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Desmosomes: New Perspectives on a Classic Kathleen J. Green1,2 and Cory L. Simpson1

Desmosomes are highly specialized anchoring junctions that link intermediate filaments to sites of intercellular adhesion, thus facilitating the formation of a supracellular scaffolding that distributes mechanical forces throughout a tissue. These junctions are thus particularly important for maintaining the integrity of tissues that endure physical stress, such as the and myocardium. The importance of the classic mechanical functions of desmosomal constituents is underscored by pathologies reported in animal models and an ever- expanding list of human mutations that target both desmosomal cadherins and their associated cytoskeletal anchoring . However, the notion that desmosomes are static structures that exist simply to glue cells together belies their susceptibility to remodeling in response to environmental cues and their important tissue- specific roles in cell behavior and signaling. Here, we review the molecular blueprint of the desmosome and models for assembling its components to form an adhesive interface and the desmosomal plaque. We also discuss emerging evidence of supra-adhesive roles for desmosomal proteins in regulating tissue morphogenesis and homeostasis. Finally, we highlight the dynamic nature of these adhesive organelles, examining mechanisms in health and disease for modulating adhesive strength and stability of desmosomes. Journal of Investigative Dermatology (2007) 127, 2499–2515; doi:10.1038/sj.jid.5701015

Introduction cytes, desmosomes have fascinated contributed by neighboring cells. The Desmosomes, named after the Greek cutaneous morphologists for almost junctions serve as tethers for cytoplas- ‘‘desmo’’, meaning bond or ligament, 150 years (Getsios et al., 2004b; mic intermediate filaments (IFs), pliant are intercellular adhesive organelles Calkins and Setzer, 2007). These highly 10 nm cytoskeletal fibers designed to that have been characterized as cellu- organized, disc-shaped junctions are withstand a high degree of mechanical lar spot welds by classical histologists. formed by mirror image, tri-partite strain without breaking (Coulombe and As the most prominent cell surface electron-dense plaques, each closely Omary, 2002). Together with their specializations in epidermal keratino- opposing the plasma membranes attached IF network, desmosomes form Editor’s Note Ever since the discovery that skin is composed of multiple and co-authors detail the basic structure and function separate cells that are able to hold themselves together, of desmosomes; Dr. Gabriele Richard and colleagues scientists have been interested in junctions describe Gap junctions; and Dr. Carien Niessen and and adhesion. With the development of microscopes, the co-authors discuss Tight Junctions and Adherens Junctions. basic structure of the desmosome was seen initially with Next month the series will continue with Dr. John McGrath light microscopes and then with the electron microscope. describing the genetic diseases of cell junctions. Over the last 3 decades, however, there has been an These Perspectives will update the cutaneous biologist explosion of knowledge regarding the molecular mechan- on our current knowledge of these critical epithelial isms that hold together (See Milestones in structures that allow keratinocytes to stand together. As Cutaneous Biology Desmosomes, http://www.nature.com/ Aesop stated in his fable The Four Oxen and the Lion, milestones/skinbio1/full/intro.html). The next two issues ‘‘United we stand, divided we fall’’. of JID Perspectives will explore keratinocyte adhesion and cell junctions in-depth. In this issue Dr. Kathy Green Russell P. Hall, III, Editor

1Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA and 2Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA Portions of this review were adapted from Progress in Dermatology, vol. 40 no. 3, 2006 Correspondence: Professor Kathleen J. Green, Northwestern University Feinberg School of Medicine, 303 E. Chicago Ave., Chicago, Illinois 60611, USA. E-mail: [email protected] Abbreviations: DP, ; DP-GFP, DP fused with green fluorescent protein; Dsc, desmocollin; Dsg, desmoglein; IF, ; PG, ; PKC, protein kinase C; PF, pemphigus foliaceus; PKP, ; PV, pemphigus vulgaris Received 17 March 2007; revised 24 April 2007; accepted 30 April 2007

& 2007 The Society for Investigative Dermatology www.jidonline.org 2499 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

a standing of the mechanisms regulating junction homeostasis and remodeling, and how these mechanisms are co- opted during pathogenic processes, leading to human heart and skin disease.

Molecular blueprint of desmosomes and variations on a theme Desmosome proteins come from three major gene families: cadherins, arma- dillo proteins, and (Figure 1). Transmembrane members of the cad- herin family, the desmogleins (Dsgs) and desmocollins (Dscs), cooperate to form the adhesive interface (reviewed b in Koch and Franke, 1994; Angst et al., 2001; Garrod et al., 2002; Getsios et al., 2004b). The cytoplasmic tails of the cadherins provide a binding plat- form for the armadillo family members plakoglobin (PG), (PKPs) 1–3, and p0071 (reviewed in Cowin and Mechanic, 1994; Hatzfeld, 1999; Anastasiadis and Reynolds, 2000; Schmidt and Jager, 2005). The family member, desmoplakin (DP), in turn, links the stress-bearing IF cytoske- leton to this specialized region of the Dsg Pg DP plasma membrane (Hatsell and Cowin, 2001) (Figure 1). Lateral interactions Dsc PKP IF among proteins in the junctional pla- que reinforce its stability (reviewed in Getsios et al., 2004b). Figure 1. Molecular blueprint of the desmosome. (a) Diagram of a ‘spot-weld’ desmosome inspired by (Staehelin and Hull, 1978) and (b) an electron micrograph onto which are super-imposed the major Variations on this molecular blue- desmosomal protein constituents from three families. Transmembrane desmosomal cadherins, Dsg and print exist in the desmosomes of Dsc, bind the armadillo family protein PG, which in turn anchors the plakin family member DP and PKP. different cell types. These variations The cytoplasmic plaque, which is further stabilized by lateral interactions among these proteins, anchors correlate with structural differences, the IF to the desmosome. revealed by electron microscopy, showing that desmosomes in different cells and distinct layers of the epider- a supracellular scaffolding that func- cent evidence has emerged to suggest mis vary in appearance and size. For tions to distribute the forces of physical that these junctions are more struc- instance, during epidermal differentia- stress throughout the tissues. Their turally and functionally complex than tion, smaller, less-organized desmo- important contribution to cell and once thought. In this review, we somes in cells of the basal layer are tissue architecture is revealed in pa- describe how recent findings have replaced by larger, more electron- tients with diseases of the skin and reshaped our perspective of the classic dense desmosomes suprabasally. These heart whose desmosomes are dysfunc- textbook model, allowing us to gain a compositional and structural differ- tional owing to inherited mutations or new appreciation of the structural and ences are thought to tailor junctions attack by autoimmune antibodies or functional variability that exists among to suit specialized functions in different bacterial toxins (for recent reviews, see desmosomes. We challenge the con- organs and stages of differentiation. Cheng and Koch, 2004; Getsios et al., cept that this intercellular organelle is In the case of the adhesive ‘‘core’’, 2004b). simply a mechanical weld that tethers four Dsg (Dsg1–4) and three Dsc genes Although a majority of studies to IF to the membrane, providing evi- (Dsc1–3) have been identified. The Dsc date have focused on identifying the dence that it is a more central partici- genes give rise to two differentially structure and function of the molecular pant in the processes of differentiation spliced isoforms, Dsc ‘‘a’’ and ‘‘b’’, the building blocks that are the basis of the and tissue morphogenesis. Finally, we latter of which is a shorter version desmosomes’ physical properties, re- discuss recent advances in our under- lacking the PG-binding site. Simple

2500 Journal of Investigative Dermatology (2007), Volume 127 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

instance, PKP1, one of the three des- Dsg4 CO mosomal PKPs, is concentrated in the desmosomes of suprabasal cells, and its GR loss due to human mutations results in Dsg1 Dsc1 a that is accom- panied by a loss of DP from junctions and retraction of IF (McGrath et al., SP Involucrin / K1 1999). Other specialized molecules such as pinin (Ouyang and Sugrue, Dsc2 Dsc3 Dsg3 1996; Shi and Sugrue, 2000) and the BA

K14 -binding protein kazrin Dsg2 (Groot et al., 2004) have been impli- Figure 2. Isoform-specific expression pattern of desmosomal cadherins in the epidermis. A schematic of cated in strengthening adhesive inter- the epidermis (right) demonstrates the basal (BA), spinous (SP), granular (GR), and cornified (CO) layers. actions by tailoring the IF cytoskeleton Tightly controlled expression levels and localization of the Dsgs and Dscs (left) within the epidermal deep in the plaque or contributing to layers establish a complex, isoform-specific patterning of desmosomal cadherins that likely contributes to cornified envelope structure, respec- tissue morphogenesis and differentiation. Mouse models (see Table 1) have been utilized to examine cutaneous functions of desmosomal cadherins by targeted ablation or misexpression utilizing tively. Clearly, coordination of the epidermal-specific promoters (far right) that specify basal (e.g., 14) or suprabasal (e.g., keratin 1, various functions of desmosome mem- involucrin) expression. brane and plaque components is a necessary part of orchestrating proper development and homeostasis of com- plex tissues such as the epidermis. epithelia express only the Dsg2/Dsc2 molecule that is a transcriptional target Components originally defined as pair, whereas stratified complex epithe- of p63 (Ihrie et al., 2005). Perp is ‘‘desmosomal’’ can also be found in lia, such as the epidermis, express not expressed in all tissues, but it is non-classical desmosome-related junc- primarily Dsc1/3 and Dsg1/3 with low tempting to speculate that other Perp- tions in other tissues, where they differ levels of Dsg2/Dsc2 in the basal layers like membrane proteins are widely considerably in ultrastructure from and Dsg4 concentrated in the granular present in desmosomes, where they epithelial desmosomes. For instance, and cornified layers (Figure 2) (Garrod may play a more general role in DP is a component of specialized et al., 2002; Mahoney et al., 2006; regulating junctional assembly or func- vascular and lymphatic endothelial cell Dusek et al., 2007). The special attri- tion. The modified desmosomes of junctions called complexus adhaer- butes and developmental contributions suprabasal cornified layers, sometimes entes, which contain vascular endothe- of the differentially expressed desmo- referred to as corneodesmosomes, lial cadherin along with PG and DP somal cadherins are not fully under- contain a secreted glycoprotein (Borrmann et al., 2006; Franke et al., stood, but the highly patterned called , which has 2006; Hammerling et al., 2006). In distribution of different adhesion mole- been reported to have homophilic addition, recent immunoelectron mi- cules may ensure maintenance of cell adhesive properties (Jonca et al., croscopical analysis of cardiac muscle relationships during morphogenesis of 2002). During the final stages of tissue has provided a new perspective multilayered tissues. Consistent with differentiation, the desmosome mem- on the composition of the intercalated this idea, cell adhesion recognition brane complex contributes to the disc, a specialized region of the cardi- peptides that interfere with the adhe- construction of the crosslinked cornified omyocyte membrane classically sive functions of Dsg2/Dsc2 and Dsg3/ cell envelope by serving as a substrate thought to contain both desmosomes Dsc3 prevent proper sorting of lumenal for transglutaminases, likely contribut- and adherens junctions (fascia adherens), and myoepithelial cells, which express ing to some of the unique morpho- in addition to gap junctions. Franke different desmosomal cadherins within logical features of corneodesmosomes et al., (2006) made the surprising three-dimensional mammary cultures revealed by ultrastructural studies observation that desmosome molecules (Runswick et al., 2001). Further, it has (Steinert and Marekov, 1999). intermingle intimately with classic been proposed that the differentiation- Desmosomal plaque proteins, such cadherin components in both junc- specific pattern and ratios of desmoso- as the armadillo family members, PKPs tion types, independent of ultra- mal cadherins may regulate epidermal and PG, and plakin family members, structural appearance. For this reason, development and differentiation (Gar- including , , and intercalated discs have been re- rod, 1996; Chidgey et al., 1997; Ishii periplakin, could also be used to classified as the ‘‘area composita’’ of and Green, 2001). customize desmosomes during differ- adhering junctions. Loss of DP from While the desmosomal cadherins entiation in ways that likely translate these junction types may explain have been thought to be the primary into differences in desmosome function the observed defects in heart and transmembrane molecules within in different tissues or histological layers microvasculature development in desmosomes, others have been (Hatzfeld, 1999; DiColandrea et al., tetraploid rescued DP-null embryos reported, including Perp, a tetraspanin 2000; Getsios et al., 2004b). For (Gallicano et al., 2001).

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Mechanical functions of desmosomes at approximately embryonic day 11 (for Human diseases provide support for revealed: breaking them down and review, see Cheng et al., 2005). In PG- mechanical functions. While investiga- building them up null embryos surviving until birth, skin tors in the lab probed the function of Experimental and spontaneous animal fragility was also observed (Bierkamp desmosome genes by engineering their models reveal desmosomal functions et al., 1996). Similarly, a conditional deletion and mutation over the past in adhesion and tissue integrity. The knockout of DP in skin yielded animals decade, representatives of each of the crucial importance of desmosomes with severe epidermal fragility and loss three major gene families as well as during embryogenesis and in the adult of barrier function leading to perinatal accessory molecules found in the des- is highlighted by observations from lethality (Vasioukhin et al., 2001). mosome have been revealed as targets engineered and spontaneous mutations While DP-null animals were previously for mutation in human diseases of skin in animal models and further supported reported not to assemble desmosomes, and heart. In addition to DP mutations by evidence from human diseases of intercellular junctions were still present causing skin, hair, and/or heart defects, skin, heart, and hair (Cheng and Koch, in the epidermis of the -Cre human mutations have also been iden- 2004; McGrath, 2005). Mutations in mice with targeted ablation of DP, but tified in PG (skin, hair, heart), PKP1 both the transmembrane adhesive mole- lacked an inner plaque and could not (skin), PKP2 (heart), Dsg1 (skin), Dsg4 cules and the plaque components that anchor IF properly. (hair), Dsg2 (heart), and Dsc2 (heart) anchor the IF cytoskeleton lead to a More recent reports are revealing a (reviewed in McGrath, 2005; Pilichou spectrum of phenotypes that have been similar range of phenotypes owing to et al., 2006; Syrris et al., 2006). These largely attributed to mechanical de- ablation of or mutations in desmosomal observations suggest that mutational fects, but, as will be discussed later, the cadherins. Engineered and sponta- attack on any individual component resulting pathologies appear to encom- neous mutations in desmosomal cad- of the complex is sufficient to unravel pass defects that go beyond the struc- herins that are expressed in a more or compromise junctional structures, tural roles of this organelle. limited distribution in stratified epithe- resulting in defects in tissue integrity, DP-null embryos do not survive lia or epidermal appendages have development, and differentiation. Des- beyond embryonic day 6.5, the time restricted phenotypes, including skin mosomal component mutations can be during when the egg cylinder normally fragility and barrier defects (reviewed inherited as both dominant and reces- expands (Gallicano et al., 1998). Not in Cheng and Koch, 2004) (Figure 2 sive alleles, and the defects range from only did /18 IF appear dis- and Table 1). In contrast, ablation of relatively minor skin conditions, result- organized in these developing em- Dsg2, which is expressed in very early ing from haploinsufficiencies and point bryos, but also the number and embryogenesis and throughout epithe- mutations, to severe fragility and mor- structure of desmosomes were im- lial tissues, exhibited an embryonic phogenetic defects. In some cases, paired. These observations led the lethal phenotype, dying shortly after other phenotypic characteristics are authors to suggest that desmosomes implantation (Eshkind et al., 2002). observed, including arrhythmogenic are not simply uncoupled from IF in Moreover, the authors suggested that right ventricular cardiomyopathy and DP-deficient animals, but their assem- this cadherin is necessary for normal woolly hair. bly is also altered. This same group embryonic stem cell proliferation be- The spectrum of mutations and followed up their initial studies by fore the appearance of cell junctions. disease phenotypes supports a general performing a tetraploid rescue experi- Surprisingly, loss of Dsc3, which was conclusion that desmosomes and their ment in which extraembryonic func- originally reported as being present molecular building blocks are impor- tions were rescued, allowing an specifically in stratified epithelia (Chid- tant for the proper function of the heart analysis of specific embryonic defects gey et al., 1997; King et al., 1997), and skin, but leaves us with some owing to loss of DP (Gallicano et al., leads to an even earlier, pre-implanta- puzzling unanswered questions. There 2001). The resulting animals were tion lethal phenotype in mice (Den does not seem to be a particularly unable to survive much past gastrula- et al., 2006). The authors reported that strong correlation between the location tion owing to dysfunctions of the heart, embryos die within the first 2 days of of a mutation and phenotypic charac- neuroepithelium, and skin. As men- development, before the appearance of teristics that arise in families in the case tioned above, defects in microvascula- mature desmosomes, and suggested of DP. In some cases, haploinsuffi- ture, in which the endothelial cells that this cadherin may operate inde- ciency of DP leads to skin disease are joined by complexus adhaerentes pendently of desmosomes in early alone (Armstrong et al., 1999) and in instead of desmosomes, were also embryonic adhesion or other functions. others to heart defects alone (Norman observed. Interestingly, a truncated form of Dsc1 et al., 2005; Yang et al., 2006). It is also Conditional ablation of DP in the lacking the PG- and PKP-binding tail was unclear why defects in internal epithe- heart (Garcia-Gras et al., 2006) or null shown to be sufficient to support a lial tissues have not been reported mutations of the armadillo proteins PG normal epidermal phenotype in vivo in patients. For instance, one might or PKP2 (Bierkamp et al., 1996; Ruiz (Cheng et al., 2004). The possibility that speculate that alterations in the intest- et al., 1996; Grossmann et al., 2004) desmosome molecules play desmosome- inal epithelium, which is another con- yield animals with cardiac abnorma- independent functions will be devel- stantly renewing tissue like epidermis lities and associated lethality beginning oped further in the sections that follow. that is exposed to its own set of

