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Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

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

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Related in: MedlinePlus

Pkp3 deficiency leads to aberrant coalescence of DP at cell–cell borders. (A) Representative still images taken from the Supplemental Videos S1 and S2 showing appearance of DP at the borders at times indicated. In control cells, indicated times coincide with the wound closure. In Pkp3KD cells, white arrow indicates the closing wound starting at 14 min. White solid rectangles to the left delineate regions enlarged at the right. DP cytoplasmic and membrane particles in control and Pkp3 KD cells, respectively, were colorized as follows: pink, purple, orange, red, and blue to highlight cytoplasmic DP precursors migrating into the cell–cell border in control cells; red, dark green, orange, yellow, pink, blue, and purple to highlight membrane DP coalescing at the sites of cell–cell contacts in Pkp3KD cells; and green to highlight DP appearing, then coalescing, at the site of newly forming cell–cell border in Pkp3KD cells. Scale bar, 20 μm. (B) Graph representing average fluorescent pixel intensities of DP present at the sites of representative cell–cell contacts, measured every 5 min in A431 cells establishing new cell–cell junctions upon wound closure. Blue, green, and orange brackets indicate phases 1–3 in control cells, respectively. (C) Vertical scatter plot representing the number of DP particles present at individual borders at times indicated during live-cell imaging experiments. Number of borders analyzed: control, 16; Pkp3 KD, 18. Fewer borders were analyzed for 3-h time point, as not all border formation was followed by live-cell imaging for 3 h: control, N = 12; Pkp3 for KD, N = 17. Bars represent mean ± SEM. ns, p > 0.5, ***p < 0.0001 (ANOVA, Bonferroni).
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Figure 4: Pkp3 deficiency leads to aberrant coalescence of DP at cell–cell borders. (A) Representative still images taken from the Supplemental Videos S1 and S2 showing appearance of DP at the borders at times indicated. In control cells, indicated times coincide with the wound closure. In Pkp3KD cells, white arrow indicates the closing wound starting at 14 min. White solid rectangles to the left delineate regions enlarged at the right. DP cytoplasmic and membrane particles in control and Pkp3 KD cells, respectively, were colorized as follows: pink, purple, orange, red, and blue to highlight cytoplasmic DP precursors migrating into the cell–cell border in control cells; red, dark green, orange, yellow, pink, blue, and purple to highlight membrane DP coalescing at the sites of cell–cell contacts in Pkp3KD cells; and green to highlight DP appearing, then coalescing, at the site of newly forming cell–cell border in Pkp3KD cells. Scale bar, 20 μm. (B) Graph representing average fluorescent pixel intensities of DP present at the sites of representative cell–cell contacts, measured every 5 min in A431 cells establishing new cell–cell junctions upon wound closure. Blue, green, and orange brackets indicate phases 1–3 in control cells, respectively. (C) Vertical scatter plot representing the number of DP particles present at individual borders at times indicated during live-cell imaging experiments. Number of borders analyzed: control, 16; Pkp3 KD, 18. Fewer borders were analyzed for 3-h time point, as not all border formation was followed by live-cell imaging for 3 h: control, N = 12; Pkp3 for KD, N = 17. Bars represent mean ± SEM. ns, p > 0.5, ***p < 0.0001 (ANOVA, Bonferroni).

Mentions: To enhance the temporal resolution of DP trafficking to sites of newly forming cell–cell contacts, we next performed live-cell imaging on well-characterized doxycycline-inducible, green fluorescent protein (GFP)–tagged DP-expressing A431 cells (Godsel et al., 2005) coming into contact after scratch wounding (Figure 4 and Supplemental Videos S1 and S2). In control cells, the relative fluorescence intensity of DP increased both during initial phase 1 coalescence at the membrane and as precursors incorporated into the maturing border during phase 3 (Figure 4, A and B). In Pkp3 KD, the initial phase occurs, but the corresponding increase in DP accumulation during junction maturation appeared to be abolished (Figure 4A), resulting in a plateauing of the DP fluorescence intensity after phase 1 (Figure 4B). However, this leveling off belies the striking difference in DP particle dynamics at the borders of Pkp3 KD cells compared with control. Phase 3, during which particles assembled in the cytoplasm in phase 2 translocate and incorporate into the cell–cell border (Godsel et al., 2005), starts ∼40 min upon initial cell–cell contact. In contrast, in Pkp3 KD cells, this phase is missing. Instead of steady growth in both the numbers and fluorescence intensity of junctional DP particles observed in the control, Pkp3 KD cells exhibit extensive lateral DP particle coalescence and concomitant decrease in number (Figure 4, A–C, and Supplemental Video S2). Large structures formed in this manner may correspond to the clustered desmosomes observed by electron microscopy (Figure 1C).


Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Pkp3 deficiency leads to aberrant coalescence of DP at cell–cell borders. (A) Representative still images taken from the Supplemental Videos S1 and S2 showing appearance of DP at the borders at times indicated. In control cells, indicated times coincide with the wound closure. In Pkp3KD cells, white arrow indicates the closing wound starting at 14 min. White solid rectangles to the left delineate regions enlarged at the right. DP cytoplasmic and membrane particles in control and Pkp3 KD cells, respectively, were colorized as follows: pink, purple, orange, red, and blue to highlight cytoplasmic DP precursors migrating into the cell–cell border in control cells; red, dark green, orange, yellow, pink, blue, and purple to highlight membrane DP coalescing at the sites of cell–cell contacts in Pkp3KD cells; and green to highlight DP appearing, then coalescing, at the site of newly forming cell–cell border in Pkp3KD cells. Scale bar, 20 μm. (B) Graph representing average fluorescent pixel intensities of DP present at the sites of representative cell–cell contacts, measured every 5 min in A431 cells establishing new cell–cell junctions upon wound closure. Blue, green, and orange brackets indicate phases 1–3 in control cells, respectively. (C) Vertical scatter plot representing the number of DP particles present at individual borders at times indicated during live-cell imaging experiments. Number of borders analyzed: control, 16; Pkp3 KD, 18. Fewer borders were analyzed for 3-h time point, as not all border formation was followed by live-cell imaging for 3 h: control, N = 12; Pkp3 for KD, N = 17. Bars represent mean ± SEM. ns, p > 0.5, ***p < 0.0001 (ANOVA, Bonferroni).
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Figure 4: Pkp3 deficiency leads to aberrant coalescence of DP at cell–cell borders. (A) Representative still images taken from the Supplemental Videos S1 and S2 showing appearance of DP at the borders at times indicated. In control cells, indicated times coincide with the wound closure. In Pkp3KD cells, white arrow indicates the closing wound starting at 14 min. White solid rectangles to the left delineate regions enlarged at the right. DP cytoplasmic and membrane particles in control and Pkp3 KD cells, respectively, were colorized as follows: pink, purple, orange, red, and blue to highlight cytoplasmic DP precursors migrating into the cell–cell border in control cells; red, dark green, orange, yellow, pink, blue, and purple to highlight membrane DP coalescing at the sites of cell–cell contacts in Pkp3KD cells; and green to highlight DP appearing, then coalescing, at the site of newly forming cell–cell border in Pkp3KD cells. Scale bar, 20 μm. (B) Graph representing average fluorescent pixel intensities of DP present at the sites of representative cell–cell contacts, measured every 5 min in A431 cells establishing new cell–cell junctions upon wound closure. Blue, green, and orange brackets indicate phases 1–3 in control cells, respectively. (C) Vertical scatter plot representing the number of DP particles present at individual borders at times indicated during live-cell imaging experiments. Number of borders analyzed: control, 16; Pkp3 KD, 18. Fewer borders were analyzed for 3-h time point, as not all border formation was followed by live-cell imaging for 3 h: control, N = 12; Pkp3 for KD, N = 17. Bars represent mean ± SEM. ns, p > 0.5, ***p < 0.0001 (ANOVA, Bonferroni).
Mentions: To enhance the temporal resolution of DP trafficking to sites of newly forming cell–cell contacts, we next performed live-cell imaging on well-characterized doxycycline-inducible, green fluorescent protein (GFP)–tagged DP-expressing A431 cells (Godsel et al., 2005) coming into contact after scratch wounding (Figure 4 and Supplemental Videos S1 and S2). In control cells, the relative fluorescence intensity of DP increased both during initial phase 1 coalescence at the membrane and as precursors incorporated into the maturing border during phase 3 (Figure 4, A and B). In Pkp3 KD, the initial phase occurs, but the corresponding increase in DP accumulation during junction maturation appeared to be abolished (Figure 4A), resulting in a plateauing of the DP fluorescence intensity after phase 1 (Figure 4B). However, this leveling off belies the striking difference in DP particle dynamics at the borders of Pkp3 KD cells compared with control. Phase 3, during which particles assembled in the cytoplasm in phase 2 translocate and incorporate into the cell–cell border (Godsel et al., 2005), starts ∼40 min upon initial cell–cell contact. In contrast, in Pkp3 KD cells, this phase is missing. Instead of steady growth in both the numbers and fluorescence intensity of junctional DP particles observed in the control, Pkp3 KD cells exhibit extensive lateral DP particle coalescence and concomitant decrease in number (Figure 4, A–C, and Supplemental Video S2). Large structures formed in this manner may correspond to the clustered desmosomes observed by electron microscopy (Figure 1C).

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus