Desmosomes are adhesive complexes found at sites of intercellular contact that are essential for mediating cell-cell adhesion. These junctions undergo regulated assembly and reorganization during processes such as embryogenesis and wound healing. Plakophilins (PKPs) are armadillo family members related to the classic cadherin-associated protein p120ctn. PKPs localize to the cytoplasmic plaque of intercellular junctions and participate in linking the intermediate filament (IF)-binding protein desmoplakin (DP) to desmosomal cadherins. During desmosome assembly PKP2 associates with DP in plaque precursors that form in the cytoplasm in response to cell-cell contact, which translocate to nascent desmosomes. Here we provide evidence that PKP2 governs DP assembly dynamics by scaffolding a DP-PKP2-PKCalpha complex, which is disrupted by PKP2 knockdown. The behavior of a phosphorylation-deficient DP mutant that associates more tightly with IF is mimicked by PKP2- and PKCalpha knockdown and PKC pharmacological inhibition, all of which impair junction assembly. siRNA-mediated PKP2 knockdown is accompanied by increased phosphorylation of PKC substrates, raising the possibility that global alterations in PKC signaling may contribute to pathogenesis of congenital defects caused by PKP2 deficiency. An intact actin cytoskeleton is required for the efficient assembly of these plaque precursors, which closely associate with both actin and IF during assembly. However, the molecular mechanism underlying actin involvement in the assembly of desmosomes is not well understood. Here we demonstrate that PKP2 directs myosin-dependent actin contractility and actin reorganization. Cells deficient in PKP2 exhibit constitutive myosin activation accompanied by blunted Rho GTPase membrane localization and activation, and the inability to properly reorganize the actin cytoskeleton. These studies demonstrate that Rho activity is required early during junction assembly but must be downregulated later in order for junction maturation to occur. Taken together, our results suggest that PKP2 regulates actin contractility signaling in a temporal and spatially restricted manner that tailors these activities to desmosome assembly-specific events. The two major desmosome compartments (membrane and plaque) undergo distinct assembly pathways. How PKPs are involved in the coordination of these two pathways is not clear. Here, I show a dramatic defect in desmosomal cadherin assembly (but not adherens junction assembly) in cells deficient for PKP2. Further, siRNA-mediated PKP2 knockdown led to an enhancement in the colocalization of desmosomal plaque component, DP, with desmosomal cadherins and other arm proteins. Taken together these data suggest that PKP2 is required for efficient desmosome assembly. From this work, I conclude that PKP2 plays an important role in the coordination of desmosomal cadherin and plaque components and in the regulation of signaling pathways important for temporal and spatial control of actin reorganization and DP-IF interactions during desmosome assembly.

Last modified

• 09/20/2018

Creator

• Amanda Elisabeth Bass-Zubek

DOI

• https://doi.org/10.21985/N2XQ7S

Subject

• Integrated Graduate Program in the Life Sciences

Keyword

• desmosome
• cytoskeleton
• cell-cell adhesion
• armadillo
• plakophilin

Date created

• 2008-09-18

Resource type

• Dissertation

Rights statement

• In Copyright