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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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Desmoplakin cytoplasmic particles are found in close proximity to cortical actin and border localization of desmoplakin involves myosin II contractility. (A) SCC9 cells grown overnight in low-calcium medium (0.05 mM) were switched to normal calcium levels for 30 min to induce junction assembly. Cells were fixed with 4% formal saline followed by 0.2% Triton X-100 extraction. Cells were then incubated with primary antibodies against desmoplakin (DP) followed by incubation with Alexa Fluor 568–conjugated secondary antibody and Alexa Fluor 488–labeled phalloidin to visualize actin. DP containing precursor particles are found in close proximity with the actomyosin network (arrows). One XY plane is shown along with the orthogonal planes for the captured Z-stack. Bar, 5 μm. (B) SCC12f cells coexpressing DP.GFP and actin.mCherry were wounded and imaged at 1-min intervals after cell–cell contact. DP precursor particles (white arrows, 1–3) move toward contact sites in coordination with the reorganizing junctional actin. The white box in the full field of view (left) is enlarged and shown for six time points illustrating particle movement and actin reorganization. Bar, 10 μm. See also Supplemental Video 1. (C–E) Cells cultured overnight in low-calcium medium were preincubated for 10 min in the presence of 25 μM blebbistatin or DMSO carrier before switching them to normal calcium containing 25 μM blebbistatin for 20-, 40, and 60-min time points. Cells were fixed with anhydrous methanol and incubated with primary antibodies against DP (C), E-cadherin (Ecad; D), or desmoglein 2 (Dsg2) followed by incubation with Alexa Fluor 488–conjugated secondary antibodies. Bar, 20 μm (E) Average pixel intensities for DP, Ecad, or Dsg2 were quantified along sites of cell–cell contact within the population. Quantitative analyses of immunostained images captured from >10 fields and >30 borders per condition were assessed using Metamorph software; *p < 0.05. The experiments shown are representative of three or more experiments. Although the border localization of Dsg2 and E-cad were unaffected by blebbistatin treatment, DP localization at borders was decreased about four-fold by the inhibition of myosin II.
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Figure 1: Desmoplakin cytoplasmic particles are found in close proximity to cortical actin and border localization of desmoplakin involves myosin II contractility. (A) SCC9 cells grown overnight in low-calcium medium (0.05 mM) were switched to normal calcium levels for 30 min to induce junction assembly. Cells were fixed with 4% formal saline followed by 0.2% Triton X-100 extraction. Cells were then incubated with primary antibodies against desmoplakin (DP) followed by incubation with Alexa Fluor 568–conjugated secondary antibody and Alexa Fluor 488–labeled phalloidin to visualize actin. DP containing precursor particles are found in close proximity with the actomyosin network (arrows). One XY plane is shown along with the orthogonal planes for the captured Z-stack. Bar, 5 μm. (B) SCC12f cells coexpressing DP.GFP and actin.mCherry were wounded and imaged at 1-min intervals after cell–cell contact. DP precursor particles (white arrows, 1–3) move toward contact sites in coordination with the reorganizing junctional actin. The white box in the full field of view (left) is enlarged and shown for six time points illustrating particle movement and actin reorganization. Bar, 10 μm. See also Supplemental Video 1. (C–E) Cells cultured overnight in low-calcium medium were preincubated for 10 min in the presence of 25 μM blebbistatin or DMSO carrier before switching them to normal calcium containing 25 μM blebbistatin for 20-, 40, and 60-min time points. Cells were fixed with anhydrous methanol and incubated with primary antibodies against DP (C), E-cadherin (Ecad; D), or desmoglein 2 (Dsg2) followed by incubation with Alexa Fluor 488–conjugated secondary antibodies. Bar, 20 μm (E) Average pixel intensities for DP, Ecad, or Dsg2 were quantified along sites of cell–cell contact within the population. Quantitative analyses of immunostained images captured from >10 fields and >30 borders per condition were assessed using Metamorph software; *p < 0.05. The experiments shown are representative of three or more experiments. Although the border localization of Dsg2 and E-cad were unaffected by blebbistatin treatment, DP localization at borders was decreased about four-fold by the inhibition of myosin II.

