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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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Pkp3 mediates recruitment of soluble cytoplasmic DP to the sites of cell–cell contacts. (A) Western blot showing the difference in the presence of DP in cytoplasmic (saponin soluble) and membrane/cytoskeleton-bound (urea soluble) fractions in Pkp3 KD cells compared with control and Pkp2 KD cells. GAPDH and keratin 18 (K18) were used as loading controls for their respective fractions. (B) Western blot showing the levels of DP in the cytoplasmic (saponin soluble) cell fraction under conditions of calcium switch. GAPDH- normalized DP band intensity for each condition was determined using ImageJ. Ratio between soluble cytoplasmic DP in control and Pkp3 KD cells shows a rapid decrease in cytoplasmic DP in control cells. (C) Representative immunofluorescence images of DP appearing at cell borders after calcium switch from low to calcium concentration permissive for cell–cell junction formation, after the time indicated. “High Ca2+” represents cells that were switched overnight and are identical to steady-state conditions. White solid rectangles in the images to the left delineate areas enlarged at the right. Scale bar, 20 μm. (D) Ratio between the average fluorescence pixel intensities of cell–cell border and cytoplasmic DP and average total DP fluorescence intensity in the conditions depicted in C shows that shift of DP from cytoplasm to cell–cell border is largely absent in Pkp3 KD cells. Average total fluorescence intensity was normalized to represent 100% in each condition. The p values for control-to-Pkp3 KD comparisons are as follows (ANOVA, Bonferroni): 30 min cytoplasmic DP: ns, p > 0.05. All other cytoplasmic and cell–cell border DP measurements: p < 0.001. (E) Scatter plot quantifying the increase in DP particle size at the cell–cell borders of Pkp3KD cells compared to controls, as shown in part C, high Ca2+ condition. Horizontal lines represent mean ± SEM. ***p < 0.001 (t test). Number of individual particles analyzed is 1066 for control and 1148 for Pkp3 KD.
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Figure 3: Pkp3 mediates recruitment of soluble cytoplasmic DP to the sites of cell–cell contacts. (A) Western blot showing the difference in the presence of DP in cytoplasmic (saponin soluble) and membrane/cytoskeleton-bound (urea soluble) fractions in Pkp3 KD cells compared with control and Pkp2 KD cells. GAPDH and keratin 18 (K18) were used as loading controls for their respective fractions. (B) Western blot showing the levels of DP in the cytoplasmic (saponin soluble) cell fraction under conditions of calcium switch. GAPDH- normalized DP band intensity for each condition was determined using ImageJ. Ratio between soluble cytoplasmic DP in control and Pkp3 KD cells shows a rapid decrease in cytoplasmic DP in control cells. (C) Representative immunofluorescence images of DP appearing at cell borders after calcium switch from low to calcium concentration permissive for cell–cell junction formation, after the time indicated. “High Ca2+” represents cells that were switched overnight and are identical to steady-state conditions. White solid rectangles in the images to the left delineate areas enlarged at the right. Scale bar, 20 μm. (D) Ratio between the average fluorescence pixel intensities of cell–cell border and cytoplasmic DP and average total DP fluorescence intensity in the conditions depicted in C shows that shift of DP from cytoplasm to cell–cell border is largely absent in Pkp3 KD cells. Average total fluorescence intensity was normalized to represent 100% in each condition. The p values for control-to-Pkp3 KD comparisons are as follows (ANOVA, Bonferroni): 30 min cytoplasmic DP: ns, p > 0.05. All other cytoplasmic and cell–cell border DP measurements: p < 0.001. (E) Scatter plot quantifying the increase in DP particle size at the cell–cell borders of Pkp3KD cells compared to controls, as shown in part C, high Ca2+ condition. Horizontal lines represent mean ± SEM. ***p < 0.001 (t test). Number of individual particles analyzed is 1066 for control and 1148 for Pkp3 KD.

Mentions: The presence of diffuse cytoplasmic DP staining in Pkp3-silenced cells raises the possibility that Pkp3 may regulate DP incorporation into cytoplasmic desmosome precursors that are subsequently transported to the sites of cell–cell contact. To test this hypothesis, we subjected cells to fractionation and then analyzed the DP content in saponin-soluble cytosolic and saponin-insoluble fractions. In steady-state conditions there is a clear shift of DP from the keratin 18 (intermediate filament cytoskeleton component)–containing, saponin-insoluble fraction into the cytosol (Figure 3A). Moreover, this shift failed to occur in the Pkp2-ablated cells. To discern whether DP fractionation was affected during desmosome assembly, we incubated SCC9 cells in low-calcium medium to allow for existing desmosomes to disassemble and then switched them back to high calcium (calcium switch) to monitor desmosome assembly over time. Upon calcium switch, a steady decrease of DP in the saponin-soluble cytosolic fraction was observed in the control cells over time following the switch. In contrast, in Pkp3-ablated cells, DP remained primarily in the saponin-soluble fraction throughout the time course (Figure 3B). These results suggest that whereas most of the cytoplasmic DP in control cells is recruited into desmosomes and desmosome precursors, in Pkp3-silenced cells most of it remains in cytosol. We next visualized DP distribution in the cells by immunofluorescence. Whereas in the control cells the amount of DP at the borders steadily increases at the expense of cytoplasmic DP after calcium-induced desmosome assembly initiation, DP failed to be cleared from the cytoplasm in Pkp3 KD cells (Figure 3, C and D). The analysis of the size of the DP particles at cell–cell borders in steady-state conditions reveals an increase (Figure 3E), indicating possible aberrant coalescence in the Pkp3 KD cells (see later discussion).


