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Home , Catenin, Desmoplakin, Desmosome, Intermediate filament, Keratin 14, Plakin

ORIGINAL ARTICLE

Plakoglobin Is Required for Effective Anchorage to Devrim Acehan1, Christopher Petzold1, Iwona Gumper2, David D. Sabatini2, Eliane J. Mu¨ller3, Pamela Cowin2,4 and David L. Stokes1,2,5

Desmosomes are adhesive junctions that provide mechanical coupling between cells. (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the . We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured . Although knockout keratinocytes form substantial numbers of -like junctions and have a relatively normal intercellular distribution of desmosomal , their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, b- appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of -1 and to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength. Journal of Investigative Dermatology (2008) 128, 2665–2675; doi:10.1038/jid.2008.141; published online 22 May 2008

INTRODUCTION dense plaque that is further from the membrane and that Desmosomes are large macromolecular complexes that mediates the binding of intermediate filaments. represent a major category of intercellular junction (Garrod Desmoplakin is a member of the family, which also et al., 2002). They impart mechanical strength to a wide includes , , bullous pemphigoid anti- range of tissues and their importance is underscored by their gen-1, and . Generally speaking, these play a prevalence in tissues that experience shear stress, such as the role in anchoring membrane-associated complexes and . The cadherins ( and ) to intermediate filaments (Leung et al., 2002). Sequence are transmembrane glycoproteins that form extracellular predictions indicate that all family members have a long, bonds with their counterparts from the neighboring . On a-helical rod flanked by globular domains at their N- and the intracellular membrane surface, these cadherins nucleate C-termini. In the case of desmoplakin, the N-terminal, a dense plaque of proteins designed to couple the extra- globular domain targets to the outer dense plaque (Stappen- cellular bond with the network of intermediate filaments that beck et al., 1993; Bornslaeger et al., 1996; Smith and Fuchs, course throughout the cell. Plakoglobin (PG) and plakophilin 1998), where it is proposed to cluster PG and cadherin are the major components of the outer dense plaque that is complexes into discrete patches (Kowalczyk et al., 1997; proximal to the membrane. Desmoplakin composes an inner North et al., 1999). The C-terminal end has a characteristic plakin-repeat domain and is thought to interact directly with intermediate filaments (Green et al., 1992; Choi et al., 2002). 1Skirball Institute for Biomolecular Medicine, New York University School of PG and plakophilin are members of the armadillo (ARM) Medicine, New York, New York, USA; 2Department of , protein family, which includes b-catenin and p120ctn that are New York University School of Medicine, New York, New York, USA; found in the cytoplasmic plaque of the related adherens 3Vetsuisse Faculty, Institute of Pathology, University of Bern, Bern, Switzerland; 4Department Dermatology, New York University School of junction. Both PG and b-catenin associate with a conserved Medicine, New York, New York, USA and 5New York Structural Biology cytoplasmic region of various cadherins via the 12 ARM Center, New York, New York, USA repeats that characterize this family. Classical cadherins bind Correspondence: Dr David L. Stokes, Skirball Institute 3-13, New York to ARM repeats 5–9 (Huber and Weis, 2001), whereas University School of Medicine, 540 First Avenue, New York, New York 10012, USA. desmosomal cadherins bind to repeats flanking this central E-mail: [email protected] region (Witcher et al., 1996). b-Catenin has an important Abbreviations: ARM, Armadillo; EM, electron microscopy; PG, plakoglobin secondary role in WNT signaling, which also involves Received 4 January 2008; revised 17 March 2008; accepted 4 April 2008; interactions of the ARM repeats, this time with Lef/Tcf published online 22 May 2008 transcription factors and the elements of the so-called

& 2008 The Society for Investigative Dermatology www.jidonline.org 2665 D Acehan et al. Tomography of Plakoglobin-Knockout Desmosomes

destruction complex (Nelson and Nusse, 2004). PG has high for the PG , or homozygous (PG/) for the knockout both in the central ARM repeats and in allele. Cell cultures were initially seeded at low Ca2 þ the N-terminal regulatory motif that is targeted by the concentrations (0.05 mM) and, after reaching confluence, 2 þ destruction complex (Hatsell et al., 2003), and recent studies were switched to 1.4 mM Ca for periods up to 48 hours to support an analogous role in transcriptional regulation by PG induce desmosome formation (Hennings and Holbrook, (Miravet et al., 2003; Muller-Tidow et al., 2004; Williamson 1983). et al., 2006). Plakophilin has a similar architecture, although Genotypes were confirmed by PCR (Figure 1a). DNA from with fewer ARM repeats and a distinctive kink in the middle PG þ / þ cells produced an B650-bp band corresponding (Choi and Weis, 2005). Despite structural homology, no to the wild-type allele, whereas DNA from PG/ cells binding partners have been identified for the plakophilin produced an B450-bp band corresponding to the targeted ARM domain. Instead, a multitude of interactions involve PG allele. The expression levels of PG in the cultured cells the globular N-terminal domain, which is unrelated to PG were assessed by immunoblotting of extracts from cultured (Hatzfeld, 2007). cells prepared 15 hours after inducing desmosome formation 2 þ Within the desmosomal plaque, these constituents engage by increasing the Ca concentration to 1.4 mM (Figure 1b). in a number of complex interactions. In particular, the The resulting data verified that PG was not present in desmoplakin N-terminal domain interacts with the cytoplas- homozygous, knockout cells. mic tails of desmocollin ‘‘a’’ isoforms, the N-terminal head of The subcellular distributions of various desmosomal plakophilin-1, and the central ARM repeats of PG (Kapprell proteins in wild type and knockout cells were characterized et al., 1988; Troyanovsky et al., 1996; Kowalczyk et al., by immunofluorescence 15 hours after inducing desmosome 1997). Plakophilin and PG, in turn, interact with each formation. As expected, PG/ cells were unlabeled other and promote clustering of desmosomal cadherins (Figure 2b), demonstrating the specificity of the PG anti- (Bornslaeger et al., 2001; Chen et al., 2002; Bonne et al., bodies. The same antibodies labeled cell borders in PG þ / þ 2003). Plakophilin is reported to bind to intermediate cells (Figure 2a), reflecting a concentration of PG in both filaments, but desmoplakin appears to be primarily respon- desmosomes and adherens junctions. Immunolabeling for sible for linking the desmosomal plaque to the cytoskeleton plakophilin-1 (Figure 2c and d) and desmoplakin (Figure 2e (Kapprell et al., 1988; Smith and Fuchs, 1998; Bornslaeger and f) was concentrated at the periphery of both cell types. et al., 2001). At first glance there appears to be redundancy Desmoplakin produced a punctate distribution in wild-type and competition among cadherins, PG, and plakophilin for cells, characteristic of its desmosomal localization, but desmoplakin binding. Nevertheless, a full complement of produced a diffuse component of staining both around the interactions seems to be required for full adhesive function plasma membrane and within the in cultured (Bierkamp et al., 1996; Ruiz et al., 1996; McGrath et al., PG/ cells, as seen previously in (Bierkamp et al., 1997; Gallicano et al., 1998; McKoy et al., 2000; Yin and 1999). -14 antibody labeling (Figure 2g and h) Green, 2004). To date, many questions remain about the revealed intermediate filaments coursing through the cyto- physical architecture of the desmosomal plaque, the specific plasm of cells regardless of their PG genotype. A transcellular nature of these diverse molecular interactions, and the way desmosomal assembly is regulated. M +/–+/– –/– –/– –/– M +/+ +/+ Genetic knockout of the PG gene is lethal during the embryonic period, with severe heart and skin defects, 2,000 presumably due to malfunction of desmosomes and the 1,500 intercalated disk (Bierkamp et al., 1996; Ruiz et al., 1996). Unlike in wild-type , b-catenin was detected in 1,000 epidermal desmosomes from these knockout embryos, 750 suggesting that this highly homologous ARM family member attempts to substitute for Pg, but has limited functionality 500 (Bierkamp et al., 1999). In this study, we have used PG- knockout cells to shed light on the physical architecture and 300 molecular interactions within the intracellular plaque. We (bp) have used electron tomography to determine the three- M –/– –/– –/– dimensional structures of desmosomes and have used +/+ +/+ +/+ 200 immunolabeling to document changes in the composition 130 of the plaque. 85 (kDa) PGβ-Cat E-cad RESULTS Characterization of cell cultures by PCR, immunoblotting, Figure 1. Characterization of cell cultures. (a) PCR amplified distinct bands and immunofluorescence for DNA from wild type and PG-knockout alleles. As expected, DNA from heterozygous cells (PG þ /) produced both bands. Size markers are shown in To investigate the effects of PG on the cytoplasmic plaque of lanes marked ‘‘M’’. (b) Immunoblotting verified expression of PG in wild type, desmosomes, we have used long-term cell cultures of kera- but not in knockout cells. Control experiments involved blotting the same þ / þ tinocytes derived from mice that were wild type (PG ) protein extracts with antibodies to either b-catenin or E-cadherin.

