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Plakophilin 2 couples actomyosin remodeling to desmosomal plaque assembly via RhoA.

Godsel LM, Dubash AD, Bass-Zubek AE, Amargo EV, Klessner JL, Hobbs RP, Chen X, Green KJ - Mol. Biol. Cell (2010)

Bottom Line: Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome.We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization.Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP-IF interactions required for normal desmosome assembly.

View Article: PubMed Central - PubMed

Affiliation: Northwestern University Feinberg School of Medicine, Department of Pathology, Chicago, IL 60611, USA.

ABSTRACT
Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome. We previously showed that PKP2 loss prevents the incorporation of desmosome precursors enriched in the plaque protein desmoplakin (DP) into newly forming desmosomes, in part by disrupting PKC-dependent regulation of DP assembly competence. On the basis of the observation that DP incorporation into junctions is cytochalasin D-sensitive, here we ask whether PKP2 may also contribute to actin-dependent regulation of desmosome assembly. We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization. Our data suggest that these defects result from the failure of activated RhoA to localize at intercellular interfaces after cell-cell contact and an elevation of cellular RhoA, stress fibers, and other indicators of contractile signaling in squamous cell lines and atrial cardiomyocytes. Consistent with these observations, RhoA activation accelerated DP redistribution to desmosomes during the first hour of junction assembly, whereas sustained RhoA activity compromised desmosome plaque maturation. Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP-IF interactions required for normal desmosome assembly.

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Related in: MedlinePlus

PKP2 is present at cell borders early after cell–cell contact. (A) SCC9s were subjected to calcium switch and then fixed with 4% formal saline followed by 0.2% Triton X-100 extraction at 10 or 60 min and incubated with primary antibodies against PKP2 or E-cadherin (Ecad), followed by incubation with 568 Alexa–conjugated secondary antibodies and phalloidin. Bar, 20 μm. PKP2, Ecad, and actin are all localized at nascent cell–cell contacts within 10 min. (B) SCC9 cells expressing pairs of the junction molecules with GFP- or mCherry-tags were imaged during junction formation. Whisker diagrams demonstrate the temporal sequence of PKP2, Ecad, and DP appearance at nascent cell–cell contacts. On average, Ecad appears at nascent contact sites within 3 min, followed closely by PKP2 at 4 min, whereas DP appears ∼10 min after initial cell–cell contact. The experiments shown are a compilation of 39 total experiments. (C) Stills from movies of cells expressing Ecad-mCherry and DP-GFP (top panels) or Ecad-GFP and PKP2-mCherry (bottom panels). Insets, magnification of early cell–cell contacts. Bar, 10 μm. See also Videos 2 and 3. In the Ecad/DP pairing, Ecad appears at sites of cell–cell contact as small puncta within 3 min, whereas DP appears later, within 10 min. In the Ecad/PKP2 pairing Ecad appears at 1½ min after cell contact with PKP2 appearing shortly thereafter at 3 min.
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Figure 2: PKP2 is present at cell borders early after cell–cell contact. (A) SCC9s were subjected to calcium switch and then fixed with 4% formal saline followed by 0.2% Triton X-100 extraction at 10 or 60 min and incubated with primary antibodies against PKP2 or E-cadherin (Ecad), followed by incubation with 568 Alexa–conjugated secondary antibodies and phalloidin. Bar, 20 μm. PKP2, Ecad, and actin are all localized at nascent cell–cell contacts within 10 min. (B) SCC9 cells expressing pairs of the junction molecules with GFP- or mCherry-tags were imaged during junction formation. Whisker diagrams demonstrate the temporal sequence of PKP2, Ecad, and DP appearance at nascent cell–cell contacts. On average, Ecad appears at nascent contact sites within 3 min, followed closely by PKP2 at 4 min, whereas DP appears ∼10 min after initial cell–cell contact. The experiments shown are a compilation of 39 total experiments. (C) Stills from movies of cells expressing Ecad-mCherry and DP-GFP (top panels) or Ecad-GFP and PKP2-mCherry (bottom panels). Insets, magnification of early cell–cell contacts. Bar, 10 μm. See also Videos 2 and 3. In the Ecad/DP pairing, Ecad appears at sites of cell–cell contact as small puncta within 3 min, whereas DP appears later, within 10 min. In the Ecad/PKP2 pairing Ecad appears at 1½ min after cell contact with PKP2 appearing shortly thereafter at 3 min.

Mentions: Cells were seeded onto collagen-coated glass coverslips for at least 24 h before any siRNA or cDNA transfections were performed. Forty-eight to 72 h after transfection coverslips were washed in PBS and fixed for immunofluorescence. Cells shown in Figure 1, C and D, and Figure 5C were fixed in anhydrous methanol for 2 min at −20°C and air-dried briefly followed by processing with HECD-1, NW6, or 6D8. For immunofluorescence detection of RhoA shown in Figure 8 and Supplemental Figure 3, cells were fixed in ice-cold 10% trichloroacetic acid in water for 15 min followed by a 20-min extraction in ice cold 0.2% Triton X-100 as described in Hayashi et al. (1999) and Yonemura et al. (2004) before processing for immunofluorescence. RhoA localization detected by this method is thought to represent predominantly active RhoA (Takaishi et al., 1995; Yonemura et al., 2004; Piekny et al., 2005). For studies that included the analysis of the actin cytoskeleton in Figures 1, 2, 4, 5, and 7, as well as Figures 3 and 8E, and Supplemental Figure 4, cells were fixed for 10 min in 4% methanol-free formal saline followed by a 20-min extraction in ice cold 0.2% Triton X-100 in PBS before processing for immunofluorescence.