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)

Desmoplakin cytoplasmic particles are found in close proximity to cortical actin and border localization of desmoplakin involves myosin II contractility. (A) SCC9 cells grown overnight in low-calcium medium (0.05 mM) were switched to normal calcium levels for 30 min to induce junction assembly. Cells were fixed with 4% formal saline followed by 0.2% Triton X-100 extraction. Cells were then incubated with primary antibodies against desmoplakin (DP) followed by incubation with Alexa Fluor 568–conjugated secondary antibody and Alexa Fluor 488–labeled phalloidin to visualize actin. DP containing precursor particles are found in close proximity with the actomyosin network (arrows). One XY plane is shown along with the orthogonal planes for the captured Z-stack. Bar, 5 μm. (B) SCC12f cells coexpressing DP.GFP and actin.mCherry were wounded and imaged at 1-min intervals after cell–cell contact. DP precursor particles (white arrows, 1–3) move toward contact sites in coordination with the reorganizing junctional actin. The white box in the full field of view (left) is enlarged and shown for six time points illustrating particle movement and actin reorganization. Bar, 10 μm. See also Supplemental Video 1. (C–E) Cells cultured overnight in low-calcium medium were preincubated for 10 min in the presence of 25 μM blebbistatin or DMSO carrier before switching them to normal calcium containing 25 μM blebbistatin for 20-, 40, and 60-min time points. Cells were fixed with anhydrous methanol and incubated with primary antibodies against DP (C), E-cadherin (Ecad; D), or desmoglein 2 (Dsg2) followed by incubation with Alexa Fluor 488–conjugated secondary antibodies. Bar, 20 μm (E) Average pixel intensities for DP, Ecad, or Dsg2 were quantified along sites of cell–cell contact within the population. Quantitative analyses of immunostained images captured from >10 fields and >30 borders per condition were assessed using Metamorph software; *p < 0.05. The experiments shown are representative of three or more experiments. Although the border localization of Dsg2 and E-cad were unaffected by blebbistatin treatment, DP localization at borders was decreased about four-fold by the inhibition of myosin II.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921118&req=5

Figure 1: Desmoplakin cytoplasmic particles are found in close proximity to cortical actin and border localization of desmoplakin involves myosin II contractility. (A) SCC9 cells grown overnight in low-calcium medium (0.05 mM) were switched to normal calcium levels for 30 min to induce junction assembly. Cells were fixed with 4% formal saline followed by 0.2% Triton X-100 extraction. Cells were then incubated with primary antibodies against desmoplakin (DP) followed by incubation with Alexa Fluor 568–conjugated secondary antibody and Alexa Fluor 488–labeled phalloidin to visualize actin. DP containing precursor particles are found in close proximity with the actomyosin network (arrows). One XY plane is shown along with the orthogonal planes for the captured Z-stack. Bar, 5 μm. (B) SCC12f cells coexpressing DP.GFP and actin.mCherry were wounded and imaged at 1-min intervals after cell–cell contact. DP precursor particles (white arrows, 1–3) move toward contact sites in coordination with the reorganizing junctional actin. The white box in the full field of view (left) is enlarged and shown for six time points illustrating particle movement and actin reorganization. Bar, 10 μm. See also Supplemental Video 1. (C–E) Cells cultured overnight in low-calcium medium were preincubated for 10 min in the presence of 25 μM blebbistatin or DMSO carrier before switching them to normal calcium containing 25 μM blebbistatin for 20-, 40, and 60-min time points. Cells were fixed with anhydrous methanol and incubated with primary antibodies against DP (C), E-cadherin (Ecad; D), or desmoglein 2 (Dsg2) followed by incubation with Alexa Fluor 488–conjugated secondary antibodies. Bar, 20 μm (E) Average pixel intensities for DP, Ecad, or Dsg2 were quantified along sites of cell–cell contact within the population. Quantitative analyses of immunostained images captured from >10 fields and >30 borders per condition were assessed using Metamorph software; *p < 0.05. The experiments shown are representative of three or more experiments. Although the border localization of Dsg2 and E-cad were unaffected by blebbistatin treatment, DP localization at borders was decreased about four-fold by the inhibition of myosin II.
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