2666 Journal of Investigative Dermatology (2008), Volume 128 D Acehan et al. Tomography of Plakoglobin-Knockout Desmosomes

PG PP1 DP K14 E-cad β-Cat a c e g i k

b d f h j l PG–/– PG+/+

Figure 2. Immunofluorescence images from PG þ / þ (top row) and PG/ (bottom row) cell cultures. The primary antibody target is indicated at the top margin. (a, b) As expected, PG is only present in PG þ / þ cells and is concentrated at the cell boundaries. (c, d) Plakophilin-1 staining is similar for both cell types. (e, f) Desmoplakin is concentrated at cell boundaries with a less punctate appearance and greater cytoplasmic distribution in PG/ cells. (g, h) Keratin-14 labeling is similar in both cell types, although the transcell network visible in PG/ cells was a relatively rare occurrence. (i, j) E-cadherin served as a control and, as expected, produces a similar labeling pattern in both cell types. (k, l) b-Catenin is concentrated along the cell surface in both cell types, but has a more punctate appearance in PG/ cells. Bar ¼ 20 mm. network was observed frequently in wild-type cells, but only ab occasionally in mutant cells. The distribution of E-cadherin, a component of adherens junctions but not of desmosomes, was comparable in both knockout and wild-type cells (Figure 2i and j), and b-catenin produced a somewhat more punctate labeling in knockout cells (Figure 2k) as compared with PG þ / þ cells (Figure 2l). These results suggest that the absence of PG had significant effects on the distribution of certain associated proteins, although an ultrastructural study was necessary to evaluate the specific effects on the desmosomal architecture.

The number and size of desmosomes / When Transwell filters were used to support cells throughout Figure 3. Broken desmosomes from PG cells. (a,b) Ruptured cells are / the rapid freezing/freeze-substitution protocol used for our frequently observed when PG cells are scraped from culture dishes during the EM preparation. Given the consequent mechanical stress, best electron microscopy (EM) preparations, parallel cell PG-deficient desmosomes frequently break off from one cell, leaving a borders were punctuated by numerous desmosomes in both fragment of the . This observation suggests that the intercellular þ / þ / PG and PG cultures. On the other hand, when fixed bond provided by the cadherin molecules is intact but that the lack of cells were scraped off culture dishes for more conventional intermediate filament attachments produces a vulnerability in EM preparations, few desmosomes remained and the that ultimately leads to cell rupture. Bars ¼ 100 nm. corresponding cell contacts were largely disrupted in PG/ cultures (Figure S1). High-magnification images revealed that PG/ desmosomes were frequently torn out of one cell after then leveled off. Notably, PG/ cells had a significant these scraping procedures (Figure 3), but not in cells that population of abnormally large (4640 nm diameter) desmo- were consistently supported on Transwell filters. Wild-type somes that were not observed in PG þ / þ cells (Figure S2). The desmosomes appeared normal in both the preparations. number and size distribution of desmosomes from PG þ / þ These results illustrate an innate susceptibility of knockout cells were comparable to that of primary cultures of cells to shear stress, as previously documented by indirect keratinocytes isolated from newborn mice (not shown). On measurements of adhesive strength (Yin et al., 2005b), but the other hand, the relative number of desmosomes observed indicate that this susceptibility is not simply due to a lack of in cultured PG/ keratinocytes after scraping was consider- desmosome formation in the absence of PG. ably lower, close to the 5% value reported by Bierkamp et al. We quantified the numbers and sizes of desmosomes in (1999) for the epidermis of PG/ embryos compared with Transwell cultures (Table 1) and found that PG/ cells have wild type. They also reported desmosomes torn from the B60% the total number of desmosomes observed in wild- plasma membrane of adjacent cells in tissue from knockout type cells. For both cell types, the number and size of animals. These lower percentages and tissue damage most desmosomes steadily increased over the first 48 hours and likely reflect increased mechanical challenge within live

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Table 1. Characterization of desmosomes in wild type and knockout cells1 3 4 Number2 Size distribution Intermediate filament associations Desmo./cell 4160 nm (%) 160–320nm (%) 320–560nm (%) 4640 nm (%) 0 1 2 3