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

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

Pkp3 mediates recruitment of soluble cytoplasmic DP to the sites of cell–cell contacts. (A) Western blot showing the difference in the presence of DP in cytoplasmic (saponin soluble) and membrane/cytoskeleton-bound (urea soluble) fractions in Pkp3 KD cells compared with control and Pkp2 KD cells. GAPDH and keratin 18 (K18) were used as loading controls for their respective fractions. (B) Western blot showing the levels of DP in the cytoplasmic (saponin soluble) cell fraction under conditions of calcium switch. GAPDH- normalized DP band intensity for each condition was determined using ImageJ. Ratio between soluble cytoplasmic DP in control and Pkp3 KD cells shows a rapid decrease in cytoplasmic DP in control cells. (C) Representative immunofluorescence images of DP appearing at cell borders after calcium switch from low to calcium concentration permissive for cell–cell junction formation, after the time indicated. “High Ca2+” represents cells that were switched overnight and are identical to steady-state conditions. White solid rectangles in the images to the left delineate areas enlarged at the right. Scale bar, 20 μm. (D) Ratio between the average fluorescence pixel intensities of cell–cell border and cytoplasmic DP and average total DP fluorescence intensity in the conditions depicted in C shows that shift of DP from cytoplasm to cell–cell border is largely absent in Pkp3 KD cells. Average total fluorescence intensity was normalized to represent 100% in each condition. The p values for control-to-Pkp3 KD comparisons are as follows (ANOVA, Bonferroni): 30 min cytoplasmic DP: ns, p > 0.05. All other cytoplasmic and cell–cell border DP measurements: p < 0.001. (E) Scatter plot quantifying the increase in DP particle size at the cell–cell borders of Pkp3KD cells compared to controls, as shown in part C, high Ca2+ condition. Horizontal lines represent mean ± SEM. ***p < 0.001 (t test). Number of individual particles analyzed is 1066 for control and 1148 for Pkp3 KD.
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Figure 3: Pkp3 mediates recruitment of soluble cytoplasmic DP to the sites of cell–cell contacts. (A) Western blot showing the difference in the presence of DP in cytoplasmic (saponin soluble) and membrane/cytoskeleton-bound (urea soluble) fractions in Pkp3 KD cells compared with control and Pkp2 KD cells. GAPDH and keratin 18 (K18) were used as loading controls for their respective fractions. (B) Western blot showing the levels of DP in the cytoplasmic (saponin soluble) cell fraction under conditions of calcium switch. GAPDH- normalized DP band intensity for each condition was determined using ImageJ. Ratio between soluble cytoplasmic DP in control and Pkp3 KD cells shows a rapid decrease in cytoplasmic DP in control cells. (C) Representative immunofluorescence images of DP appearing at cell borders after calcium switch from low to calcium concentration permissive for cell–cell junction formation, after the time indicated. “High Ca2+” represents cells that were switched overnight and are identical to steady-state conditions. White solid rectangles in the images to the left delineate areas enlarged at the right. Scale bar, 20 μm. (D) Ratio between the average fluorescence pixel intensities of cell–cell border and cytoplasmic DP and average total DP fluorescence intensity in the conditions depicted in C shows that shift of DP from cytoplasm to cell–cell border is largely absent in Pkp3 KD cells. Average total fluorescence intensity was normalized to represent 100% in each condition. The p values for control-to-Pkp3 KD comparisons are as follows (ANOVA, Bonferroni): 30 min cytoplasmic DP: ns, p > 0.05. All other cytoplasmic and cell–cell border DP measurements: p < 0.001. (E) Scatter plot quantifying the increase in DP particle size at the cell–cell borders of Pkp3KD cells compared to controls, as shown in part C, high Ca2+ condition. Horizontal lines represent mean ± SEM. ***p < 0.001 (t test). Number of individual particles analyzed is 1066 for control and 1148 for Pkp3 KD.
Mentions: The presence of diffuse cytoplasmic DP staining in Pkp3-silenced cells raises the possibility that Pkp3 may regulate DP incorporation into cytoplasmic desmosome precursors that are subsequently transported to the sites of cell–cell contact. To test this hypothesis, we subjected cells to fractionation and then analyzed the DP content in saponin-soluble cytosolic and saponin-insoluble fractions. In steady-state conditions there is a clear shift of DP from the keratin 18 (intermediate filament cytoskeleton component)–containing, saponin-insoluble fraction into the cytosol (Figure 3A). Moreover, this shift failed to occur in the Pkp2-ablated cells. To discern whether DP fractionation was affected during desmosome assembly, we incubated SCC9 cells in low-calcium medium to allow for existing desmosomes to disassemble and then switched them back to high calcium (calcium switch) to monitor desmosome assembly over time. Upon calcium switch, a steady decrease of DP in the saponin-soluble cytosolic fraction was observed in the control cells over time following the switch. In contrast, in Pkp3-ablated cells, DP remained primarily in the saponin-soluble fraction throughout the time course (Figure 3B). These results suggest that whereas most of the cytoplasmic DP in control cells is recruited into desmosomes and desmosome precursors, in Pkp3-silenced cells most of it remains in cytosol. We next visualized DP distribution in the cells by immunofluorescence. Whereas in the control cells the amount of DP at the borders steadily increases at the expense of cytoplasmic DP after calcium-induced desmosome assembly initiation, DP failed to be cleared from the cytoplasm in Pkp3 KD cells (Figure 3, C and D). The analysis of the size of the DP particles at cell–cell borders in steady-state conditions reveals an increase (Figure 3E), indicating possible aberrant coalescence in the Pkp3 KD cells (see later discussion).

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