2502 Journal of Investigative Dermatology (2007), Volume 127 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

Table 1. Mouse models demonstrating desmosomal cadherin function Gene Author Targeting/mis-expression Observed phenotype

Dsc1 Chidgey et al. (2001) Targeted Dsc1-null mutation Epidermal flaking, defective barrier function, hyperproliferation, and granular layer acantholysis Dsc1 Henkler et al. (2001) Dsc1 transgene driven by keratin 14 promoter (basal) Normal epidermis Dsc1 Cheng et al. (2004) Targeted C-terminal truncation of Dsc1a/b Normal epidermis Dsc3 Hardman et al. (2002) Dsc3 transgene driven by keratin 1 promoter Ventral alopecia, dermal cysts, epidermal (suprabasal) hyperproliferation and hyperkeratosis, and altered b- stability/signaling Dsc3 Den et al. (2006) Targeted Dsc3-null mutation Lethal before implantation Dsg2 Eshkind et al. (2002) Targeted Dsg2-null mutation Lethal shortly after implantation; decreased proliferation of embryonic stem cells Dsg2 Brennan et al. (2007) Dsg2 transgene driven by involucrin promoter Epidermal hyperkeratosis, hyperplasia, and (suprabasal) hyperproliferation, with increased resistance to apoptosis Dsg3 Allen et al. (1996) N-terminally truncated Dsg3 transgene driven by keratin Epidermal flaking, hyper- and para-keratosis, and 14 promoter (basal) autoamputation of tail Dsg3 Koch et al. Targeted Dsg3-null mutation Runting, erosions of oral mucosa, supra-basilar (1997, 1998) epidermal acantholysis, and hair loss Dsg3 Elias et al. (2001) Dsg3 transgene driven by involucrin promoter Post-natal death with increased trans-epidermal water (suprabasal) loss and scaling of stratum corneum with lamellar structure as in mucous membranes Dsg3 Merritt et al. (2002) Dsg3 transgene driven by keratin 1 promoter Epidermal flaking and pustules, hyper-proliferation, (suprabasal) altered differentiation with hyper- and para-keratosis, and hair loss Dsg4 Kljuic et al. (2003) Spontaneous Dsg4 mutations (lanceolate hair mouse) Defective hair keratinization and premature differentiation of keratinocytes with hyperproliferation

environmental stresses, would be ob- dermal layers and different body sites those seen in PF (Amagai et al., 2000). served in patients with DP or desmo- (Stanley, 1995; Amagai, 1999), which Again, exactly how Dsg1 proteolysis somal cadherin mutations. Possible correlate with the expression patterns leads to generation of subcorneal defects in the processes of wound of Dsg3 and Dsg1, respectively (Shimi- blisters, and how the effects of exfolia- healing and neovascularization, the zu et al., 1995; Amagai et al., 1996). tive toxin A compare functionally latter requiring remodeling of DP- Although there has been controversy with those of anti-Dsg1 antibodies in expressing endothelia, might also be regarding their role in pathogenesis PF to produce identical epidermal predicted. Thus it is clear that (Nguyen et al., 2000; Amagai et al., pathologies is unknown. Collectively, additional studies into the molecular 2006), compelling evidence supports however, these autoimmune and in- consequences of specific desmosomal the idea that Dsg autoantibodies fectious diseases support the idea that mutations will be necessary to provide contribute to disease (Amagai et al., desmosomes make a key contribution further functional insight into these 1995, 1994a). Whether acantholysis to keratinocyte adhesion required for phenotypic differences. is caused by direct interference with epidermal integrity. Desmosome membrane glycopro- Dsg-dependent adhesion or antibody- teins are not only subject to defects triggered cellular signaling events re- Reconstituting desmosomes in a test tube due to gene mutation, but also to quired for blister development is a The adhesive interface. Previous stu- inactivation by autoimmune antibodies matter of current debate (Kottke et al., dies have demonstrated that classic and toxins produced by infectious 2006). Recent reports have also shown cadherins, which make up the core of bacteria (Payne et al., 2004; Stanley that the pathogenic agent produced by adherens junctions, can impart Ca2 þ - and Amagai, 2006). The targets in this Staphylococcus aureus in Staphylo- dependent adhesion onto normally case are primarily members of the Dsg coccal scalded-skin syndrome and non-adherent fibroblasts. Thus, it was subfamily of desmosomal cadherins. bullous impetigo, exfoliative toxin A, logical to ask whether desmosomal The presence of Dsg autoantibodies in is a serine protease that specifically cadherins are similarly sufficient to patients with pemphigus vulgaris (PV) cleaves the ectodomain of Dsg1. confer adhesive properties on cells. In and pemphigus foliaceus (PF) is asso- This protease results in epidermal spite of the fact that homophilic adhe- ciated with blistering in distinct epi- blisters that are virtually identical to sive or ‘‘trans’’ interactions between

