK1 or p150glued. (right) A similar anti-GFP IP from GFP-AK1B–expressing cells is shown. Black lines separate parts from different gels. (F) MTs were polymerized from RPE-1 cell lysates expressing GFP-AK1 or GFP-AK1B in the presence of exogenous tubulin and taxol. After centrifugation through a sucrose cushion, supernatants (SP) and pellets (MT) were analyzed by WB for GFP, α-tubulin, and p150glued as a positive control. (G) A summary of properties of AKAP450 and the N-terminal AK1 and AK1B fragments. αtub, α-tubulin; γTuSC, γ-tubulin small complex.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Both AK1 and AK1B contain the GA-binding GM130-interacting domain, but only AK1 binds MTs. (A) GFP-AK1–, GFP-AK1B–, and GFP-AK2–expressing cell extracts were immunoprecipitated with the anti-GM130 antibody, and blots were revealed for GM130 and GFP. Ctl, control; SP, supernatant. (B) Co-IP from flag-AK1B and YFP-GM130 double-transfected cell extracts using an anti-flag antibody. Blots were revealed for flag and GM130. (C) Control (left) or GM130-depleted (right) cells were transfected with the flag-AK1B construct and then NZ treated to fragment the GA into stacks. Labelings were for flag, GM130, and GMAP210 as indicated. Merged images are shown. Enlarged boxes on the right show single labelings or merges corresponding to outlined areas. T, transfected; D, depleted. Arrows indicate GA ministacks. Bars, 5 µm. (D) Cells coexpressing GFP- and flag-tagged versions of AK1 (left) or AK1B (right) were immunoprecipitated with an anti-GFP antibody, and blots were revealed for GFP and flag. (E) GFP-AK1–transfected cell extracts were immunoprecipitated either with anti-p150glued or anti-GFP antibodies (left). IPs were then tested for the presence of GFP-AK1 or p150glued. (right) A similar anti-GFP IP from GFP-AK1B–expressing cells is shown. Black lines separate parts from different gels. (F) MTs were polymerized from RPE-1 cell lysates expressing GFP-AK1 or GFP-AK1B in the presence of exogenous tubulin and taxol. After centrifugation through a sucrose cushion, supernatants (SP) and pellets (MT) were analyzed by WB for GFP, α-tubulin, and p150glued as a positive control. (G) A summary of properties of AKAP450 and the N-terminal AK1 and AK1B fragments. αtub, α-tubulin; γTuSC, γ-tubulin small complex.
Mentions: Endogenous GM130 was able to pull down both GFP-AK1 and GFP-AK1B (Fig. 3 A). A reciprocal coimmunoprecipitation (IP; co-IP) experiment showed an interaction between GFP-AK1B and YFP-GM130 in double-transfected cells (Fig. 3 B). No interaction was detected with the other partial constructs that do not target the GA, namely GFP-AK2 (Fig. 3 A), GFP-AK3, or GFP-AK4 fragments (Fig. S4 A). These experiments demonstrate a specific interaction of both AK1 and AK1B with GM130.

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