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Activation of Galphai3 triggers cell migration via regulation of GIV.

Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG - J. Cell Biol. (2008)

Bottom Line: We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate.A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane.Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Galphai3 binding partner. We demonstrate that in mammalian cells Galphai3 controls the functions of GIV during cell migration. We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate. A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Galphai3 serves as a molecular switch that triggers dissociation of Gbetagamma and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

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Gαi3 and GIV localize at the centrosome/MTOC and Gαi3 is necessary for centrosome repositioning. (A) Deconvolved images through the centrosomes demonstrating the localization of Gαi3 and GIV on centrosomes. HeLa and Cos7 cells were permeabilized, fixed, and costained with a centrosome marker (γ-tubulin or pericentrin) and Gαi3 (a–f), GIV (j–l), or overexpressed V5-GIV (g–i). Bar,10 μm. (B) Schematic illustration (bottom) of the centrosome repositioning assay performed on HeLa cells expressing GFP-Centrin1. Cells located at the edge of a wound are scored positive in which the centrosome (GFP-Centrin1, green) and Golgi (Man II, red) are positioned in front of the nucleus (DAPI, blue) within the 120° trident facing the direction of the wound (arrow). The white box indicates the area magnified below. Bar, 10 μm. (C) Bar graph showing the percentage of cells at the wound edge that achieved centrosome repositioning by 8 h after wounding. Gαi3-depleted cells failed to reposition their centrosomes and transfection of Gαi3wt restored this defect. GIV or Gαs-depleted cells were similar to controls (64%). Results (200–400 cells per experiment; n = 3) are shown as mean ± SEM. *, P < 0.001; #, P > 0.001 (compared with scr siRNA cells).
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fig3: Gαi3 and GIV localize at the centrosome/MTOC and Gαi3 is necessary for centrosome repositioning. (A) Deconvolved images through the centrosomes demonstrating the localization of Gαi3 and GIV on centrosomes. HeLa and Cos7 cells were permeabilized, fixed, and costained with a centrosome marker (γ-tubulin or pericentrin) and Gαi3 (a–f), GIV (j–l), or overexpressed V5-GIV (g–i). Bar,10 μm. (B) Schematic illustration (bottom) of the centrosome repositioning assay performed on HeLa cells expressing GFP-Centrin1. Cells located at the edge of a wound are scored positive in which the centrosome (GFP-Centrin1, green) and Golgi (Man II, red) are positioned in front of the nucleus (DAPI, blue) within the 120° trident facing the direction of the wound (arrow). The white box indicates the area magnified below. Bar, 10 μm. (C) Bar graph showing the percentage of cells at the wound edge that achieved centrosome repositioning by 8 h after wounding. Gαi3-depleted cells failed to reposition their centrosomes and transfection of Gαi3wt restored this defect. GIV or Gαs-depleted cells were similar to controls (64%). Results (200–400 cells per experiment; n = 3) are shown as mean ± SEM. *, P < 0.001; #, P > 0.001 (compared with scr siRNA cells).

Mentions: Because Gαi3-depleted cells failed to undergo directional migration, we looked for defects in centrosome reorientation at the wound edge using HeLa cells stably expressing the centrosome marker Centrin1-GFP and quantified as described previously (Fig. 3 B; Kupfer et al., 1982; Etienne-Manneville and Hall, 2001). We observed that although ∼64% of control siRNA-treated cells repositioned the centrosome in front of the nucleus looking toward the wound, only ∼34% of Gαi3-depleted cells (as was expected in the absence of polarization) achieved the same phenotype (Fig. 3 C). GIV or Gαs-depleted cells were similar to controls. When rGαi3wt was introduced into Gαi3-depleted cells, the defect in centrosome repositioning was reversed. Using deconvolution microscopy to visualize the plane of the centrosome, we found that both endogenous Gαi3 (Fig. 3 A, a–f) and endogenous (Fig. 3 A, j–l) or overexpressed GIV (Fig. 3 A, g–i) localized at the centrosome with bona fide centrosomal proteins (pericentrin or γ-tubulin) in HeLa and Cos7 cells. Our finding that both Gαi3 and GIV localize to the centrosome region and only Gαi3 is required for centrosomal repositioning suggests that the latter is a GIV-independent function of Gαi3.