PG+/+ 15 hours 3.3 51 47 2 0 0 2% 45% 53% 30 hours 5.8 37 51 12 0 0 0 39% 61%

PG/ 15 hours 2.3 71 2 10 0 82% 14% 4% 0% 30 hours 3.4 36 0 15 6 96% 4% 0% 0% 48 hours 4.3 26 55 15 4 79% 11% 10% 0% PG, plakoglobin. 1Desmosomes were identified and characterized in low-magnification projection images of cultured cells representing various time intervals in highCa2+ medium. 2Average number of desmosomes per cell. The presence of the was used to quantify the number of cells represented in any given field. About 100 desmosomes were counted for each category. 3Length of individual desmosomes were measured and binned into four size categories. Percentages refer to total population from a given row (that is, cell type and time point) and each represents an average of about 100 desmosomes. 4Each desmosome was assigned a score from 0–3, where 0 corresponds to no intermediate filaments visible near the desmosome, 1 corresponds to ambiguous density near the intracellular plaque, 2 corresponds to a small number of intermediate filament bundles, and 3 corresponds to strong intermediate filament bundles associated with the plaque. Each population included B50 desmosomes.

tissue or during specimen preparation for EM and reflect a is required before applying this method to knockout weakness in the desmosomal structure. desmosomes. Projection images show that desmosomes either in Ultrastructure of desmosomes primary cultures (Figure 4b) or in long-term The fine structures of wild-type and PG-deficient desmo- cultures from wild type and PG þ / keratinocytes (Figure 4c–f) somes were compared by conventional EM and electron were structurally similar to those in newborn mouse skin tomography of freeze-substituted samples. Whereas conven- (Figure 4a) (He et al., 2003). In contrast, PG knockout tional projection images suffer from superposition of features resulted in distinct changes in desmosomal architecture throughout the thickness of the section, tomography allows (Figure 4g–i). Although intercellular densities associated computational slicing through the three-dimensional struc- with cadherins appeared to be similar in projection images tures, thus allowing assessment of molecular interactions in a (Figure 4f and i) and tomographic slices (Figure 5c and f) from dense macromolecular assembly like the desmosome. The both cell types, the outer dense plaque was narrower and less characteristic features include a dense group of strand-like densely packed than in wild-type desmosomes (Figures 4 and densities (cadherin molecules) extending across the inter- 5, and see more tomographic images in Figure S3). In cellular gap and associating with one another within the addition, tomographic slices clearly show that the inner so-called midline that appears halfway between the two dense plaque, which in wild-type desmosomes comprises a membranes, a dense intracellular plaque, and bundles of network of densities, was either disorganized or completely intermediate filaments. In projection images, it is generally absent in knockout cells (Figure 5d and e; Figures S2 and S3; difficult to distinguish individual components or their specific and Movie S2). associations, due either to superposition or to excessive These inner plaque densities generally associate with frayed fixation and staining. By using freeze substitution for speci- intermediate filament bundles that lie in close proximity to the men preservation and tomography for imaging, we have been inner plaque of wild-type desmosomes (Figures 5a, b and 7; able to delineate individual cadherin molecules in the Figure S2; and Movies S1 and S3). Although such intermediate intercellular gap (He et al., 2003) as well as individual filament bundles were occasionally visible in the general intermediate filament associations with the intracellular vicinity of desmosomes in knockout cells, they rarely plaque (see below). A potential alternative is to image associated with these desmosomes 15–48hours after inducing 2 þ frozen-hydrated sections, as in a recent report addressing their formation with 1.4 mM Ca medium. In addition to the cadherin organization in epidermis (Al-Amoudi et al., examples shown in Figures 4 and 5 and Figure S3, we have 2007). However, the resulting images have very low contrast scored B50 desmosomes from each cell type. The results and extensive image averaging is therefore required to (Table 1) indicate almost no intermediate filament connections reveal features within the intercellular gap. Although to knockout desmosomes at 15 and 30 hours time points, with frozen, unstained tissue provides better specimen preserva- 10% of knockout desmosomes having only limited intermedi- tion, thorough characterization of the intracellular plaque ate filament connections after 48 hours. In many wild-type

2668 Journal of Investigative Dermatology (2008), Volume 128 D Acehan et al. Tomography of Plakoglobin-Knockout Desmosomes

a b c

d e f

g h i PG–/– PG+/+

Figure 4. Projection images of desmosomes. (a) Newborn mouse skin. (b) Primary culture of keratinocytes from newborn mouse skin. (c)PGþ / cell culture. (d–f)PGþ / þ cell culture. (g–i)PG/ cell culture. Note that magnifications are variable, but that all scale bars correspond to 100 nm. The outer dense plaque (ODP, large arrowheads) and inner dense plaque (IDP, small arrowheads) are labeled in panel (f) and indicated in panels (d),(e), and (i). Intermediate filaments are indicated by arrows in panel (d). The greater section thickness used for tomography and the retention of soluble cytoplasmic components in freeze- substituted samples cause features to appear less distinct relative to projection images in some previous publications. Similarly, the appearance of cadherins in the intercellular region depends on the precise angle of view, with a better defined midline produced when the section is cut precisely normal to the membrane plane. Taking this effect into account, the intercellular cadherin network appears similar in all these cells, but the PG/ desmosomes are characterized by a narrower, less dense cytoplasmic plaque with severely reduced connections to the intermediate filament network.

desmosomes, the intermediate filament bundles were more desmosomes, PG normally wins this competition and strongly visible on one side of a given desmosome, undoubt- excludes b-catenin (Lewis et al., 1997; Bierkamp et al., edly because they run at right angles to the plane of the section 1999), but in the absence of PG, b-catenin apparently has in the opposing cell. This observation suggests that there may sufficient affinity for the cytoplasmic tails of desmosomal be coordination between the intermediate filament networks cadherins to account for their observed clustering even in the in adjacent cells, which might be mediated in some, as yet absence of PG. undefined, way by the desmosomal architecture. Although We next addressed whether absence of PG affected the miscellaneous cellular constituents are excluded from the disposition of desmoplakin within the desmosomal plaques. immediate vicinity of wild-type desmosomes, In particular, we used antibodies specific for the desmoplakin and were seen in close proximity to the desmo- N- or C-terminus to determine not only the relative amount of somes in knockout cells (Figure 5; Figures S2, S3), probably desmoplakin, but also its orientation within the plaque. In reflecting their failure to build an organized inner plaque. PG þ / þ keratinocytes, the desmoplakin N-terminus was localized to the outer plaque (Figure 6k), whereas C-terminal Immunolabeling of desmosomal components labeling was found in the inner plaque where intermediate Immunoelectron microscopy was used to localize individual filaments join the desmosome (Figure 6j), as expected from proteins in wild type and PG-knockout desmosomes. First, earlier studies (North et al., 1999). Interestingly, we found several different antibodies were used to localize PG in wild- that this general orientation was maintained in PG/ type desmosomes (Figure 6a–c) and to verify its absence desmosomes (Figure 6n and o) even though they lacked in PG/ cells (Figure 6e–g). Second, we confirmed that, connections to the intermediate filament network. Specifi- in these long-term keratinocyte cultures, antibodies against cally, in a statistical analysis of the distribution of desmopla- b-catenin labeled desmosomes in PG/ but not PG þ / þ cells kin labels (Figure 6l), we found that the mean distance of the (Figure 6d and h). In adherens junctions, PG and b-catenin desmoplakin N-terminus from the central midline was coexist (Peifer et al., 1992) and presumably compete for B17 nm in both PG þ / þ (n ¼ 100, SEM ¼ 0.77) and PG/ binding to the cytoplasmic tail of the relevant cadherins. In (n ¼ 41, SEM ¼ 1.3) desmosomes. Although the C-terminus

