Intercellular anchoring junctions are highly specialized regions of the plasma membrane where members of the cadherin family of transmembrane adhesion molecules on opposing cells interact through their extracellular domains, and through their cytoplasmic domains serve as a platform for organizing cytoskeletal anchors and remodelers. Here we focus on assembly of so-called "anchoring" or "adhering" junctions-adherens junctions (AJs) and desmosomes (DSMs), which associate with actin and intermediate filaments, respectively. We will examine how the assembly and function of AJs and DSMs are intimately connected during embryogenesis and in adult cells and tissues, and in some cases even form specialized "mixed" junctions. We will explore signaling and trafficking machineries that drive assembly and remodeling and how these mechanisms are co-opted in human disease.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by progressive degeneration of the right ventricular myocardium, ventricular arrhythmias, fibrous-fatty replacement, and increased risk of sudden death. Mutations in 6 genes, including 4 encoding desmosomal proteins (Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin 2, and Desmoglein 2), have been identified in patients with ARVD/C. Mutation analysis of 66 probands identified 4 variants in DSP; V30M, Q90R, W233X, and R2834H. To establish a cause and effect relationship between those DSP missense mutations and ARVD/C, we performed in vitro and in vivo analyses of the mutated proteins. Unlike wild-type (WT) DSP, the N-terminal mutants (V30M and Q90R) failed to localize to the cell membrane in desomosome-forming cell line and failed to bind to and coimmunoprecipitate JUP. Multiple attempts to generate N-terminal DSP (V30M and Q90R) cardiac-specific transgenes have failed: analysis of embryos revealed evidence of profound ventricular dilation, which likely resulted in embryonic lethality. We were able to develop transgenic (Tg) mice with cardiac-restricted overexpression of the C-terminal mutant (R2834H) or WT DSP. Whereas mice overexpressing WT DSP had no detectable histologic, morphological, or functional cardiac changes, the R2834H-Tg mice had increased cardiomyocyte apoptosis, cardiac fibrosis, and lipid accumulation, along with ventricular enlargement and cardiac dysfunction in both ventricles. These mice also displayed interruption of DSP-desmin interaction at intercalated discs (IDs) and marked ultra-structural changes of IDs. These data suggest DSP expression in cardiomyocytes is crucial for maintaining cardiac tissue integrity, and DSP abnormalities result in ARVD/C by cardiomyocyte death, changes in lipid metabolism, and defects in cardiac development.

By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF–membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF–membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.

Summary Armadillo family proteins known as plakophilins have been characterized as structural components of desmosomes that stabilize and strengthen adhesion by enhancing attachments with the intermediate filament cytoskeleton. However, plakophilins and their close relatives are emerging as versatile scaffolds for multiple signaling and metabolic processes that not only facilitate junction dynamics but also more globally regulate diverse cellular activities. While perturbation of plakophilin functions contribute to inherited diseases and cancer pathogenesis, the functional significance of the multiple PKP isoforms and the mechanisms by which their behaviors are regulated remain to be elucidated.

The mechanisms by which proteins are targeted to flagella and cilia are poorly understood. We set out to determine the basis for the specific localization of a 24 kDa flagellar calcium-binding protein (FCaBP) expressed in all life cycle stages of Trypanosoma cruzi. Through the study of trypanosome transfectants expressing various FCaBP deletion mutants, we found that the N-terminal 24 amino acids of the protein are necessary and sufficient for flagellar localization. Subsequent experiments revealed that FCaBP is myristoylated and palmitoylated and, in fact, is one of very few proteins in the cell possessing these acyl modifications. Both fatty acids are required for flagellar localization, suggesting that FCaBP localization may be mediated through association with the flagellar plasma membrane. Indeed, FCaBP associates with the flagellar membrane in a calcium-dependent manner, reminiscent of the recoverin family of calcium-myristoyl switch proteins. Thus, FCaBP is a novel member of the calcium-acyl switch protein family and is the only member described to date that requires two fatty acid modifications for specific membrane association. Its unique localization mechanism is the first described for any flagellar protein. The existence of such a protein in this protozoan suggests that acylation and calcium switch mechanisms for regulated membrane association are conserved among eukaryotes.