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Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins.

Lopez-Sanchez I, Kalogriopoulos N, Lo IC, Kabir F, Midde KK, Wang H, Ghosh P - Mol. Biol. Cell (2015)

Bottom Line: As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi.Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs.Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA 92093 inmalopezsanchez@hotmail.com prghosh@ucsd.edu.

No MeSH data available.


Related in: MedlinePlus

GIV localizes to FAs and is essential for the integrity of these structures. (A, B) Cos7 cells grown on collagen-coated coverslips were fixed and costained with GIV (green), vinculin (red), and DAPI (nuclei; blue; A) or with vinculin (green), phalloidin–Texas red (F-actin, red), and DAPI (nuclei; blue; B) and analyzed by confocal microscopy. Representative images. Bar, 10 μm. (C) Cos7 cells grown on either noncoated (left) or collagen-coated (right) glass coverslips, fixed, and costained with phospho-Tyr-1764-GIV (pYGIV; red), vinculin (green), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Arrowheads indicated focal complexes; arrows indicate mature FAs. Representative images. Bar, 10 μm. (D) Cos7 cells were fixed and costained with phospho-Tyr-1764-GIV (pYGIV; green), phalloidin–Texas red (F-actin; red), paxillin (far red; pseudocolored purple), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Representative images are shown. Bar, 25 μm. (E) Schematic summarizing the findings in A–D. (F, G) Control (sh Control) or GIV-depleted (sh GIV) Cos7 cells were fixed and stained for vinculin (red, F) or paxillin (red, G) and DAPI (nuclei; blue) and analyzed by confocal microscopy. In GIV-depleted cells we found that vinculin was present on vesicular structures (arrowheads). Representative images are shown. Bar, 25 μm. Depletion of GIV was confirmed by immunoblotting (Supplemental Figure S1D). (H) Cos7 cells in F and G were analyzed for collagen-induced haptotactic cell motility using Transwell assays. Bar graphs show quantification of the number of migrated cells/high-power fields (HPF). Error bars represent mean ± SD; n = 3; *p < 0.05.
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Figure 1: GIV localizes to FAs and is essential for the integrity of these structures. (A, B) Cos7 cells grown on collagen-coated coverslips were fixed and costained with GIV (green), vinculin (red), and DAPI (nuclei; blue; A) or with vinculin (green), phalloidin–Texas red (F-actin, red), and DAPI (nuclei; blue; B) and analyzed by confocal microscopy. Representative images. Bar, 10 μm. (C) Cos7 cells grown on either noncoated (left) or collagen-coated (right) glass coverslips, fixed, and costained with phospho-Tyr-1764-GIV (pYGIV; red), vinculin (green), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Arrowheads indicated focal complexes; arrows indicate mature FAs. Representative images. Bar, 10 μm. (D) Cos7 cells were fixed and costained with phospho-Tyr-1764-GIV (pYGIV; green), phalloidin–Texas red (F-actin; red), paxillin (far red; pseudocolored purple), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Representative images are shown. Bar, 25 μm. (E) Schematic summarizing the findings in A–D. (F, G) Control (sh Control) or GIV-depleted (sh GIV) Cos7 cells were fixed and stained for vinculin (red, F) or paxillin (red, G) and DAPI (nuclei; blue) and analyzed by confocal microscopy. In GIV-depleted cells we found that vinculin was present on vesicular structures (arrowheads). Representative images are shown. Bar, 25 μm. Depletion of GIV was confirmed by immunoblotting (Supplemental Figure S1D). (H) Cos7 cells in F and G were analyzed for collagen-induced haptotactic cell motility using Transwell assays. Bar graphs show quantification of the number of migrated cells/high-power fields (HPF). Error bars represent mean ± SD; n = 3; *p < 0.05.

Mentions: We previously showed that GIV modulates growth factor RTK signaling via its ability to bind ligand-activated RTKs and couple them to activation of G proteins at the PM (Midde et al., 2015). To study specifically the localization of GIV at the PM without the noise contributed by the larger cytosolic pool, we carried out immunofluorescence studies on cells fixed using a low-temperature methanol-fixation treatment that leads to improved accessibility of the intracellular antigens and reduces cytosolic background (Schnell et al., 2012). GIV localized to the Golgi, the nucleus, and actin stress fibers (Figure 1A), all previously reported by us and others (Enomoto et al., 2005; Ghosh et al., 2008). A pool of actin-bound GIV colocalized with vinculin, indicating that GIV localizes also on PM patches where actin caps associate with FAs (Figure 1B); this pattern is specific, because it was lost in GIV-depleted cells (Supplemental Figure S1A). When we immunostained for the tyrosine-phosphorylated pool of “active” GIV (pYGIV), as determined by a diagnostic-grade antibody previously validated and confirmed to specifically detect phospho-Tyr-1764-GIV (see Materials and Methods), we found it to be almost exclusively colocalized with vinculin in FAs: in the absence of collagen, pYGIV and vinculin colocalized predominantly in the peripherally located “environment-probing” focal complexes (Figure 1C, left), whereas in the presence of collagen, they colocalized primarily within mature FAs (Figure 1C, right). Localization of GIV at FAs was further confirmed using paxillin, another marker of FAs (Figure 1D and Supplemental Figure S1B). Finally, tyrosine-phosphorylated GIV localized to FAs in cells stimulated exclusively with collagen in the absence of serum (Supplemental Figure S1C), suggesting that integrin signaling is sufficient for both tyrosine phosphorylation and localization of GIV at FAs. These results indicate that a pool of total GIV and the majority of “active” pYGIV localize discretely to nascent and mature FAs (Figure 1E).


Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins.