www.jidonline.org 2669 D Acehan et al. Tomography of Plakoglobin-Knockout Desmosomes

a b c

IDP ODP

d e f PG–/– PG+/+

Figure 5. Tomographic imaging of desmosomes. (a–c)PGþ / þ .(d–f)PG/. Grayscale images correspond to 7-A˚ thick slices through the tomographic volume and color has been applied to objects representing the three-dimensional segmentation of selected components of the desmosome. The outer dense plaque (ODP) and inner dense plaque (IDP) are indicated in panel (a) and appear to be substantially more developed in wild type desmosomes compared to the knockout. Cell membranes are shown in cyan, desmoplakin in yellow and intermediate filaments in blue. In knockout desmosomes, ribosomes (red) and microtubules (purple) are seen more frequently in the region of the IDP. Close-up views (c, f) show that extracellular cadherin interactions are comparable in the two cell types, although the corresponding densities are not as well preserved as in epidermal tissue (for example, Figures 3a and 7). Animated representations of these tomographic reconstructions are shown in Movies S1 and S2.

was decidedly further from the membrane in both the cases, associating with non-desmosomal regions of the membrane, there was a significant difference in the distance measured in but a much lower density at the desmosomes. This result is wild type versus knockout (Po0.02): 70 nm in PG þ / þ consistent with the membrane localization of plakophilin (n ¼ 83, SEM ¼ 2.6) and 60 nm in PG/ (n ¼ 59, SEM ¼ 3.6) immunofluorescence in both cell types and suggests that desmosomes, respectively. Furthermore, we observed that the plakophilin associates with the membrane prior to incorpora- number of desmoplakin molecules localized to PG/ tion into the desmosomal plaque. desmosomes was 2–3 times lower compared with PG þ / þ desmosomes, which is consistent with previous biochemical DISCUSSION data from whole-cell lysates (Caldelari et al., 2001; Yin et al., In this report, we have compared desmosomes produced by 2005a). These findings indicate that, in PG-knockout desmo- long-term keratinocyte cultures derived from wild type and somes, cadherins, plakophilin, and/or b-catenin continue to PG-knockout mice. Although this knockout has been orient desmoplakin correctly within the cytoplasmic plaque. characterized in previous studies (Bierkamp et al., 1996, However, the location of the desmoplakin C-terminus is 1999; Ruiz et al., 1996; Caldelari et al., 2001; Yin et al., altered, perhaps reflecting the defect in anchoring intermedi- 2005a), electron tomography allowed us to evaluate the ate filaments to PG/ desmosomes. Specifically, an apparent specific structural defect in desmosomal architecture. In shortening of the desmoplakin molecule may be due to a lack particular, we documented that 15–48hours after inducing of tension within this inner dense plaque that normally desmosome formation with the Ca2 þ switch, cadherin–cad- accompanies its linkage to the cytoskeleton. herin interactions in the intercellular gap of PG/ junctions Finally, we investigated the distribution of plakophilin-1 in appear to be similar to those in wild-type desmosomes, wild type and knockout cells. Although labeling was present in indicating that cadherin molecules are clustered normally desmosomes in both cell types, we observed an B6-fold within the cell membrane even in the absence of PG. decrease in the number of plakophilin-1 labels associated However, we found that this knockout results in a thinner, with PG/ desmosomes compared with PG þ / þ desmosomes sparser outer dense plaque and furthermore that the inner (Figure 6i and m). We also calculated the ratio of plakophilin dense plaque seems to be disorganized or missing altogether. labels in desmosomal versus non-desmosomal regions of the In addition, the desmosomes seen by electron tomography plasma membrane. In wild-type cells, this ratio was 1.5, are defective in anchoring the intermediate filament network indicating that plakophilin preferentially associated with to the membrane and the cohesion of corresponding cell desmosomes. In contrast, the ratio was 0.17 in knockout cells, cultures is more susceptible to mechanical stress. Immuno- reflecting a comparable density of plakophilin molecules gold labeling confirmed previous reports that b-catenin was

2670 Journal of Investigative Dermatology (2008), Volume 128 D Acehan et al. Tomography of Plakoglobin-Knockout Desmosomes

PG 11E4 PG 1407 PG 1408 β-Catenin a b c d PG +/+

efg h PG –/–

i j k l PG+/+ PG–/– % 50 40 30 20 10

5 152535 DP N-term nm m no % 50 40 30 20

PG –/– PG +/+ 10 10 30 50 70 90 120 DP C-term nm

Plakophilin1 DP-C DP-N

Figure 6. Immunogold localization of desmosomal proteins. Cell type is indicated along the left-hand margin, whereas antibody targets are indicated at the top and bottom. Lower frequency gold labels are marked with arrowheads. (a–c) Three different PG antibodies resulted in robust labeling in PG þ / þ desmosomes, but did not label PG/ desmosomes at all (e–g). (d, h) b-Catenin was absent from PG þ / þ desmosomes, but present in PG/ desmosomes. (i, m) Plakophilin-1 labeling was observed for both cells types, although levels were considerably lower for the knockout cells. (j, k, n, o) Two different antibodies targeted the N- and C-terminal domains of desmoplakin. Although both localized to the cytoplasmic plaque, the N-terminus was significantly closer to the membrane than the C-terminus. Also, fewer desmoplakin antibodies were observed in knockout cells. (l) Histograms illustrate the distribution of antibodies for the two ends of desmoplakin. Student’s t-test indicated a significant difference between N- and C-terminal locations in both cell lines (Po0.01) and also a significant difference between C-terminal locations in PG þ / þ and PG/ cells (Po0.02).