Activation of Galphai3 triggers cell migration via regulation of GIV.

Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG - J. Cell Biol. (2008)

Gαi3 and GIV localize at the centrosome/MTOC and Gαi3 is necessary for centrosome repositioning. (A) Deconvolved images through the centrosomes demonstrating the localization of Gαi3 and GIV on centrosomes. HeLa and Cos7 cells were permeabilized, fixed, and costained with a centrosome marker (γ-tubulin or pericentrin) and Gαi3 (a–f), GIV (j–l), or overexpressed V5-GIV (g–i). Bar,10 μm. (B) Schematic illustration (bottom) of the centrosome repositioning assay performed on HeLa cells expressing GFP-Centrin1. Cells located at the edge of a wound are scored positive in which the centrosome (GFP-Centrin1, green) and Golgi (Man II, red) are positioned in front of the nucleus (DAPI, blue) within the 120° trident facing the direction of the wound (arrow). The white box indicates the area magnified below. Bar, 10 μm. (C) Bar graph showing the percentage of cells at the wound edge that achieved centrosome repositioning by 8 h after wounding. Gαi3-depleted cells failed to reposition their centrosomes and transfection of Gαi3wt restored this defect. GIV or Gαs-depleted cells were similar to controls (64%). Results (200–400 cells per experiment; n = 3) are shown as mean ± SEM. *, P < 0.001; #, P > 0.001 (compared with scr siRNA cells).
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fig3: Gαi3 and GIV localize at the centrosome/MTOC and Gαi3 is necessary for centrosome repositioning. (A) Deconvolved images through the centrosomes demonstrating the localization of Gαi3 and GIV on centrosomes. HeLa and Cos7 cells were permeabilized, fixed, and costained with a centrosome marker (γ-tubulin or pericentrin) and Gαi3 (a–f), GIV (j–l), or overexpressed V5-GIV (g–i). Bar,10 μm. (B) Schematic illustration (bottom) of the centrosome repositioning assay performed on HeLa cells expressing GFP-Centrin1. Cells located at the edge of a wound are scored positive in which the centrosome (GFP-Centrin1, green) and Golgi (Man II, red) are positioned in front of the nucleus (DAPI, blue) within the 120° trident facing the direction of the wound (arrow). The white box indicates the area magnified below. Bar, 10 μm. (C) Bar graph showing the percentage of cells at the wound edge that achieved centrosome repositioning by 8 h after wounding. Gαi3-depleted cells failed to reposition their centrosomes and transfection of Gαi3wt restored this defect. GIV or Gαs-depleted cells were similar to controls (64%). Results (200–400 cells per experiment; n = 3) are shown as mean ± SEM. *, P < 0.001; #, P > 0.001 (compared with scr siRNA cells).
Mentions: Because Gαi3-depleted cells failed to undergo directional migration, we looked for defects in centrosome reorientation at the wound edge using HeLa cells stably expressing the centrosome marker Centrin1-GFP and quantified as described previously (Fig. 3 B; Kupfer et al., 1982; Etienne-Manneville and Hall, 2001). We observed that although ∼64% of control siRNA-treated cells repositioned the centrosome in front of the nucleus looking toward the wound, only ∼34% of Gαi3-depleted cells (as was expected in the absence of polarization) achieved the same phenotype (Fig. 3 C). GIV or Gαs-depleted cells were similar to controls. When rGαi3wt was introduced into Gαi3-depleted cells, the defect in centrosome repositioning was reversed. Using deconvolution microscopy to visualize the plane of the centrosome, we found that both endogenous Gαi3 (Fig. 3 A, a–f) and endogenous (Fig. 3 A, j–l) or overexpressed GIV (Fig. 3 A, g–i) localized at the centrosome with bona fide centrosomal proteins (pericentrin or γ-tubulin) in HeLa and Cos7 cells. Our finding that both Gαi3 and GIV localize to the centrosome region and only Gαi3 is required for centrosomal repositioning suggests that the latter is a GIV-independent function of Gαi3.

Bottom Line: We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate.A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane.Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Galphai3 binding partner. We demonstrate that in mammalian cells Galphai3 controls the functions of GIV during cell migration. We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate. A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Galphai3 serves as a molecular switch that triggers dissociation of Gbetagamma and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

Show MeSH
Related in: MedlinePlus