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desmosomal cadherins can occur in principles governing IF anchorage. While cell culture, in vitro overlay, vitro using recombinant molecules or Protein interaction studies have pro- and yeast two/three hybrid experiments peptides (Syed et al., 2002; Waschke vided a picture of desmosomes that all support the idea that DP associates et al., 2005), a series of studies establishes a linear chain in which the with IF, there is still uncertainty demonstrated that individual desmoso- Dsc and Dsg tails all associate with PG regarding the relative contributions of mal cadherins are unable to confer via an intracellular cadherin-type seg- the plakin repeat domains themselves adhesive properties comparable to ment domain resembling the classic and the flexible linking regions that those of epithelial cells on non-adhe- cadherin-binding site for b-catenin. In divide and terminate these domains. rent fibroblasts (Amagai et al., 1994b; turn, PG binds via its central armadillo Co-sedimentation analyses are consis- Chidgey et al., 1996; Kowalczyk et al., repeat domain to the N-terminus of DP. tent with the idea that plakin repeat 1996; Marcozzi et al., 1998). Finally, through its C-terminus, DP domains are sufficient for binding to Collectively, these studies suggest not completes the link to the IF cytoskele- . These studies also suggest only that both Dsgs and Dscs ton. In general, PKPs exhibit a much that the more the plakin repeat are required to reconstitute intercellu- more complex repertoire of interac- domains, the better the binding: B plus lar adhesion (Kowalczyk et al., 1996; tions, associating with desmosomal C, or a protein containing all three Marcozzi et al., 1998; Tselepis et al., cadherins, other armadillo proteins plakin repeat domains, co-sediment 1998), but also that the ratio of (both classic and desmosomal), the N- more vimentin than individual domains these cadherins must be carefully terminus of DP, and IF directly, thus (Choi et al., 2002). Other studies regulated or else adhesion will not likely contributing to a complex web of suggest that the plakin repeat domains occur (Getsios et al., 2004a). In lateral stabilizing interactions in the cannot bind in the absence of addition, co-immunoprecipitation plaque (Hatzfeld, 2006). While high- associated flexible regions, and also experiments demonstrated that ectopi- resolution structural data, such as those underscore the important contribution cally expressed Dsc and Dsg from recently obtained for the arm repeats of of the terminal tail (Fontao et al., 2003). neighboring cells can interact hetero- PKP1, are likely to aid in defining how Collectively, however, the studies philically (Chitaev and Troyanovsky, these family members engage in agree that different sequences within 1997). These experiments strongly sup- interactions, so far all the identified the linkers and tail fine-tune and port the idea that both Dsgs and Dscs partnerships occur through the PKP specify interactions with different IF are needed for normal adhesive func- head domain (Choi and Weis, 2005; types (Green et al., 1990; Stappenbeck tion, but stop short of detailing how Hatzfeld, 2006). et al., 1993a; Kouklis et al., 1994; they are organized in situ, and whether The anchorage of IF to the desmo- Meng et al., 1997; Choi et al., 2002; interactions at the molecular level are somal plaque is thought to occur Fontao et al., 2003; Lapouge et al., homophilic, heterophilic, or both. largely through interactions of the C- 2006). Further, interaction of the Recent tomographic studies have terminus of DP with keratin, , or C-terminus of DP with IF is modulated attempted to address how desmosomal vimentin IF (Stappenbeck and Green, from strong to weak by serine cadherins are organized in situ. Based 1992; Stappenbeck et al., 1993b; phosphorylation at residue 2849 on the analysis of cryosections of Kouklis et al., 1994; Meng et al., (Stappenbeck et al., 1994), and a desmosomes from mouse epidermal 1997; Fontao et al., 2003). This point mutation at this site (S2849G) tissue, the authors interpreted their data IF-binding domain comprises three impairs DP assembly into desmosomes to suggest that the organization of subdomains, each consisting of 4.5 (Godsel et al., 2005). The recent desmosomal cadherin ectodomains is copies of a 38-residue plakin repeat identification of additional serine sites consistent with a previously proposed domain (Green et al., 1990; Choi et al., located in key linking regions surround- model for classic cadherin ectodomain 2002). The subdomains are separated ing the plakin repeat domain and interaction. However, the identity of by flexible linking regions and the tail suggests further opportunities for contributing partners was not ad- final ‘‘C’’ plakin repeat domain is regulating the conformation and IF- dressed in this study. Advances in followed by a terminal 68-residue tail. binding properties of this domain biochemical crosslinking approaches, Based on the pattern of charged (Beausoleil et al., 2004). similar to those employed to analyze residues in the C-terminal domain, Our understanding of DP–interme- classic cadherins (Troyanovsky, 2005), we previously hypothesized that the diate filament interactions is limited by may provide a strategy for sorting out C-terminus of DP interacts with IF a lack of knowledge about the IF end of how these cadherins are organized in (Green et al., 1990). Molecular model- the partnership. Type I and II keratin IF, cells. ing based on more recent structural which are most frequently associated analysis suggested that approximately with DP in desmosomes of epithelial The plaque. A combination of in vitro four turns of an a-helix, such as those cells, are known to form obligate hetero- protein–protein interaction studies and present in the rod domains of IF, might polymers in vivo (Coulombe and Omary, reconstitution strategies has also been be able to interact with a shallow 2002). Early work suggested that forma- used to try to understand the minimal positively charged groove present with- tion of the heterodimer is not required for requirements for assembling a desmo- in each plakin repeat domain (Choi IF binding, at least not in the case of somal plaque and for establishing et al., 2002). epidermal (Kouklis et al., 1994;

2504 Journal of Investigative Dermatology (2007), Volume 127 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

Meng et al., 1997). However, more and cardiovascular tissues. However, of the hair follicle, it was not possible recent in vitro studies suggest that both desmosome molecule functions are not in this initial study to differentiate type III IF and keratins, including epider- limited to their traditional roles in between adhesion-dependent and ad- mal keratins, require the presence of the desmosomes or in providing mechan- hesion-independent defects. A some- a-helical rod domain for DP association ical integrity to tissues, but extend to what similar impairment in the (Fontao et al., 2003), although head supra-adhesive functions in vivo. Con- transition from the basal proliferative and/or tail sequences may be required sistent with a role for desmosomal to suprabasal differentiating keratino- for optimal binding. cadherins in morphogenesis and tissue cyte compartments was observed in How all of these individual interac- homeostasis, misexpression or targeted organotypic epidermal ‘‘rafts’’ in which tions are harnessed by the cell to deletion of desmosomal cadherins in Dsg1 expression was limited using construct the plaque is not well under- mice affects keratinocyte homeostasis shRNA-mediated knockdown (Getsios stood, and efforts to deconstruct the and differentiation (Allen et al., 1996; S, Simpson CL, Green KJ, unpublished plaque have been hampered by Chidgey et al., 2001; Elias et al., 2001; data). In Dsg1-deficient raft cultures, the highly insoluble nature of the des- Merritt et al., 2002; Cheng and Koch, the intermediate layers and markers of mosome. In an extension of the ap- 2004; Hardman et al., 2005) (Table 1). spinous differentiation were under-re- proaches used to reconstitute adhesion Forced suprabasal expression (via the presented, suggesting that this cadherin in a test tube, investigators endeavored involucrin promoter) of Dsg2, which is is required for normal epidermal mor- to construct plaques in vitro by expres- normally only present at low levels deep phogenesis. Interestingly, specific re- sing a combination of armadillo pro- in the epidermis, led to epidermal moval of the Dsg1 adhesive teins and DP in concert with full-length hyperplasia and the development of ectodomain by chronic exposure desmosomal cadherins or chimeric pre-cancerous papillomas (Brennan to exfoliative toxin A did not inhibit membrane molecules containing either et al., 2007). These changes were stratification or intermediate layer Dsc or Dsg tail domains. While these accompanied by an increase in the morphogenesis, suggesting that Dsg1’s experiments are subject to a number activity of multiple cell growth and role in this process is adhesion- of interpretations, some common survival pathways. These findings are independent. themes have emerged. These include particularly interesting in light of the How might desmosomes be in- the following: (1) the complexity and previous observation that Dsg2 is im- volved in adhesion-independent signal- insolubility of the desmosomal plaque portant for proliferation of embryonic ing? One potential mechanism is increases with introduction of each stem cells (Eshkind et al., 2002). How- through PG, which is an armadillo class of components (Bornslaeger ever, the underlying molecular mechan- family member and the closest relative et al., 2001); (2) in concert with PG isms directly linking Dsg2 to cell growth of b-catenin, a well-known regulator of and the DP N-terminus, the desmoso- and survival have not been determined. the canonical Wnt/wingless signaling mal cadherin tails are sufficient to Suprabasal expression of Dsg3 and pathway (Zhurinsky et al., 2000; Huels- nucleate the formation of an electron- Dsc3 driven by the keratin 1 promoter ken and Birchmeier, 2001; Yin and dense plaque (Kowalczyk et al., 1997); similarly resulted in epidermal hyper- Green, 2004). Because PG is capable but (3) the proper clustering and/or proliferation, but a different outcome of associating with, and perhaps com- segregation of plaque components into was observed when Dsg3 was misex- peting for, many of the same partners as punctate plaque structures requires the pressed suprabasally via the involucrin b-catenin, its ability to modulate the coexpression of a PKP and PG (Born- promoter. In this case, Dsg3 expression Wnt pathway has been thought by slaeger et al., 2001; Koeser et al., resulted in a Dsg profile similar to that some to be indirect, by interfering with 2003); and (4) the extent of plaque seen in normal mucous membranes and b-catenin’s turnover and/or transcrip- length is regulated by PG end domains conversion to an epithelium with histo- tional activity. This idea is supported by (Palka and Green, 1997). logical and functional attributes of a one study in which a stabilized form of While these biochemical studies non-keratinized mucosal epithelium PG (DN122-PG) led to the formation of have laid a foundation for our under- (Elias et al., 2001). This observation additional hair germs and hyperplastic standing of how desmosomes are built supported the idea initially proposed by hair follicles (Teuliere et al., 2004), from the ground up, understanding Garrod that the tightly regulated expres- similar to those seen in mice with assembly in cells and tissues will sion profile of desmosomal cadherins elevated b-catenin (Gat et al., 1998); require different strategies, such as live may be required for proper patterning of however, this effect is seen only in a cell imaging, which will be discussed stratified tissues. b-catenin wild-type, not null, back- below. In hair follicles from mice with Dsg4 ground. The notion that altering des- mutations, Kljuic et al. (2003) observed mosome function could impact on Non-traditional roles of desmosome an abrupt, rather than gradual, transi- b-catenin signaling is supported by the proteins in differentiation and disease tion from the proliferative to differen- observed ability of the b-catenin bind- The studies described so far underscore tiating zones of follicular cells, a defect ing partner PKP2 to modulate b-cate- the importance of desmosomes as inter- resulting in a lanceolate hair pheno- nin-dependent TOP-FLASH reporter cellular adhesive organelles that are type. Although this observation sug- activity in vitro (Chen et al., 2002); in required for the integrity of epithelial gests a role for Dsg4 in morphogenesis addition, forced suprabasal expression