present in knockout desmosomes (Bierkamp et al., 1999) and and consummated by coupling to the intermediate filament presumably responsible for cadherin clustering. However, we network. also documented substantially reduced levels of desmoplakin and plakophilin-1, suggesting that recruitment of plaque Interactions in the outer dense plaque components is secondary to cadherin clustering and depen- In wild-type desmosomes, the outer dense plaque is dent on unique properties of PG. The consequent defect in composed of PG, plakophilin, and the N-terminal domains anchoring intermediate filaments provides a definitive of desmoplakin. Specifically, the ARM repeats of PG interact structural explanation for the failure of PG/ tissue adhesion. with the N-terminal domain of desmoplakin, thus anchoring Thus, our observations suggest an orderly sequence for it to the desmosome (Kowalczyk et al., 1997). In PG/ desmosome assembly initiated by cadherin clustering, desmosomes, b-catenin is not expected to bind desmoplakin; mediated by either PG or b-catenin, followed by recruitment instead, the presence of desmoplakin most likely reflects its of plakophilin and desmoplakin to the intracellular plaque, interaction with desmocollin-1a (Troyanovsky et al., 1994;

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Smith and Fuchs, 1998), albeit at the lower levels as indicated a by our immunolabeling studies and supported by previous IDP analyses of protein levels in cell lysates (Caldelari et al., ODP 2001; Yin et al., 2005a). Plakophilin-1 is reported to bind ODP desmoglein-1, desmocollin-1, and desmoplakin (Kapprell et al., 1988; Smith and Fuchs, 1998; Kowalczyk et al., IDP 1999; Hatzfeld et al., 2000), but our observation of a sixfold / reduction of plakophilin-1 in PG desmosomes suggests bc def that the desmoplakin N-terminal domain plays a larger role than desmosomal cadherins in recruiting plakophilin-1 to the outer dense plaque.

Linkage with the inner dense plaque Linkage of cadherins, PG, and plakophilin in the outer dense plaque to the intermediate filament network occurs via an inner plaque, which is composed primarily of desmoplakin. We have extended previous immunolabeling of desmoplakin in wild-type desmosomes (North et al., 1999) to demonstrate the disposition of desmoplakin in knockout desmosomes. In Figure 7. Segmentation of the inner dense plaque of a desmosome from both wild type and knockout cells, the N-terminal domain wild-type mouse epidermis. (a) Overview of this desmosome, which has of desmoplakin lies close to the membrane, whereas the relatively low density of cadherins and intracellular plaque proteins, making C-terminal domain is 40–50nm away at the inner margin of segmentation easier. Segmented area is framed; the inner dense plaque (IDP) the cytoplasmic plaque. This disposition is consistent with the and outer dense plaque (ODP) are indicated. (b–e) Sections from lower, role of this C-terminal domain in anchoring intermediate middle and upper portions of the tomogram framed in panel (a). filaments (Stappenbeck et al., 1993; Kouklis et al., 1994; (f) Segmentation of the densities for the membrane (cyan), cadherins (purple and pink), desmoplakin (yellow and orange), and intermediate Meng et al., 1997; Smith and Fuchs, 1998; Choi et al., 2002), filaments (blue). which localize to the same area. In our tomograms from wild-type keratinocytes, desmoplakin appears as strands of density running across this region. In PG/ desmosomes, compelling explanation for the susceptibility of this knockout these strands are either absent or much attenuated, which is cell line to mechanical stress and for the embryonic lethality consistent with the 2–3 reduction in immunolabeling of PG and desmoplakin knockouts in mice, although the of desmoplakin in the knockout. Figure 7 shows three- earlier and more severe phenotype of the latter (Gallicano dimensional segmentation of a desmosome from wild-type et al., 1998) suggests additional critical roles of desmoplakin mouse epidermis. Several consecutive slices are shown in in desmosome assembly. Figure 7b–e in which individual strands are visible connecting Our work extends previous observations of desmosomes in the lower membrane to the intermediate filaments running at epidermis of PG-knockout mice (Bierkamp et al., 1999). the very bottom of the panels (see also Movie S3) and the These authors described increased intercellular spaces segmentation in Figure 7f gives a general sense of the three- between cells in the upper and lower spinous layers as well dimensional distribution of these desmoplakin densities. On as missing cytoplasmic plaques and few associated inter- the basis of their size and shape, as well as previous evidence mediate filament bundles, although their conventional for a dimeric assembly (Green et al., 1992), we hypothesize electron micrographs were not able to define the detailed that individual strands correspond to desmoplakin dimers. desmosomal architecture. In addition, they reported broken These appear to interact with the lateral edge of individual desmosomes with fragments left attached to the neighboring intermediate filaments frayed from a larger bundle that runs cell, analogous to the images we present in Figure 3. On the roughly parallel to the membrane surface. other hand, transcellular networks of intermediate filaments We found that the number of desmosomes observed in and immunoprecipitation of desmoglein by keratin-14 knockout cells depends strongly on the preparation method antibodies in knockout cell cultures seem to reflect intact used for electron microscopy in a way that reflects a connections between intermediate filaments and cadherins vulnerability of these mutant desmosomes to mechanical (Caldelari et al., 2001). Although these latter observations stress. Even though immunofluorescence images indicate an appear to be inconsistent with our observations by tomo- intact intermediate filament network in PG/ cells, electron graphy, it is possible that longer periods of desmosome tomography reveals that desmosomal connections are defec- formation produce a subpopulation of knockout desmosomes tive. Indeed, under conditions of increased mechanical stress, that form limited associations with the intermediate filament PG/ cells had a preponderance of desmosome-associated network (c.f., Table 1). membrane fragments torn from an adjacent cell (Figure 3), graphically illustrating the consequences of this defect even Regulation of desmoplakin binding to intermediate filaments when the cadherin-mediated intercellular bond remains Our data are consistent with a sequential model of desmo- intact. Failure of intermediate filament coupling offers a some assembly: clustering of cadherins by PG followed by

