Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.
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.
Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.Show MeSH
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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).
Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.