www.jidonline.org 2505 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

of Dsc3 enhanced b-catenin signaling study has shown that loss of DP from nyavsky et al., 2007). These authors activity and altered epidermal differen- junctions can result in PG translocation suggest further that Dsgs are not the tiation in transgenic mice (Hardman into the nucleus of cardiac myocytes sole or even primary mediators of this et al., 2005). (Garcia-Gras et al., 2006). This resulted cascade, based on its continued stimu- On the other hand, recent reports in a reduction in canonical Wnt signal- lation in cells with siRNA-mediated have demonstrated PG’s ability to ing through Tcf/Lef1, decreases in b- knockdown of Dsg1 or 3. Associated signal in a b-catenin-deficient back- catenin target genes c-Myc and cyclin armadillo proteins may be important ground, providing support for b-cate- D1, and an increase in adipogenic contributors to cellular responses lead- nin-independent functions in signaling . In animals, these ing to acantholysis, as pathogenic PV (Maeda et al., 2004; Teuliere et al., alterations were accompanied by ex- antibodies are unable to induce keratin 2004; Williamson et al., 2006). These cess adipocytes and fibrosis in the IF retraction in PG-null keratinocytes included a regulatory role in differen- heart, increased myocyte apoptosis, (Caldelari et al., 2001). Further, PV tiation and proliferation of keratino- cardiac dysfunction, and ventricular antibodies appear to reduce nuclear cytes within the interfollicular arrhythmias, similar to the pathologies accumulation of PG, thus alleviating epidermis of mice expressing stabilized observed in patients with arrhythmo- PG-dependent inhibition of c-Myc trans- PG in a b-catenin-null background genic right ventricular cardiomyopathy. cription, which was proposed to con- (Teuliere et al., 2004). Another study These data shed new light on the tribute to an increase in proliferation demonstrated that ectopically ex- previous proposal that the pathogenesis and loss of intercellular adhesion in PV pressed PG suppressed hair growth in arrhythmogenic right ventricular patients (Williamson et al., 2006). and follicle development even in a cardiomyopathy is due to transdiffer- While PG has been implicated in wild-type b-catenin background (Char- entiation of cardiac myocytes to adipo- several ‘‘non-traditional’’ roles, non- pentier et al., 2000). The notion that PG cytes in response to stress (d’Amati desmosomal functions of the PKP sub- has functions distinct from b-catenin is et al., 2000). In fact, this transdiffer- family of armadillo proteins are just also supported by cell culture studies entiation may be due more directly to beginning to emerge. In addition to its showing that PG-null keratinocytes dis- alterations in PG signaling. These data roles in junctions, PKP2 may play a play reduced motility, even of single also shed light on the ‘‘woolly hair’’ more general role in regulation of cells, and reduced sensitivity to UV- phenotype observed in humans with transcriptional machinery as part of induced apoptosis (Yin et al., 2005b; certain mutations in either PG or DP, the RNA polymerase III holoenzyme Dusek et al., 2007). raising the possibility that similar to b- complex (Mertens et al., 2001). Its Post-translational modification of catenin, PG may be important in fate access to the transcriptional machinery PG has been shown to regulate its determination during hair follicle mor- is regulated by C-TAK1-dependent protein partnerships and subcellular phogenesis, and DP mutations alter this phosphorylation and 14-3-3 binding, distribution, and this ultimately has an function (MacRae et al., 2006). which determine whether it can gain impact on the available signaling pool. Signaling pathways linked to desmo- entrance to the nucleus (Muller et al., Tyrosine phosphorylation has been somes have also been revealed by 2003). The family members PKP1 and 3 reported to promote PG’s translocation cellular responses to pemphigus anti- have also been found in association into the nucleus in keratinocytes, re- bodies directed against Dsgs, including with RNA-binding proteins, and may sulting in its increased association with increases in inositol triphosphate, mo- likewise participate in processes of LEF/TCF transcription factors and in- bilization of intracellular calcium with translation and RNA metabolism (Hof- hibition of b-catenin-dependent repor- concomitant activation of protein ki- mann et al., 2006). The PKP-related ter activity (Hu et al., 2003). PG can be nase C (PKC) (Esaki et al., 1995; protein p0071 (sometimes referred phosphorylated by multiple protein Seishima et al., 1995; Osada et al., to as PKP4) was recently shown to tyrosine kinases with different effects 1997), and phosphorylation of the be required for spatially localizing depending on the site of phosphoryla- Dsg3 cytoplasmic domain accompa- RhoA during cytokinesis through its tion, which modulates both its associa- nied by loss of PG binding (Aoyama association with the Rho GTPase ex- tion with intercellular junctions and et al., 1999). Further, antibody-induced change factor Ect2 (Wolf et al., 2006). availability for regulation of LEF/TCF interference with RhoA and activation Finally, the periplakin-binding protein, signaling (Miravet et al., 2003). For of p38 mitogen-activated protein kazrin, associates with the nuclear instance, Y549 phosphorylation in- kinase have been reported to be re- matrix in a cell cycle-dependent creases PG binding to adherens junc- quired for loss of keratinocyte adhesion manner before blastocyst formation tion components, resulting in an mediated by pathogenic antibodies during embryogenesis (Gallicano increase in the transcriptional activity (Berkowitz et al., 2005, 2006; Waschke et al., 2005). Although kazrin is not a of the b-catenin–Tcf4 complex (Miravet et al., 2005, 2006). p38 mitogen- PKP protein, it shares plakin-binding et al., 2003). Whereas EGF receptor- activated protein kinase activation has properties with PKP family members. dependent tyrosine phosphorylation of recently been proposed to occur late in Collectively, these studies raise the PG can impair DP recruitment to a signaling cascade involving the up- possibility that plakin-binding proteins junctions, weakening desmosomal ad- stream activation of EGF receptor and have diverse nuclear functions outside hesion (Yin et al., 2005a), a recent Src in response to PV antisera (Cher- of junctions.