Plakophilin 2 couples actomyosin remodeling to desmosomal plaque assembly via RhoA.

Godsel LM, Dubash AD, Bass-Zubek AE, Amargo EV, Klessner JL, Hobbs RP, Chen X, Green KJ - Mol. Biol. Cell (2010)

PKP2 is present at cell borders early after cell–cell contact. (A) SCC9s were subjected to calcium switch and then fixed with 4% formal saline followed by 0.2% Triton X-100 extraction at 10 or 60 min and incubated with primary antibodies against PKP2 or E-cadherin (Ecad), followed by incubation with 568 Alexa–conjugated secondary antibodies and phalloidin. Bar, 20 μm. PKP2, Ecad, and actin are all localized at nascent cell–cell contacts within 10 min. (B) SCC9 cells expressing pairs of the junction molecules with GFP- or mCherry-tags were imaged during junction formation. Whisker diagrams demonstrate the temporal sequence of PKP2, Ecad, and DP appearance at nascent cell–cell contacts. On average, Ecad appears at nascent contact sites within 3 min, followed closely by PKP2 at 4 min, whereas DP appears ∼10 min after initial cell–cell contact. The experiments shown are a compilation of 39 total experiments. (C) Stills from movies of cells expressing Ecad-mCherry and DP-GFP (top panels) or Ecad-GFP and PKP2-mCherry (bottom panels). Insets, magnification of early cell–cell contacts. Bar, 10 μm. See also Videos 2 and 3. In the Ecad/DP pairing, Ecad appears at sites of cell–cell contact as small puncta within 3 min, whereas DP appears later, within 10 min. In the Ecad/PKP2 pairing Ecad appears at 1½ min after cell contact with PKP2 appearing shortly thereafter at 3 min.
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Related In: Results  -  Collection

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Figure 2: PKP2 is present at cell borders early after cell–cell contact. (A) SCC9s were subjected to calcium switch and then fixed with 4% formal saline followed by 0.2% Triton X-100 extraction at 10 or 60 min and incubated with primary antibodies against PKP2 or E-cadherin (Ecad), followed by incubation with 568 Alexa–conjugated secondary antibodies and phalloidin. Bar, 20 μm. PKP2, Ecad, and actin are all localized at nascent cell–cell contacts within 10 min. (B) SCC9 cells expressing pairs of the junction molecules with GFP- or mCherry-tags were imaged during junction formation. Whisker diagrams demonstrate the temporal sequence of PKP2, Ecad, and DP appearance at nascent cell–cell contacts. On average, Ecad appears at nascent contact sites within 3 min, followed closely by PKP2 at 4 min, whereas DP appears ∼10 min after initial cell–cell contact. The experiments shown are a compilation of 39 total experiments. (C) Stills from movies of cells expressing Ecad-mCherry and DP-GFP (top panels) or Ecad-GFP and PKP2-mCherry (bottom panels). Insets, magnification of early cell–cell contacts. Bar, 10 μm. See also Videos 2 and 3. In the Ecad/DP pairing, Ecad appears at sites of cell–cell contact as small puncta within 3 min, whereas DP appears later, within 10 min. In the Ecad/PKP2 pairing Ecad appears at 1½ min after cell contact with PKP2 appearing shortly thereafter at 3 min.
Mentions: Cells were seeded onto collagen-coated glass coverslips for at least 24 h before any siRNA or cDNA transfections were performed. Forty-eight to 72 h after transfection coverslips were washed in PBS and fixed for immunofluorescence. Cells shown in Figure 1, C and D, and Figure 5C were fixed in anhydrous methanol for 2 min at −20°C and air-dried briefly followed by processing with HECD-1, NW6, or 6D8. For immunofluorescence detection of RhoA shown in Figure 8 and Supplemental Figure 3, cells were fixed in ice-cold 10% trichloroacetic acid in water for 15 min followed by a 20-min extraction in ice cold 0.2% Triton X-100 as described in Hayashi et al. (1999) and Yonemura et al. (2004) before processing for immunofluorescence. RhoA localization detected by this method is thought to represent predominantly active RhoA (Takaishi et al., 1995; Yonemura et al., 2004; Piekny et al., 2005). For studies that included the analysis of the actin cytoskeleton in Figures 1, 2, 4, 5, and 7, as well as Figures 3 and 8E, and Supplemental Figure 4, cells were fixed for 10 min in 4% methanol-free formal saline followed by a 20-min extraction in ice cold 0.2% Triton X-100 in PBS before processing for immunofluorescence.

Bottom Line: Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome.We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization.Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP-IF interactions required for normal desmosome assembly.

View Article: PubMed Central - PubMed

Affiliation: Northwestern University Feinberg School of Medicine, Department of Pathology, Chicago, IL 60611, USA.

ABSTRACT
Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome. We previously showed that PKP2 loss prevents the incorporation of desmosome precursors enriched in the plaque protein desmoplakin (DP) into newly forming desmosomes, in part by disrupting PKC-dependent regulation of DP assembly competence. On the basis of the observation that DP incorporation into junctions is cytochalasin D-sensitive, here we ask whether PKP2 may also contribute to actin-dependent regulation of desmosome assembly. We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization. Our data suggest that these defects result from the failure of activated RhoA to localize at intercellular interfaces after cell-cell contact and an elevation of cellular RhoA, stress fibers, and other indicators of contractile signaling in squamous cell lines and atrial cardiomyocytes. Consistent with these observations, RhoA activation accelerated DP redistribution to desmosomes during the first hour of junction assembly, whereas sustained RhoA activity compromised desmosome plaque maturation. Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP-IF interactions required for normal desmosome assembly.

Show MeSH
Related in: MedlinePlus