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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 redistributes to the cell periphery during cell migration and is necessary for cell migration. (A) Distribution of Gαi3 in quiescent (a–f) versus migrating (g–j) HeLa cells after scratch wounding. In quiescent cells, endogenous Gαi3 colocalizes with βGalT in the Golgi (a–c) but not actin (d–f). In migrating cells, Gαi3 is found in puncta that partially colocalize with actin at the leading edge (g–i). Arrows denote direction of migration. Boxed area in i is enlarged in j. HeLa cells were aldehyde fixed 0 (left) and 8 (right) h after scratch wounding, as shown in the diagram, and stained as indicated. Bars,10 μm. (B) Depletion of Gαi3 (g and h) or GIV (e and f), but not Gαs (c and d), impairs wound healing (compare with scrambled siRNA [scr siRNA] controls; a and b). Repletion of hGαi3 by rat Gαi3 (i and j), but not vector alone (k and l), restores this defect. HeLa cells treated as indicated were subjected to scratch wounding and examined immediately after wounding (0 h) or 16 h later (n = 5). (C) Cell lysates from cells treated as in B were immunoblotted to assess the efficiency of siRNA depletion of Gαs (∼90%), GIV (∼85%), or Gαi3 (∼95%) and the expression of rGαi3. Several GIV bands are identified that most likely are different posttranslationally modified forms of GIV because they are specifically depleted using GIV siRNA. (D) Comparative trajectories of Gαi3-depleted cells versus those transfected with Gαi3-YFP were followed by live cell imaging for 8 h after wounding. Representative cells at the advancing edge of the wound (dashed line) were traced using a cell tracker application on videos obtained of transfected (solid squiggles) or untransfected (circles) cells through the YFP channel (b) or a simultaneously recorded DIC channel (a; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200712066/DC1). (E) Bar graphs showing the distance covered by cells in D analyzed by the cell-tracker application (top graph) and the percent of untransfected Gαi3-depleted cells versus Gαi3-depleted cells transfected with rGαi3-YFP that displayed directional migration (bottom graph). Results are shown as mean ± SEM (n = 3).
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fig1: Gαi3 redistributes to the cell periphery during cell migration and is necessary for cell migration. (A) Distribution of Gαi3 in quiescent (a–f) versus migrating (g–j) HeLa cells after scratch wounding. In quiescent cells, endogenous Gαi3 colocalizes with βGalT in the Golgi (a–c) but not actin (d–f). In migrating cells, Gαi3 is found in puncta that partially colocalize with actin at the leading edge (g–i). Arrows denote direction of migration. Boxed area in i is enlarged in j. HeLa cells were aldehyde fixed 0 (left) and 8 (right) h after scratch wounding, as shown in the diagram, and stained as indicated. Bars,10 μm. (B) Depletion of Gαi3 (g and h) or GIV (e and f), but not Gαs (c and d), impairs wound healing (compare with scrambled siRNA [scr siRNA] controls; a and b). Repletion of hGαi3 by rat Gαi3 (i and j), but not vector alone (k and l), restores this defect. HeLa cells treated as indicated were subjected to scratch wounding and examined immediately after wounding (0 h) or 16 h later (n = 5). (C) Cell lysates from cells treated as in B were immunoblotted to assess the efficiency of siRNA depletion of Gαs (∼90%), GIV (∼85%), or Gαi3 (∼95%) and the expression of rGαi3. Several GIV bands are identified that most likely are different posttranslationally modified forms of GIV because they are specifically depleted using GIV siRNA. (D) Comparative trajectories of Gαi3-depleted cells versus those transfected with Gαi3-YFP were followed by live cell imaging for 8 h after wounding. Representative cells at the advancing edge of the wound (dashed line) were traced using a cell tracker application on videos obtained of transfected (solid squiggles) or untransfected (circles) cells through the YFP channel (b) or a simultaneously recorded DIC channel (a; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200712066/DC1). (E) Bar graphs showing the distance covered by cells in D analyzed by the cell-tracker application (top graph) and the percent of untransfected Gαi3-depleted cells versus Gαi3-depleted cells transfected with rGαi3-YFP that displayed directional migration (bottom graph). Results are shown as mean ± SEM (n = 3).

Mentions: Both GIV and Gαi3 are localized on Golgi membranes and the plasma membrane (PM; Stow et al., 1991; Denker et al., 1996; Enomoto et al., 2005; Le-Niculescu et al., 2005). We reported previously that GIV is predominantly on vesicles near the Golgi during quiescence (Le-Niculescu et al., 2005), and others reported that during cell migration it accumulates at the leading edge where it interacts with Akt and participates in actin remodeling (Enomoto et al., 2005). Because key participants in cell migration (GIV, PI3K, Akt, and actin; Merlot and Firtel, 2003; Enomoto et al., 2005) are enriched at the leading edge, we asked if Gαi3 behaves similarly. We subjected confluent monolayers of HeLa cells to scratch wounding to induce unidirectional cell migration (Kupfer et al., 1982) and examined the distribution of endogenous Gαi3 by immunofluorescence (IF). In quiescent cells far from the wound, Gαi3 showed a predominant Golgi localization based on colocalization with a Golgi marker, β1-4 galactosyltransferase (β-GALT; Fig. 1 A, a–c), and was almost undetectable at the cell periphery marked by actin (Fig. 1 A, d–f). In contrast, migrating cells at the edge of the wound showed peripheral Gαi3-stained puncta within lamellipodial extensions. Some of these puncta colocalized with actin at the leading edge (Fig. 1 A, g–j). Thus, Gαi3 showed different distributions depending on the migratory state of the cell.


Activation of Galphai3 triggers cell migration via regulation of GIV.