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recruitment of plaque proteins followed by linkage of of wild-type keratinocytes (Hennings and Holbrook, 1983) were intermediate filaments. To coordinate this assembly, binding established by treating mouse skin with dispase for 4–12hours on of desmoplakin to intermediate filaments must be regulated ice, which allowed separation of the epidermis from the dermis. Use in order to prevent binding at non-desmosomal sites and of mice was approved by the authors’ Institutional Review Board. perhaps also to control desmosome assembly/disassembly The epidermis was digested with 0.5% trypsin at 37 1C, treated with during cell division or migration. Although the mechanisms 25 mg ml1 DNAse, and sheared by vigorous pipetting with a Pasteur have not been precisely defined, PG appears to play a pivotal pipette. The resulting solution was filtered through several layers of role. The observed distance between desmoplakin N- and gauze and washed with DMEM and centrifuged at 1,000 r.p.m. to C-termini in desmosomes is considerably smaller than the collect primary keratinocytes. Long-term cultures were grown in length of isolated, metal-shadowed desmoplakin molecules CnT2 growth medium (CellnTec Advanced Cell Systems, Bern, (up to 180 nm, O’Keefe et al., 1989). This discrepancy Switzerland) and primary keratinocytes were grown in keratinocyte suggests that the central a-helical rod of desmoplakin (Green growth medium (Cambrex Bio Science, Walkersville MD) for 3–5 et al., 1992) is either highly flexible or folded up within the days in order to reach confluence in the absence of calcium. In the intracellular plaque. Thus, one mechanism for regulating case of the knockout, cells are hyperproliferative and, to compen- assembly could be an autoinhibitory interaction between sate, they were seeded at a lower concentration than that used for N- and C-terminal domains, as seen for example in smooth- wild-type cells (Williamson et al., 2006). Polycarbonate Transwell muscle (Burgess et al., 2007). Recruitment to the filters (Corning Inc., Corning, NY) were used for electron tomo- desmosome could disrupt this interaction, either by steric graphy and plastic Petri dishes were used for other experiments. hindrance of PG binding to the N-terminal domain or by After reaching confluence, desmosome formation was initiated by

PG-mediated . With respect to phosphory- adding 1.4 mM CaCl2 to the culture media. lation, phosphorylation of the C-terminal GSR domain For PCR, primers A(neo)f 50-GCCTTCTATCGCCTTCTTGAC-30 of desmoplakin has been implicated in the regulation of and B(ex5)r 50-CTCAGGAGTTAGGAGCACTG-30 were used to intermediate filament binding (Stappenbeck et al., 1994); also recognize the neomycin cassette in the targeted allele; C(ex3)f epidermal growth factor stimulation results in tyrosine 50-AGCTGCTCAACGATGAGGAC-30 and D(ex4)r 50-AGCATTCGG phosphorylation of PG, loss of desmoplakin from desmo- ACTAGGGCAGG-30 were used to recognize exon-3 and exon-4 somes, and decreased adhesive strength (Yin et al., 2005a). of the wild-type gene. For western blots, cells were washed with Furthermore, evidence is building for the participation of PG phosphate-buffered saline buffer, scraped off of Petri dishes, and in a signaling pathway that mediates the response to the collected by centrifugation. The resulting cell pellet was resus- , which results in dramati- pended in radioimmunoprecipitation buffer (50 mM Tris-HCl, 1 cally weakened desmosomal adhesion (Caldelari et al., 2001; 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 mgml Aprotinin, Williamson et al., 2006; de Bruin et al., 2007). 5 mgml1 Leupeptin, 1% Triton X-100, 1% sodium deoxycholate, A rather different view is that productive binding of 0.1% SDS, pH 7.4), pipetted vigorously through a 22-G needle, and intermediate filaments requires a threshold concentration of incubated on ice for 1 hour. Cell lysates were centrifuged at 15,000 g desmoplakin molecules. It is clear from our tomograms that for 20 minutes at 4 1C and the resulting supernatant was run on 10% the inner dense plaque in wild-type cells consists of a protein SDS polyacrylamide gels, transferred to polyvinylidene difluoride network that is dense enough to exclude extraneous cellular membranes, and blotted with antibodies for PG (5172, mouse constituents such as ribosomes and microtubules, reflecting a monoclonal), E-cadherin (mouse monoclonal; BD Biosciences, San very high local concentration of desmoplakin within this Jose, CA) and b-catenin (mouse monoclonal from BD Biociences, region. In PG/ desmosomes, this network is absent and Franklin Lakes, NJ). immunolabeling indicated reduced amounts of desmoplakin consistent with a considerably lower local concentration that Immunofluorescence and immunogold labeling appears to be ineffective in binding intermediate filaments. For immunofluorescence light microscopy, cells were grown on Such a threshold requirement could reflect weak binding of glass slides, fixed with methanol at 20 1C, and incubated for 1 hour desmoplakin to intermediate filaments, which is overcome by on ice with primary antibodies listed above as well as the following: a binding cooperativity manifested only in a large ensemble. desmoplakin (2.15, mouse monoclonal), plakophilin (mouse mono- Thus, although transient, individual interactions could occur clonal; Abcam Inc., Cambridge, MA), and keratin-14 (rabbit in the bulk cytoplasm, only after clustering molecules within polyclonal; Covance Inc., Princeton, NJ). After washing with the desmosomal plaque would desmoplakin be effective in phosphate-buffered saline buffer, cells were incubated with fluores- anchoring the cytoskeleton. A similar mechanism has been cently labeled anti rabbit (cy3) or anti mouse (Alexa488, Texas Red) proposed as the basis for cadherin–cadherin selectivity, with secondary antibodies for 1 hour on ice and visualized at room the sum of numerous weak interactions explaining their temperature using a 63 Plano Apochromat objective with a Zeiss specificity as well as the adhesive strength of the overall Axoplan 2 model LSM510 confocal fluorescence microscope. ensemble (Chen et al., 2005). For immunogold electron microscopy, we used primary anti- bodies NW6 (desmoplakin C-terminal, rabbit polyclonal), NW161 MATERIALS AND METHODS (desmoplakin N-terminal, rabbit polyclonal), 11E4 (PG, mouse Cell culture, genotyping, and immunoblotting monoclonal), 1407, and 1408 (PG chicken polyclonal), which were Long-term cultures of PG þ / þ and PG/ keratinocytes have been a gift from Dr Kathleen J Green Laboratory (Northwestern University, described previously (Caldelari et al., 2000, 2001). Primary cultures Feinberg School of Medicine). In addition, b-catenin (BD Biosciences)