2506 Journal of Investigative Dermatology (2007), Volume 127 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

While we have focused so far on sition of insolubility correlating with Based on electron microscopy ana- non-desmosomal signaling and nuclear junction assembly. The evidence sug- lysis, the cytoplasmic particles range in functions in this section, recently an gested that the plaque and membrane size, the largest frequently exceeding unexpected role for DP in regulating pools exist in spatially and biochemi- the diameter of a large keratin IF the distribution of the cally separate compartments until the bundle (Jones and Goldman, 1985; cytoskeleton during epidermal differ- final assembly steps at the plasma Godsel et al., 2005). While their entiation was observed. membrane, and that DP is associated biochemical nature is poorly under- were shown to shift away from with the IF cytoskeleton whereas mem- stood, immunogold electron micro- a radially arrayed, centrosome- brane proteins are associated with the scopy and ‘‘retrospective’’ associated organization in basal cells microtubule cytoskeleton (Figure 3). immunofluorescence microscopy of to accumulation in the cortical region Early ultrastructural studies also sug- cells first imaged live and then counter- near cell–cell junctions within supraba- gested that the DP–IF scaffold is closely stained for other junctional molecules sal keratinocytes (Lechler and Fuchs, associated with the cortical cy- revealed that the DP-associated protein 2007). Along with this, there is a toskeleton (Green et al., 1987). How- PKP2 is uniquely associated with as- redistribution of the centrosomal pro- ever, further analysis has been sembly-competent cytoplasmic parti- tein called ninein to desmosomes. This hampered by the insoluble nature of cles in the cells used in this study. switch in ninein distribution requires desmosomes and desmosome precur- Membrane components such as Dsc DP. Although desmosomal localization sors (Pasdar et al., 1991). Recent and Dsg were not associated with of another microtubule-associated pro- developments in optical microscopy anterograde moving particles, only tein, CLIP170, was reported years ago have helped overcome these limita- retrogradely moving particles, which (Wacker et al., 1992), until now a tions. Using fluorescently tagged DP may have represented endocytosed or functional association between these and desmosomal cadherins, several engulfed desmosomes. junctions and microtubule organization studies have revealed previously un- The mechanism of translocation of had not been revealed. This finding appreciated dynamic behaviors of both the precursor particles is unclear. How- opens up new avenues for examining plaque and membrane proteins (Wind- ever, the observation that the micro- the role of desmosomes in regulating offer et al., 2002; Gloushankova et al., filament poison, cytochalasin D, cell polarity and cytoarchitecture. 2003; Godsel et al., 2005). significantly impairs DP particle move- ment (Godsel et al., 2005) suggests that Desmosome dynamics and regulation in Assembly/regulation of the desmoso- contribute to the trans- health and disease mal plaque. Using DP fused with green location process. As previous studies Much has been done to catalog the fluorescent protein (DP-GFP), we re- have suggested that the formation of protein–protein interactions of desmo- cently mapped the behavior of DP in adherens junctions is functionally some molecules, but how these interac- three dimensions during cell contact- linked to desmosome assembly (Lewis tions are coordinated temporally and initiated desmosome assembly (Godsel et al., 1997), it is tempting to speculate spatially in living cells to regulate assem- et al., 2005). These studies suggested that events associated with maturation bly, disassembly, and adhesive strength is that assembly occurs in three tempo- of adherens junction-associated corti- less well understood. Characterizing these rally overlapping phases, beginning cal actin may be required for proper key determinants of junctional stability with the rapid accumulation of DP at formation of desmosomes upon cell–- will be important not only for under- contacting borders during the first few cell contact. Interestingly, PKPs can standing desmosome homeostasis and minutes following contact. Contact associate with actin, providing a po- remodeling, but also for discovering produces a signal that triggers the tential link between the DP–IF network how mechanisms that regulate assembly formation of fluorescent bright non- and actin-dependent desmosome as- state are co-opted in human disease. membrane-bound DP-containing parti- sembly (Hatzfeld et al., 2000). cles in the cortical region of the cell Although previous studies have sug- Assembly. A series of papers in the (phase II) that subsequently translocate gested that IF are not required for DP 1980s mapped out alterations in the at a very slow rate (slower than most accumulation and desmosome assem- localization, biosynthesis, and fate of conventional microtubule-based mo- bly, the behavior of DP-GFP mutants the desmosomal plaque and membrane tors) to cell–cell borders, where they with compromised or enhanced bind- components following the elevation of join the progressively brightening clus- ing to IF suggests that they play a extracellular calcium levels to trigger ters that formed during the earlier regulatory role in the biphasic en- junction assembly (e.g., Watt et al., phase. This observation is consistent hancement of DP at cell–cell borders 1984; Jones and Goldman, 1985; Mat- with earlier work, suggesting that at revealed by live cell imaging (Godsel tey and Garrod, 1986; Penn et al., least some endogenous DP particles et al., 2005). A C-terminal phosphor- 1987; Pasdar and Nelson, 1988a,b). observed in the cytoplasm are desmo- ylation-deficient serine-to-glycine mu- Collectively, these studies demon- some precursors (Jones and Goldman, tant, DP-S2849G, exhibits delayed strated that desmosomal components 1985), rather than all being remnants of outward trafficking, in part owing to are synthesized and initially recruited previously internalized junctions (Mat- its aberrant retention on IF, to which it into a soluble pool, followed by acqui- tey and Garrod, 1986). binds with higher affinity (Stappenbeck

www.jidonline.org 2507 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

a Cyto D

Membrane components Plaque components b

9 minutes 19 minutes 49 minutes 64 minutes

c 350 DMSO 300 DP If Cytochalasin D 250 Dsc Microtubule 200 Dsg Actin 150 100 PG MT-based 50 motor PKP Border intensity/length 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time (minutes)

Figure 3. Model of desmosome assembly from distinct membrane and plaque compartments. (a) On the left, Dsg- and/or Dsc-containing vesicles bud from the Golgi (1) and appear to be transported along microtubules (MT) (nocodazole-sensitive) by unknown motors (2), which deliver them to the cell surface (3), allowing formation of cadherin trans-dimers (4) and later incorporation into mature desmosomes (5). It is not completely clear whether Dsgs and Dscs traffic together or in separate vesicles. On the right, non-membranous DP- and PKP-containing particles appear in the cytoplasm (1), then move toward the periphery, where actin-based motility (cytochalasin D-sensitive) seems to facilitate delivery to the cell surface (2), allowing formation of immature junctions (3) and eventual incorporation into mature desmosomes (4). (b) Live imaging of wounded epithelial cells shows appearance of cytoplasmic DP-GFP particles (arrows), which move toward the cell periphery and incorporate into nascent intercellular junctions. (c) Quantification of cell–cell border fluorescence reflects a two-stage increase in DP-GFP accumulation over time, the second of which is completely inhibited by application of cytochalasin D to destabilize actin filaments (adapted in part from Godsel et al., 2005).

et al., 1994; Meng et al., 1997; Fontao (Huen et al., 2002). Further, adherens dependent desmosome formation et al., 2003). Achieving maximum junctions are also unable to mature (Hobbs RP, Hsieh SN, et al., personal intercellular adhesive strength requires properly in cells lacking DP and communication) and results in retention proper attachments of junctions to both normal IF attachments (Vasioukhin of DP along IF in a manner similar to IF and cortical actin, as intercellular et al., 2001). that observed for the S2849G DP mutant. adhesion is synergistically strengthened by Interestingly, knockdown of the Activation of PKC had previously the presence of IF and actin attachments a-isoform of PKC impairs calcium- been reported to trigger desmosome