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

Gαi3 redistributes to the cell periphery during cell migration and is necessary for cell migration. (A) Distribution of Gαi3 in quiescent (a–f) versus migrating (g–j) HeLa cells after scratch wounding. In quiescent cells, endogenous Gαi3 colocalizes with βGalT in the Golgi (a–c) but not actin (d–f). In migrating cells, Gαi3 is found in puncta that partially colocalize with actin at the leading edge (g–i). Arrows denote direction of migration. Boxed area in i is enlarged in j. HeLa cells were aldehyde fixed 0 (left) and 8 (right) h after scratch wounding, as shown in the diagram, and stained as indicated. Bars,10 μm. (B) Depletion of Gαi3 (g and h) or GIV (e and f), but not Gαs (c and d), impairs wound healing (compare with scrambled siRNA [scr siRNA] controls; a and b). Repletion of hGαi3 by rat Gαi3 (i and j), but not vector alone (k and l), restores this defect. HeLa cells treated as indicated were subjected to scratch wounding and examined immediately after wounding (0 h) or 16 h later (n = 5). (C) Cell lysates from cells treated as in B were immunoblotted to assess the efficiency of siRNA depletion of Gαs (∼90%), GIV (∼85%), or Gαi3 (∼95%) and the expression of rGαi3. Several GIV bands are identified that most likely are different posttranslationally modified forms of GIV because they are specifically depleted using GIV siRNA. (D) Comparative trajectories of Gαi3-depleted cells versus those transfected with Gαi3-YFP were followed by live cell imaging for 8 h after wounding. Representative cells at the advancing edge of the wound (dashed line) were traced using a cell tracker application on videos obtained of transfected (solid squiggles) or untransfected (circles) cells through the YFP channel (b) or a simultaneously recorded DIC channel (a; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200712066/DC1). (E) Bar graphs showing the distance covered by cells in D analyzed by the cell-tracker application (top graph) and the percent of untransfected Gαi3-depleted cells versus Gαi3-depleted cells transfected with rGαi3-YFP that displayed directional migration (bottom graph). Results are shown as mean ± SEM (n = 3).
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fig1: Gαi3 redistributes to the cell periphery during cell migration and is necessary for cell migration. (A) Distribution of Gαi3 in quiescent (a–f) versus migrating (g–j) HeLa cells after scratch wounding. In quiescent cells, endogenous Gαi3 colocalizes with βGalT in the Golgi (a–c) but not actin (d–f). In migrating cells, Gαi3 is found in puncta that partially colocalize with actin at the leading edge (g–i). Arrows denote direction of migration. Boxed area in i is enlarged in j. HeLa cells were aldehyde fixed 0 (left) and 8 (right) h after scratch wounding, as shown in the diagram, and stained as indicated. Bars,10 μm. (B) Depletion of Gαi3 (g and h) or GIV (e and f), but not Gαs (c and d), impairs wound healing (compare with scrambled siRNA [scr siRNA] controls; a and b). Repletion of hGαi3 by rat Gαi3 (i and j), but not vector alone (k and l), restores this defect. HeLa cells treated as indicated were subjected to scratch wounding and examined immediately after wounding (0 h) or 16 h later (n = 5). (C) Cell lysates from cells treated as in B were immunoblotted to assess the efficiency of siRNA depletion of Gαs (∼90%), GIV (∼85%), or Gαi3 (∼95%) and the expression of rGαi3. Several GIV bands are identified that most likely are different posttranslationally modified forms of GIV because they are specifically depleted using GIV siRNA. (D) Comparative trajectories of Gαi3-depleted cells versus those transfected with Gαi3-YFP were followed by live cell imaging for 8 h after wounding. Representative cells at the advancing edge of the wound (dashed line) were traced using a cell tracker application on videos obtained of transfected (solid squiggles) or untransfected (circles) cells through the YFP channel (b) or a simultaneously recorded DIC channel (a; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200712066/DC1). (E) Bar graphs showing the distance covered by cells in D analyzed by the cell-tracker application (top graph) and the percent of untransfected Gαi3-depleted cells versus Gαi3-depleted cells transfected with rGαi3-YFP that displayed directional migration (bottom graph). Results are shown as mean ± SEM (n = 3).
Mentions: Both GIV and Gαi3 are localized on Golgi membranes and the plasma membrane (PM; Stow et al., 1991; Denker et al., 1996; Enomoto et al., 2005; Le-Niculescu et al., 2005). We reported previously that GIV is predominantly on vesicles near the Golgi during quiescence (Le-Niculescu et al., 2005), and others reported that during cell migration it accumulates at the leading edge where it interacts with Akt and participates in actin remodeling (Enomoto et al., 2005). Because key participants in cell migration (GIV, PI3K, Akt, and actin; Merlot and Firtel, 2003; Enomoto et al., 2005) are enriched at the leading edge, we asked if Gαi3 behaves similarly. We subjected confluent monolayers of HeLa cells to scratch wounding to induce unidirectional cell migration (Kupfer et al., 1982) and examined the distribution of endogenous Gαi3 by immunofluorescence (IF). In quiescent cells far from the wound, Gαi3 showed a predominant Golgi localization based on colocalization with a Golgi marker, β1-4 galactosyltransferase (β-GALT; Fig. 1 A, a–c), and was almost undetectable at the cell periphery marked by actin (Fig. 1 A, d–f). In contrast, migrating cells at the edge of the wound showed peripheral Gαi3-stained puncta within lamellipodial extensions. Some of these puncta colocalized with actin at the leading edge (Fig. 1 A, g–j). Thus, Gαi3 showed different distributions depending on the migratory state of the cell.

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