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and plakophilin-1 (Abcam Inc.) antibodies were obtained commer- SUPPLEMENTARY MATERIAL cially. Cells were grown on Petri dishes and then fixed for 2 hours Figure S1. Influence of different methods for EM preparation. with 2% paraformaldehyde and 0.2% glutaraldehyde. Cells were Figure S2. Tomographic images of desmosomes. scraped with a Teflon spatula and centrifuged to form a pellet that þ þ Figure S3. A gallery of tomographic slices from PG / and PG / cells. was infused with 2.3 M sucrose in phosphate-buffered saline and Movie S1. Animated series of slices through the tomographic volume in frozen on a microtome chuck prior to cryosectioning. Sections Figure 5a. (80-nm thick) were cut with a cryo-ultramicrotome and collected on Movie S2. Animated series of slices through the tomographic volume in nickel EM grids. For labeling, sections were incubated overnight at Figure 5d. 4 1C with primary antibodies diluted in 1% BSA in phosphate- Movie S3. Animated series of slices through the tomographic volume in buffered saline. After several washes to remove unbound primary Figure 7a. antibodies, sections were incubated with 10 nm gold-conjugated protein-A for 30 minutes at room temperature. Monoclonal antibodies REFERENCES required additional 30 minutes of incubation with rabbit or chicken Al-Amoudi A, Diez DC, Betts MJ, Frangakis AS (2007) The molecular IgG to bridge between primary antibody and protein-A gold. In the architecture of cadherins in native epidermal desmosomes. Nature case of b-catenin, the primary antibody was directly labeled with 450:832–7 10 nm gold-conjugated antimouse IgG antibody. After labeling Bierkamp C, McLaughlin KJ, Schwarz H, Huber O, Kemler R (1996) Embryonic heart and skin defects in mice lacking plakoglobin. Dev Biol reactions and washes, samples were further fixed with 1% 180:780–5 glutaraldehyde, stained with uranyl acetate at neutral pH, and Bierkamp C, Schwarz H, Huber O, Kemler R (1999) Desmosomal localization embedded in a mixture of 0.4% uranyl acetate and 1.8% methyl of beta-catenin in the skin of plakoglobin null-mutant mice. Develop- cellulose. ment 126:371–81 Bonne S, Gilbert B, Hatzfeld M, Chen X, Green KJ, van Roy F (2003) Defining Electron tomography desmosomal plakophilin-3 interactions. J Cell Biol 161:403–16 For electron tomography, cells were grown on Transwell membranes Bornslaeger EA, Corcoran CM, Stappenbeck TS, Green KJ (1996) Breaking the connection: displacement of the desmosomal plaque protein desmo- and prepared by high-pressure freezing followed by freeze substitu- plakin from cell–cell interfaces disrupts anchorage of intermediate tion. High-pressure freezing planchettes were loaded with 2 2-mm filament bundles and alters intercellular junction assembly. J Cell Biol pieces of Transwell membranes, packed with 15% dextran in culture 134:985–1001 medium, and frozen within 2 minutes after removal from growth Bornslaeger EA, Godsel LM, Corcoran CM, Park JK, Hatzfeld M, Kowalczyk medium. For freeze substitution, the planchettes were incubated in AP et al. (2001) Plakophilin 1 interferes with plakoglobin binding to 1 desmoplakin, yet together with plakoglobin promotes clustering of acetone containing 1% OsO4 at 90 C for 48–72hours, followed by desmosomal plaque complexes at cell-cell borders. J Cell Sci 114: 60 1C for 12 hours, and 30 1C for 12 hours; transitions between 727–38 1 these temperatures were made at 5 1C hour . After warming Burgess SA, Yu S, Walker ML, Hawkins RJ, Chalovich JM, Knight PJ (2007) samples to room temperature, they were washed with pure acetone, Structures of myosin and heavy meromyosin in the infiltrated with LX112 Epon resin, and polymerized at 60 1C. folded, shutdown state. J Mol Biol 372:1165–78 Sections were stained with 1–3% uranyl acetate and Sato Lead Caldelari R, de Bruin A, Baumann D, Suter MM, Bierkamp C, Balmer V et al. (2001) A central role for the armadillo protein plakoglobin in the stain, and finally coated with 5 or 10 nm colloidal gold particles for autoimmune disease . J Cell Biol 153:823–34 use as fiducial markers. Caldelari R, Suter MM, Baumann D, De Bruin A, Muller E (2000) Long-term Electron micrographs of samples tilted between 701 and þ 701 culture of murine epidermal keratinocytes. J Invest Dermatol 114:1064–5 B at 1–21 intervals were recorded at 1 mm defocus with 25–68k Chen CP, Posy S, Ben-Shaul A, Shapiro L, Honig BH (2005) Specificity of magnification on a 4 k 4 k charge-coupled device camera (Tietz cell–cell adhesion by classical cadherins: critical role for low-affinity Video Imaging Processing System GmbH, Gauting Germany) using dimerization through beta-strand swapping. Proc Natl Acad Sci USA 102:8531–6 SerialEM (Mastronarde, 2005). A second tilt series of the same area B 1 Chen X, Bonne S, Hatzfeld M, van Roy F, Green KJ (2002) Protein binding was collected after manually rotating the microscope grid by 90 . and functional characterization of plakophilin 2. Evidence for its diverse Images were binned 2 to produce sampling intervals of roles in desmosomes and beta-catenin signaling. J Biol Chem 277: 0.25–0.68nm, depending on magnification. Prior to data collection, 10512–22 sections were stabilized by pre-irradiation with B105 electrons nm2 Choi HJ, Park-Snyder S, Pascoe LT, Green KJ, Weis WI (2002) Structures of and the cumulative dose for the dual-axis tilt series was B105 elec- two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure. Nat Struct Biol 9:612–20 trons nm2. Dual-axis tomographic reconstructions were calculated Choi HJ, Weis WI (2005) Structure of the domain of using IMOD (Kremer et al., 1996) and features in selected plakophilin 1. J Mol Biol 346:367–76 tomograms were segmented for presentation using Amira (Mercury de Bruin A, Caldelari R, Williamson L, Suter MM, Hunziker T, Wyder M et al. Computer Systems Inc., Chelmsford, MA). (2007) Plakoglobin-dependent disruption of the desmosomal plaque in pemphigus vulgaris. Exp Dermatol 16:468–75 CONFLICT OF INTEREST Gallicano GI, Kouklis P, Bauer C, Yin M, Vasioukhin V, Degenstein L et al. The authors state no conflict of interest. (1998) Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage. J Cell Biol 143:2009–22 ACKNOWLEDGMENTS Garrod DR, Merritt AJ, Nie Z (2002) Desmosomal adhesion: structural basis, We thank Dr Kathleen Green for kindly providing numerous antibodies used molecular mechanism and regulation. Mol Membr Biol 19:81–94 for this work. We would like to acknowledge the New York Structural Biology Green KJ, Stappenbeck TS, Parry DA, Virata ML (1992) Structure of Center for providing electron microscopy facilities used in this work. Funding desmoplakin and its association with intermediate filaments. J Dermatol for this study was provided by NIH Grant R01 GM071044 to DLS. 19:765–9

