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Drosophila Ric-8 interacts with the Gα12/13 subunit, Concertina, during activation of the Folded gastrulation pathway.

Peters KA, Rogers SL - Mol. Biol. Cell (2013)

Bottom Line: A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events.Ric-8 mutants exhibit similar gastrulation defects to Cta mutants.We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27514.

ABSTRACT
Heterotrimeric G proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein-coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.

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Recapitulation of Fog signaling in S2R+ cells. (A) Fog-Myc is secreted into the medium of a stable cell line expressing the construct but not by untransfected control S2 cells. Fog-Myc is recognized by anti-Myc and anti-Fog by immunoblot. (B) S2R+ cells undergo cellular shape changes in response to ectopic Fog application. RNAi-mediated depletion of Cta or Ric-8 prevents Fog-induced cellular constriction. Scale bar, 10 μm. (C) Fog-induced S2R+ contraction is accompanied by an increase in active phosphorylated nonmuscle myosin II (pRLC). S2R+ cells were treated with either control or Fog-containing media and stained for actin (red), pRLC (green), and DNA (blue). Scale bar, 100 μm. (D) S2R+ cells lose their responsiveness to Fog after RNAi against known pathway components, as well as Ric-8. Percentage of cells constricting in response to Fog was measured within a population of cells (±SEM).
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Figure 1: Recapitulation of Fog signaling in S2R+ cells. (A) Fog-Myc is secreted into the medium of a stable cell line expressing the construct but not by untransfected control S2 cells. Fog-Myc is recognized by anti-Myc and anti-Fog by immunoblot. (B) S2R+ cells undergo cellular shape changes in response to ectopic Fog application. RNAi-mediated depletion of Cta or Ric-8 prevents Fog-induced cellular constriction. Scale bar, 10 μm. (C) Fog-induced S2R+ contraction is accompanied by an increase in active phosphorylated nonmuscle myosin II (pRLC). S2R+ cells were treated with either control or Fog-containing media and stained for actin (red), pRLC (green), and DNA (blue). Scale bar, 100 μm. (D) S2R+ cells lose their responsiveness to Fog after RNAi against known pathway components, as well as Ric-8. Percentage of cells constricting in response to Fog was measured within a population of cells (±SEM).

Mentions: To study the effect of Fog signaling on cell morphology, we developed a cell culture system to allow us to replicate in vivo signaling events. We began by engineering a stable S2 cell line that expresses full-length Fog tagged at its C-terminus with the Myc epitope under an inducible metallothionein promoter (S2:Fog-Myc). Costa et al. (1994) originally hypothesized that Fog is a secreted protein based on hydropathy analysis of the protein's primary sequence, which revealed the presence of an N-terminal 12–amino acid hydrophobic region predicted to function as a signal sequence. Later analysis of Fog localization in cells of the embryonic ventral furrow and posterior midgut showed that the protein localized to membrane-bound organelles targeted for the apical surface of the blastoderm epithelia (Dawes-Hoang et al., 2005). To test whether Fog is secreted from S2:Fog-Myc cells, we induced its expression with copper sulfate for 48 h, collected the conditioned medium, and concentrated it ∼20-fold. An affinity-purified antibody against the N-terminus of Fog recognized a single protein with a molecular weight of ∼150 kDa on immunoblots of conditioned medium from induced S2:Fog-Myc cells, and the same-sized band was also recognized by a monoclonal anti-Myc antibody. Neither antibody recognized the protein in conditioned medium collected from untransfected S2 cells (Figure 1A). Thus, as found in tissues in the Drosophila blastoderm preceding cellular shape change, ectopic Fog-Myc is expressed in S2 cells as a secreted protein.


Drosophila Ric-8 interacts with the Gα12/13 subunit, Concertina, during activation of the Folded gastrulation pathway.

Peters KA, Rogers SL - Mol. Biol. Cell (2013)

Recapitulation of Fog signaling in S2R+ cells. (A) Fog-Myc is secreted into the medium of a stable cell line expressing the construct but not by untransfected control S2 cells. Fog-Myc is recognized by anti-Myc and anti-Fog by immunoblot. (B) S2R+ cells undergo cellular shape changes in response to ectopic Fog application. RNAi-mediated depletion of Cta or Ric-8 prevents Fog-induced cellular constriction. Scale bar, 10 μm. (C) Fog-induced S2R+ contraction is accompanied by an increase in active phosphorylated nonmuscle myosin II (pRLC). S2R+ cells were treated with either control or Fog-containing media and stained for actin (red), pRLC (green), and DNA (blue). Scale bar, 100 μm. (D) S2R+ cells lose their responsiveness to Fog after RNAi against known pathway components, as well as Ric-8. Percentage of cells constricting in response to Fog was measured within a population of cells (±SEM).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818808&req=5

Figure 1: Recapitulation of Fog signaling in S2R+ cells. (A) Fog-Myc is secreted into the medium of a stable cell line expressing the construct but not by untransfected control S2 cells. Fog-Myc is recognized by anti-Myc and anti-Fog by immunoblot. (B) S2R+ cells undergo cellular shape changes in response to ectopic Fog application. RNAi-mediated depletion of Cta or Ric-8 prevents Fog-induced cellular constriction. Scale bar, 10 μm. (C) Fog-induced S2R+ contraction is accompanied by an increase in active phosphorylated nonmuscle myosin II (pRLC). S2R+ cells were treated with either control or Fog-containing media and stained for actin (red), pRLC (green), and DNA (blue). Scale bar, 100 μm. (D) S2R+ cells lose their responsiveness to Fog after RNAi against known pathway components, as well as Ric-8. Percentage of cells constricting in response to Fog was measured within a population of cells (±SEM).
Mentions: To study the effect of Fog signaling on cell morphology, we developed a cell culture system to allow us to replicate in vivo signaling events. We began by engineering a stable S2 cell line that expresses full-length Fog tagged at its C-terminus with the Myc epitope under an inducible metallothionein promoter (S2:Fog-Myc). Costa et al. (1994) originally hypothesized that Fog is a secreted protein based on hydropathy analysis of the protein's primary sequence, which revealed the presence of an N-terminal 12–amino acid hydrophobic region predicted to function as a signal sequence. Later analysis of Fog localization in cells of the embryonic ventral furrow and posterior midgut showed that the protein localized to membrane-bound organelles targeted for the apical surface of the blastoderm epithelia (Dawes-Hoang et al., 2005). To test whether Fog is secreted from S2:Fog-Myc cells, we induced its expression with copper sulfate for 48 h, collected the conditioned medium, and concentrated it ∼20-fold. An affinity-purified antibody against the N-terminus of Fog recognized a single protein with a molecular weight of ∼150 kDa on immunoblots of conditioned medium from induced S2:Fog-Myc cells, and the same-sized band was also recognized by a monoclonal anti-Myc antibody. Neither antibody recognized the protein in conditioned medium collected from untransfected S2 cells (Figure 1A). Thus, as found in tissues in the Drosophila blastoderm preceding cellular shape change, ectopic Fog-Myc is expressed in S2 cells as a secreted protein.

Bottom Line: A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events.Ric-8 mutants exhibit similar gastrulation defects to Cta mutants.We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27514.

ABSTRACT
Heterotrimeric G proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein-coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.

Show MeSH
Related in: MedlinePlus