2508 Journal of Investigative Dermatology (2007), Volume 127 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

formation in low-calcium conditions, herin assembly behavior has also transport of 60 nm vesicles containing or in cells lacking desmosomes owing benefited from the use of fluorescently mostly Dsc2 and lower amounts of Dsg to mutation of adherens junction pro- labeled probes. Using Dsg2 fused to and E-cadherin to the cell surface. The teins (Sheu et al., 1989; Hengel et al., GFP (Dsg2-GFP), the continual assem- second stage involves transport of Dsg, 1997). Thus, PKC may act as a rheostat bly of nascent desmosomes has been E-cadherin, PG, and b-catenin in to control both the availability of DP for observed within stable contacts located 200 nm vesicles that were specifically junction assembly and the rate at at the middle of an epithelial sheet targeted to the basolateral plasma which DP reinforcements are as- (Gloushankova et al., 2003). New GFP- membrane, rather than diffusely all sembled into particles and traffic to labeled structures appear as a group of over the cell surface, as was the case join their counterparts already building fine puncta, which after a few minutes with the smaller vesicles. Plaque pro- up the desmosomal plaque. Recently, a coalesce into a single structure. This teins such as DP I/II were added mutation located within the regulatory observation is in line with the rapid subsequently to these sites of junction terminal tail of DP, which also contains appearance of Dsg2-GFP puncta in nucleation (Burdett and Sullivan, two other serine phosphorylation sites newly contacting cells at the edge of 2002). Consistent with this, electron (Beausoleil et al., 2004), was disco- a wound (Amargo EV and Green KJ, microscopy pulse–chase experiments vered in patients with arrhythmogenic unpublished data). Desmosomal cad- demonstrated that Dsg3 first forms right ventricular cardiomyopathy (Yang herins can continue to exchange into small clusters on the surface lacking et al., 2006). It seems plausible that this stable junctions once they are formed, IF attachment, chasing into larger IF- mutation could affect phosphorylation- as demonstrated by fluorescence re- attached half desmosomes, before dependent regulation of DP either by covery after photobleaching, showing presumably incorporating into desmo- altering the phosphorylation state of the that the desmosomal cadherin Dsc2 somal junctions (Sato et al., 2000). tail or by interfering with the conforma- exchanges into existing junctions with These data suggest a multistage me- tion of this region and thus the ability of a half-life of 30 minutes (Windoffer chanism for regulating the distribution the tail to properly fine-tune interac- et al., 2002). Although the extent of and rate of junction assembly. Further tions with IF. Future studies to examine desmosome dynamics in vivo during studies will be necessary to address the role of DP phosphorylation regula- epidermal differentiation is unknown, whether Dscs and Dsgs are functionally tion in skin wound healing and cardiac clearly new desmosomes with different interdependent at any stage during the function will be important to under- compositions reflecting differentiation- secretory process, or whether their stand the physiological importance of dependent expression patterns have to proper sorting and delivery require these assembly regulatory pathways in be made either de novo during this association of specific armadillo pro- tissue function. process or remodeled or both (North teins. In addition, the potential involve- Interfering with intracellular cal- et al., 1996). ment of specific Rab GTPases and/or a cium homeostasis also results in the Previous studies suggested that Dsg unique desmosomal exocyst complex, accumulation of cytoplasmic DP in in Madin–Darby canine kidney cells similar to that emerging for classic cells derived from patients with muta- was trafficked toward the plasma mem- cadherins, is yet to be tested (Langevin tions in the gene encoding the sar- brane on microtubules (Pasdar and et al., 2005; Lock and Stow, 2005). co(endo)plasmic reticulum Ca2 þ - Nelson, 1989). Our recent data support ATPase isoform 2 pump (Sakuntabhai this idea, as dual-label live cell imaging Modulation of desmosome adhesive et al., 1999; Dhitavat et al., 2003). shows Dsg-GFP-containing particles status and desmosome disassembly. These patients present with a disorder moving rapidly toward the periphery Desmosome assembly is typically fol- called Darier’s disease, characterized on microtubules labeled with mCherry- lowed by a maturation phase charac- by skin abnormalities owing to impair- , probing the plasma membrane terized by increasing stability and ment of intercellular adhesion and region before appearing to fuse with resistance to environmental influences desmosome structure in epidermal ker- existing fluorescent puncta that ap- (Figure 4). Although this process is atinocytes (Dhitavat et al., 2004). The peared shortly after cell contact. This poorly understood, it is likely to involve authors of the report suggested that DP rapid transport occurs at a rate consis- both remodeling of the associated retention and aggregation in the cyto- tent with conventional motors, cortical cytoskeleton and other unde- plasm may be due to improper protein and is dependent on microtubules, as fined post-translational modifications. folding, but it also seems possible the motile behavior is inhibited by In tissues or cells that have been that calcium-dependent alterations in nocodazole. cultured for long periods of time, other regulatory pathways, such as The spatial relationship between desmosomes ultimately attain a hyper- those upstream of calcium-dependent Dscs and Dsgs as they traverse the adhesive state known as ‘‘calcium kinases like PKC, could contribute to secretory apparatus on their way to independence’’. In this state, desmo- the observed defects in desmosome newly forming desmosomes is not well somes are stable even in the absence of assembly. understood. Electron microscopic level extracellular calcium (Watt et al., analysis has suggested that desmosome 1984; Garrod et al., 2005; Kimura Assembly of the desmosomal cadher- formation in MDCK cells occurs in two et al., 2007), which would normally ins. Understanding desmosomal cad- stages. The first involves the early disrupt junctions and cause their rapid

www.jidonline.org 2509 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

Stable desmosomes Dynamic desmosomes (calcium-independent) (calcium-independent)

• Intact epithelia • Wounded epithelia • Reduced PKC activity • Activated PKC De-phosphorylated DP (S2849) ? Phosphorylated DP (S2849) ? PV-IgG ?

Figure 4. Desmosomal stability and remodeling. (Left) In confluent sheets of cells or tissues subjected to high levels of calcium, desmosomes are stable and insensitive to low-calcium conditions. This hyper-adhesive state is promoted by de-activation of PKCa (Wallis et al., 2000). (Right) On the other hand, in more sparse cultures or after wounding of an epithelial sheet, PKCa activation appears to promote more dynamic, less adhesive desmosomes, permitting cell mobility as required for re-epithelialization during wound healing. Moreover, desmosome stability and adhesive strength are compromised by treatment with PV-IgG, which may also work through PKC-dependent pathways to de-stabilize desmosomes (Kitajima et al., 1999). It seems plausible that PKCa activity may alter the phosphorylation state of desmosomal proteins, such as DP, affecting their interactions and association with IF, which could modulate adhesive strength and/or stability. In support of this, a PKC phosphorylation consensus site mutation in DP (S2849G) alters its affinity for keratin IF and impairs its assembly into desmosomes.

internalization (Mattey and Garrod, which tyrosine phosphorylation of the 2003; Yin et al., 2005a). Phosphoryla- 1986; Kartenbeck et al., 1991; Wind- Dsg2 tail and PG is accompanied by tion of PG at Y549 also decreases offer et al., 2002). Activation of PKCa, reduced levels of both Dsg2 and Dsc2 association with DP. Src phosphoryla- which has been shown to occur at as well as accumulation of an inter- tion of PG on Y643, on the other hand, wound edges, causes a reversion to a nalized pool of desmosomal cadherins. increases its binding to DP, and at the calcium-dependent state and has been Treatment of these cells with an EGF same time reduces PG’s ability to proposed to facilitate epithelial remo- receptor inhibitor (PKI) not only re- associate with components of the deling (Wallis et al., 2000). Although a sulted in stabilization and retention of classic cadherin complex, to which both complete understanding of how PKCa desmosomal cadherins on the cell sur- b-catenin and PG can bind. Collectively, contributes to desmosome stability is face, but also enhanced desmosome these studies put PG at a critical juncture lacking, altered phosphorylation of assembly and dramatically increased in the adhesive axis of adherens junctions desmosome and/or associated cytoske- adhesive strength of cell monolayers and desmosomes. These phosphorylation letal components may underlie the shift (Lorch et al., 2004). The effect of EGF events may also indirectly regulate PG’s to a less adhesive phenotype. In light of receptor inhibition was potent, as these distribution in nuclear and cytosolic the reported role for PKC in internaliza- increases in intercellular adhesive pools, where it is subject to regulation tion of classic cadherins (Le et al., strength occur even under conditions by the proteasomal degradation machin- 2002), it is tempting to speculate that of reduced extracellular calcium. These ery. Proteasome-dependent degradation PKC similarly regulates the balance findings provide mechanistic insight of PG is inhibited by its O-glycosylation, of desmosomal cadherin trafficking. into the therapeutic value of EGF which in turn enhances keratinocyte Finally, the fact that PKCa also plays a receptor inhibitors for patients with intercellular adhesion (Hatsell et al., positive role in desmosome assembly invasive lung and head and neck 2003; Hu et al., 2006). supports the idea that this kinase is cancers, many of which over express Emerging data suggest that cadherin important for maintaining a biolo- EGF receptor. stability and internalization are key gically ‘‘metastable’’ state, which would A number of tyrosine phosphoryla- components of pathogenesis in the allow rapid desmosome remodeling tion sites on the armadillo protein PG autoimmune disease pemphigus, as (either assembly or disassembly) in re- have been identified (Miravet et al., has recently been reviewed elsewhere sponse to environmental cues (Figure 4). 2003; Yin and Green, 2004). EGF (Kottke et al., 2006). Dsg3 is degraded Tyrosine phosphorylation of the receptor-dependent phosphorylation in response to PV autoantibody (PV- cadherin tail complex is another po- of PG on Y693, Y724, and/or Y729 IgG) binding of DJM cells, resulting in tential mechanism for modulating cad- impairs its association with DP and Dsg3-depleted desmosomes (Aoyama herin-based junctions (Gumbiner, results in decreased intercellular adhe- and Kitajima, 1999). Recent work 2005). Such a pathway is readily sive strength, presumably by impairing tracking the fate of antibody- or bio- revealed in squamous cell carcinoma association with the IF cytoskeleton tin-labeled cell surface protein is con- cells overexpressing EGF receptor, in (Gaudry et al., 2001; Miravet et al., sistent with the idea that rapid