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Hatsell S, Medina L, Merola J, Haltiwanger R, Cowin P (2003) Plakoglobin is modulates beta-catenin-mediated transcription. Mol Cell Biol 23: O-glycosylated close to the N-terminal destruction box. J Biol Chem 7391–402 278:37745–52 Muller-Tidow C, Steffen B, Cauvet T, Tickenbrock L, Ji P, Diederichs S et al. Hatzfeld M (2007) : multifunctional proteins or just regulators of (2004) Translocation products in acute myeloid leukemia activate the desmosomal adhesion? Biophys Biochym Acta 1773:69–77 in hematopoietic cells. Mol Cell Biol Hatzfeld M, Haffner C, Schulze K, Vinzens U (2000) The function of 24:2890–904 plakophilin 1 in desmosome assembly and filament organization. Nelson WJ, Nusse R (2004) Convergence of Wnt, beta-catenin, and cadherin J Cell Biol 149:209–22 pathways. Science 303:1483–7 He W, Cowin P, Stokes DL (2003) Untangling desmosomal knots with North AJ, Bardsley WG, Hyam J, Bornslaeger EA, Cordingley HC, Trinnaman electron tomography. Science 302:109–13 B et al. (1999) Molecular map of the desmosomal plaque. J Cell Sci Hennings H, Holbrook KA (1983) Calcium regulation of cell–cell contact 112:4325–36 and differentiation of epidermal cells in culture. An ultrastructural study. O’Keefe EJ, Erickson HP, Bennett V (1989) Desmoplakin I and desmoplakin II. Exp Cell Res 143:127–42 Purification and characterization. J Biol Chem 264:8310–8 Huber AH, Weis WI (2001) The structure of the beta-catenin/E-cadherin Peifer M, McCrea PD, Green KJ, Wieschaus E, Gumbiner BM (1992) The complex and the molecular basis of diverse ligand recognition by beta- adhesive junction proteins beta-catenin and plakoglobin and catenin. Cell 105:391–402 the Drosophila segment polarity gene armadillo form a multigene family Kapprell HP, Owaribe K, Franke WW (1988) Identification of a basic protein with similar properties. J Cell Biol 118:681–91 of Mr 75,000 as an accessory desmosomal plaque protein in stratified Ruiz P, Brinkmann V, Ledermann B, Behrend M, Grund C, Thalhammer C and complex epithelia. J Cell Biol 106:1679–91 et al. (1996) Targeted mutation of plakoglobin in mice reveals essential Kouklis PD, Hutton E, Fuchs E (1994) Making a connection: direct binding functions of desmosomes in the embryonic heart. J Cell Biol 135: between keratin intermediate filaments and desmosomal proteins. J Cell 215–25 Biol 127:1049–60 Smith EA, Fuchs E (1998) Defining the interactions between intermediate Kowalczyk AP, Bornslaeger EA, Borgwardt JE, Palka HL, Dhaliwal AS, filaments and desmosomes. J Cell Biol 141:1229–41 Corcoran CM et al. (1997) The amino-terminal domain of desmoplakin Stappenbeck TS, Bornslaeger EA, Corcoran CM, Luu HH, Virata ML, Green KJ binds to plakoglobin and clusters desmosomal cadherin–plakoglobin (1993) Functional analysis of desmoplakin domains: specification of the complexes. J Cell Biol 139:773–84 interaction with keratin versus intermediate filament networks. Kowalczyk AP, Hatzfeld M, Bornslaeger EA, Kopp DS, Borgwardt JE, J Cell Biol 123:691–705 Corcoran CM et al. (1999) The head domain of plakophilin-1 binds to Stappenbeck TS, Lamb JA, Corcoran CM, Green KJ (1994) Phosphorylation of desmoplakin and enhances its recruitment to desmosomes. Implications the desmoplakin COOH terminus negatively regulates its interaction for cutaneous disease. J Biol Chem 274:18145–8 with keratin intermediate filament networks. J Biol Chem 269:29351–4 Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of Troyanovsky RB, Chitaev NA, Troyanovsky SM (1996) Cadherin binding sites three-dimensional image data using IMOD. J Struct Biol 116:71–6 of plakoglobin: localization, specificity and role in targeting to adhering Leung CL, Green KJ, Liem RK (2002) : a family of versatile cytolinker junctions. J Cell Sci 109(Pt 13):3069–78 proteins. Trends Cell Biol 12:37–45 Troyanovsky SM, Troyanovsky RB, Eshkind LG, Leube RE, Franke WW (1994) Lewis JE, Wahl JK III, Sass KM, Jensen PJ, Johnson KR, Wheelock MJ (1997) Identification of amino acid sequence motifs in desmocollin, a Cross-talk between adherens junctions and desmosomes depends on desmosomal glycoprotein, that are required for plakoglobin binding plakoglobin. J Cell Biol 136:919–34 and plaque formation. Proc Natl Acad Sci USA 91:10790–4 Mastronarde DN (2005) Automated electron microscope tomography using Williamson L, Raess NA, Caldelari R, Zakher A, de Bruin A, Posthaus H et al. robust prediction of specimen movements. J Struct Biol 152:36–51 (2006) Pemphigus vulgaris identifies plakoglobin as key suppressor of c-Myc in the skin. EMBO J 25:3298–309 McGrath JA, McMillan JR, Shemanko CS, Runswick SK, Leigh IM, Lane EB et al. (1997) Mutations in the plakophilin 1 gene result in ectodermal Witcher LL, Collins R, Puttagunta S, Mechanic SE, Munson M, Gumbiner B dysplasia/. Nat Genet 17:240–4 et al. (1996) Desmosomal cadherin binding domains of plakoglobin. McKoy G, Protonotarios N, Crosby A, Tsatsopoulou A, Anastasakis A, Coonar J Biol Chem 271:10904–9 A et al. (2000) Identification of a deletion in plakoglobin in Yin T, Getsios S, Caldelari R, Godsel LM, Kowalczyk AP, Muller EJ et al. arrhythmogenic right ventricular with palmoplantar (2005a) Mechanisms of plakoglobin-dependent adhesion: desmosome- and woolly (Naxos disease). Lancet 355:2119–24 specific functions in assembly and regulation by epidermal growth factor Meng JJ, Bornslaeger EA, Green KJ, Steinert PM, Ip W (1997) Two-hybrid receptor. J Biol Chem 280:40355–63 analysis reveals fundamental differences in direct interactions between Yin T, Getsios S, Caldelari R, Kowalczyk AP, Muller EJ, Jones JC et al. (2005b) desmoplakin and cell type-specific intermediate filaments. J Biol Chem Plakoglobin suppresses keratinocyte motility through both cell–cell 272:21495–503 adhesion-dependent and -independent mechanisms. Proc Natl Acad Miravet S, Piedra J, Castano J, Raurell I, Franci C, Dunach M et al. (2003) Sci USA 102:5420–5 Tyrosine phosphorylation of plakoglobin causes contrary effects on its Yin T, Green KJ (2004) Regulation of desmosome assembly and adhesion. association with desmosomes and components and Semin Cell Dev Biol 15:665–77

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