Lopez-Sanchez I, Kalogriopoulos N, Lo IC, Kabir F, Midde KK, Wang H, Ghosh P - Mol. Biol. Cell (2015)

GIV localizes to FAs and is essential for the integrity of these structures. (A, B) Cos7 cells grown on collagen-coated coverslips were fixed and costained with GIV (green), vinculin (red), and DAPI (nuclei; blue; A) or with vinculin (green), phalloidin–Texas red (F-actin, red), and DAPI (nuclei; blue; B) and analyzed by confocal microscopy. Representative images. Bar, 10 μm. (C) Cos7 cells grown on either noncoated (left) or collagen-coated (right) glass coverslips, fixed, and costained with phospho-Tyr-1764-GIV (pYGIV; red), vinculin (green), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Arrowheads indicated focal complexes; arrows indicate mature FAs. Representative images. Bar, 10 μm. (D) Cos7 cells were fixed and costained with phospho-Tyr-1764-GIV (pYGIV; green), phalloidin–Texas red (F-actin; red), paxillin (far red; pseudocolored purple), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Representative images are shown. Bar, 25 μm. (E) Schematic summarizing the findings in A–D. (F, G) Control (sh Control) or GIV-depleted (sh GIV) Cos7 cells were fixed and stained for vinculin (red, F) or paxillin (red, G) and DAPI (nuclei; blue) and analyzed by confocal microscopy. In GIV-depleted cells we found that vinculin was present on vesicular structures (arrowheads). Representative images are shown. Bar, 25 μm. Depletion of GIV was confirmed by immunoblotting (Supplemental Figure S1D). (H) Cos7 cells in F and G were analyzed for collagen-induced haptotactic cell motility using Transwell assays. Bar graphs show quantification of the number of migrated cells/high-power fields (HPF). Error bars represent mean ± SD; n = 3; *p < 0.05.
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Related In: Results  -  Collection

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Figure 1: GIV localizes to FAs and is essential for the integrity of these structures. (A, B) Cos7 cells grown on collagen-coated coverslips were fixed and costained with GIV (green), vinculin (red), and DAPI (nuclei; blue; A) or with vinculin (green), phalloidin–Texas red (F-actin, red), and DAPI (nuclei; blue; B) and analyzed by confocal microscopy. Representative images. Bar, 10 μm. (C) Cos7 cells grown on either noncoated (left) or collagen-coated (right) glass coverslips, fixed, and costained with phospho-Tyr-1764-GIV (pYGIV; red), vinculin (green), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Arrowheads indicated focal complexes; arrows indicate mature FAs. Representative images. Bar, 10 μm. (D) Cos7 cells were fixed and costained with phospho-Tyr-1764-GIV (pYGIV; green), phalloidin–Texas red (F-actin; red), paxillin (far red; pseudocolored purple), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Representative images are shown. Bar, 25 μm. (E) Schematic summarizing the findings in A–D. (F, G) Control (sh Control) or GIV-depleted (sh GIV) Cos7 cells were fixed and stained for vinculin (red, F) or paxillin (red, G) and DAPI (nuclei; blue) and analyzed by confocal microscopy. In GIV-depleted cells we found that vinculin was present on vesicular structures (arrowheads). Representative images are shown. Bar, 25 μm. Depletion of GIV was confirmed by immunoblotting (Supplemental Figure S1D). (H) Cos7 cells in F and G were analyzed for collagen-induced haptotactic cell motility using Transwell assays. Bar graphs show quantification of the number of migrated cells/high-power fields (HPF). Error bars represent mean ± SD; n = 3; *p < 0.05.
Mentions: We previously showed that GIV modulates growth factor RTK signaling via its ability to bind ligand-activated RTKs and couple them to activation of G proteins at the PM (Midde et al., 2015). To study specifically the localization of GIV at the PM without the noise contributed by the larger cytosolic pool, we carried out immunofluorescence studies on cells fixed using a low-temperature methanol-fixation treatment that leads to improved accessibility of the intracellular antigens and reduces cytosolic background (Schnell et al., 2012). GIV localized to the Golgi, the nucleus, and actin stress fibers (Figure 1A), all previously reported by us and others (Enomoto et al., 2005; Ghosh et al., 2008). A pool of actin-bound GIV colocalized with vinculin, indicating that GIV localizes also on PM patches where actin caps associate with FAs (Figure 1B); this pattern is specific, because it was lost in GIV-depleted cells (Supplemental Figure S1A). When we immunostained for the tyrosine-phosphorylated pool of “active” GIV (pYGIV), as determined by a diagnostic-grade antibody previously validated and confirmed to specifically detect phospho-Tyr-1764-GIV (see Materials and Methods), we found it to be almost exclusively colocalized with vinculin in FAs: in the absence of collagen, pYGIV and vinculin colocalized predominantly in the peripherally located “environment-probing” focal complexes (Figure 1C, left), whereas in the presence of collagen, they colocalized primarily within mature FAs (Figure 1C, right). Localization of GIV at FAs was further confirmed using paxillin, another marker of FAs (Figure 1D and Supplemental Figure S1B). Finally, tyrosine-phosphorylated GIV localized to FAs in cells stimulated exclusively with collagen in the absence of serum (Supplemental Figure S1C), suggesting that integrin signaling is sufficient for both tyrosine phosphorylation and localization of GIV at FAs. These results indicate that a pool of total GIV and the majority of “active” pYGIV localize discretely to nascent and mature FAs (Figure 1E).

Bottom Line: As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi.Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs.Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA 92093 inmalopezsanchez@hotmail.com prghosh@ucsd.edu.

No MeSH data available.


Related in: MedlinePlus