2510 Journal of Investigative Dermatology (2007), Volume 127 KJ Green and CL Simpson Desmosomes: New Perspectives on a Classic

internalization of the soluble pool of (Aho, 2004). In contrast to classic and Andrew Kowalczyk for critical reading and feedback on the manuscript. CLS is supported by PV-IgG-bound Dsg3 occurs upon anti- cadherins, which are reported to be a Kirschstein NRSA Fellowship from the NIH F30 body ligation (Calkins et al., 2006), pro-survival factors, the presence of ES014990 and a Malkin Award from the R H Lurie consistent with previous electron mi- Dsg1 sensitizes keratinocytes to pro- Comprehensive Cancer Center. KJG was sup- croscopy pulse–chase analysis, suggest- grammed cell death (Dusek et al., ported by Grants R01AR43380, R01AR41836, R01CA122151, project no. 4 of P01DE12328, ing that PV-IgG-mediated inter- 2006a), as does the associated protein and the JL Mayberry Endowment. nalization targets peri-junctional ‘‘free’’ PG (Dusek et al., 2007). Finally, Dsg3, which may be more accessible regulated proteolysis of Dsg1 by tryptic to pathogenic antibodies (Sato et al., and chymotryptic enzymes of the REFERENCES 2000). Data regarding the fate of stratum corneum is thought to be Aho S (2004) Plakin proteins are coordinately associated PG differ, some suggesting important for normal granular layer cleaved during apoptosis but preferentially that PG dissociates from phosphory- function, as deficiency in the protease through the action of different caspases. Exp lated Dsg3, and others suggesting that inhibitor LEKTI leads to elevated de- Dermatol 13:700–7 PG, but not DP, remains associated gradation of Dsg1 accompanied by Allen E, Yu Q-C, Fuchs E (1996) Mice expressing a mutant desmosomal cadherin exhibit with the cadherin (Calkins et al., 2006); altered desquamation and impaired abnormalities in desmosomes, proliferation, more recently, it has been suggested barrier function in a mouse model of and epidermal differentiation. J Cell Biol that PV-IgG binding to Dsg3 results Netherton syndrome (Descargues et al., 133:1367–82 in a reduction in nuclear PG with 2005). Amagai M (1999) Autoimmunity against desmo- downstream transcriptional effects somal cadherins in pemphigus. J Dermatol Sci 20:92–102 (Williamson et al., 2006). Following Perspectives and prospects for the future Amagai M, Ahmed AR, Kitajima Y, Bystryn JC, internalization, Dsg3 associates with The last 20 years have seen tremendous Milner Y, Gniadecki R et al. (2006) Are endosomes and lysosomes, where it is progress in understanding the protein– desmoglein autoantibodies essential for the presumably degraded (Calkins et al., protein interactions in desmosomes immunopathogenesis of pemphigus vulgaris, or just ‘‘witnesses of disease’’? Exp Dermatol 2006). As mentioned above, several responsible for anchoring intermediate 15:815 signaling pathways, including PKC filaments to intercellular junctions as Amagai M, Hashimoto T, Green KJ, Shimizu N, activation, are initiated in response to well as the importance of this tethering Nishikawa T (1995) Antigen-specific immu- antibody binding. In light of PKC’s function for adhesive strength and noadsorption of pathogenic autoantibodies reported ability to revert desmosomes tissue integrity. The accelerated pace in pemphigus foliaceus. J Invest Derm 105:895–901 to a calcium-dependent state and to of discovery of human mutations in Amagai M, Hashimoto T, Shimizu N, Nishikawa T promote classic cadherin internaliza- desmosome proteins during the last 5 (1994a) Absorption of pathogenic autoanti- tion (Le et al., 2002), a potential years has intensified interest in inter- bodies by the extracellular domain of pem- contribution of PKC to Dsg3 internali- cellular junction assembly and function phigus vulgaris antigen (Dsg3) produced by zation in PV seems possible (Kitajima, in human heart and skin. These studies baculovirus. J Clin Invest 94:59–67 2002). PV-IgG-induced alterations in have revealed that desmosome mole- Amagai M, Karpati S, Klaus-Kovtun V, Udey MC, Stanley JR (1994b) The extracellular domain both IF attachment (Caldelari et al., cules serve not only as structural ties of pemphigus vulgaris antigen (desmoglein 2001; Calkins et al., 2006) and cortical that bind intermediate filaments to the 3) mediates weak homophilic adhesion. actin organization (Waschke et al., plasma membrane, but also as regula- J Invest Dermatol 102:402–8 2006) may also regulate this process. tors of cell signaling and differentiation. Amagai M, Koch PJ, Nishikawa T, Stanley JR Desmosomes are also subject to Recent work on cellular responses (1996) Pemphigus vulgaris antigen (desmo- glein 3) is localized in the lower epidermis, regulation by proteolysis. Dsg2 is pro- triggered by autoimmune antibodies the site of blister formation in patients. cessed by matrix metalloproteinases in against desmosomes is also prompting J Invest Dermatol 106:351–5 tumor cells, and was recently identified investigators to reconcile the diversity Amagai M, Matsuyoshi N, Wang ZH, Andl C, as a substrate for members of the of signaling responses and cellular Stanley JR (2000) Toxin in bullous impetigo ADAM family of proteases (Bech-Serra pathways affected. Further work to and staphylococcal scalded-skin syndrome targets desmoglein 1. Nat Med 6:1275–7 et al., 2006). EGF receptor and matrix expose these interdependent adhesive Anastasiadis PZ, Reynolds AB (2000) The p120 metalloproteinase inhibitors blocked networks will be important in deter- catenin family: complex roles in adhesion, Dsg shedding and strengthened cell– mining the best targets for therapeutic signaling and cancer. J Cell Sci 113:1319–34 cell adhesion in squamous cell carci- strategies to interfere with the Angst B, Marcozzi C, Magee A (2001) The noma cells (Lorch et al., 2004). Both pathogenesis of diseases targeting the cadherin superfamily: diversity in form and matrix metalloproteinase-dependent desmosome. function. J Cell Sci 114:629–41 ectodomain shedding and caspase-de- Aoyama Y, Kitajima Y (1999) Pemphigus vulgaris- IgG causes a rapid depletion of desmoglein 3 pendent processing of the intracellular CONFLICT OF INTEREST (Dsg3) from the Triton X-100 soluble pools, domains of desmosomal cadherins oc- The authors state no conflict of interest. leading to the formation of Dsg3- depleted cur during apoptosis (Weiske et al., desmosomes in a human squamous carcino- 2001; Dusek et al., 2006a). These ACKNOWLEDGMENTS ma cell line, DJM-1 cells. J Invest Dermatol 112:67–71 events are coordinated with cleavage We are grateful to colleagues in the field for their contributions to the work discussed here and Aoyama Y, Owada MK, Kitajima Y (1999) A of plaque components, and ultimately apologize to those whose work could not be cited pathogenic autoantibody, pemphigus lead to the dissolution of desmosomes because of lack of space. We thank Spiro Getsios vulgaris-IgG, induces phosphorylation of

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