Plakophilin 1 Constructs and Antibodies
To address the function of plakophilin 1 in desmosome assembly and structure, we studied targeting of its domains in epithelial cells and analyzed its direct binding partners in the yeast two-hybrid system. a summarizes the plakophilin 1 constructs tested in transfection assays and in the yeast two-hybrid system. The GFP constructs of all domains were analyzed in parallel with nontagged or T7-tagged constructs to verify that the GFP tag did not interfere with intracellular sorting.
Rabbit polyclonal antibodies against the plakophilin 1 NH2-terminal domain and the arm repeat domain were generated and tested for their specificity by Western blotting on total cellular extracts. b shows that both antibodies reacted with a single band of 80 kD, demonstrating that they did not cross-react with related proteins, such as plakophilin 2 (96 kD) and 3 (86 kD), p120ctn (various isoforms of 96–115 kD), or p0071 (130 kD). The majority of the protein was detected in the insoluble protein fraction.
Wild-Type Plakophilin 1 and Its Head Domain Associate with Desmosomes and Enhance Recruitment of Desmosomal Proteins to the Plasma Membrane in HaCaT Cells
Since plakophilin 1 has been described as a protein with dual localization in desmosomes and in the nucleus (Schmidt et al. 1997
), we analyzed intracellular targeting of the protein after overexpression. Attempts to obtain clonal cell lines that strongly overexpress plakophilin 1, or its head, or repeat domain, thus far, have been unsuccessful. This may be due to the phenotype that is caused by strong overexpression of plakophilin 1 or its fragments (see below). Therefore, we have used transient transfection studies to analyze the function of plakophilin 1 and its domains in a cellular context. Wild-type plakophilin 1, which was overexpressed in HaCaT keratinocytes, localized predominantly to the nucleus and to cell borders in confluent monolayers ( a), which is in agreement with the intracellular localization of the endogenous protein (Schmidt et al. 1997
). The balance between nuclear localization and plasma membrane association appeared similar in transfected and nontransfected cells. Double labeling with desmoplakin antibodies revealed a strong increase of endogenous desmoplakin at the plasma membrane in the transfected cells compared with nontransfected cells ( a′).
Figure 2 Expression of full-length plakophilin 1 in HaCaT cells. Cells were fixed in methanol 30 h after transfection, and double labeled with the plakophilin 1 head domain antibody (a) and the desmoplakin 2.15 antibody (a′). In confluent monolayers, plakophilin (more ...)
To identify the domains that target plakophilin 1 to desmosomes and to the nucleus, the head and the arm repeat domains of plakophilin 1 were expressed separately. Whereas the arm repeat domain colocalized with the actin cytoskeleton (see below), the head domain, like the full-length protein, was detected in the nucleus and along the cell periphery (, a–e) and strongly enhanced recruitment of desmoplakin to the plasma membrane ( a′). Costaining for other desmosomal proteins revealed that recruitment of Dsg ( b′), Dsc ( c′) and, to a lesser extent, plakoglobin ( d′) was also enhanced. The amount of recruited protein roughly correlated with the size of the membrane pool of plakophilin 1. In cells with a large membrane pool of plakophilin 1, recruited proteins were detected continuously along the plasma membrane (, a–d'). In other cells, the typical punctate pattern of individual desmosomes was retained (). Costaining for keratins showed colocalization of a small pool of these proteins to plasma membrane patches enriched for plakophilin 1 ( and ′). These data demonstrate that plakophilin 1 is able to recruit various desmosomal plaque proteins to the plasma membrane, and that this effect is mediated by its head domain.
Figure 3 Expression of the plakophilin 1 head domain in HaCaT cells. Plasmid DNAs encoding the plakophilin 1 head domain in pCMV5 were transfected into HaCaT cells. Cells were fixed in methanol and extracted in Triton X-100 and double labeled with the plakophilin (more ...)
Figure 4 Laser scanning microscopy analysis of HaCaT cells expressing the plakophilin 1 head domain. (a) Cells were stained with the plakophilin 1 head domain antibody (red fluorescence) and the anti–Pan-cadherin antibody (green fluorescence). Overlay (more ...)
In addition to its plasma membrane association, the head domain showed very strong nuclear localization. Surprisingly, some desmoplakin, Dsg, and Dsc were also detected in the nucleus, suggesting that plakophilin 1 coimported a fraction of these proteins into the nucleus.
To analyze the recruitment of desmosomal plaque proteins to the plasma membrane in more detail, we used laser scanning microscopy on HaCaT cells expressing the head domain of plakophilin 1. Whereas plakophilin 1 and E-cadherin staining overlapped only very little ( a), a high degree of overlap was found between plakophilin 1 and desmoplakin staining ( b), demonstrating that the major portion of overexpressed plakophilin 1 head domain does not localize to adherens junctions. These data indicate that plakophilin 1–mediated recruitment of proteins occurs primarily in desmosomes. To investigate the effect of the recruitment on desmosome size and number, we quantitated desmoplakin staining at cell borders by scanning along plasma membrane stretches (′ and b′′, arrows). The data are displayed as fluorescence intensity profiles below the corresponding image. The number and size of the peaks within these profiles were significantly higher when recorded along cell borders of two transfected cells ( b′), compared with the cell border between transfected and nontransfected cells ( b′′). Assuming that each peak represents a desmosome or a group of desmosomes, these data indicate that plakophilin 1–mediated recruitment of plaque proteins might result in the generation and enlargement of desmosomes.
To determine the region within the plakophilin 1 head domain responsible for desmosome association, several fragments were constructed ( a). Whereas all of them were still able to associate with desmosomes in HaCaT cells (), only the ΔN1, ΔN2, and ΔC1 fragments were capable of significantly enhancing the recruitment of endogenous desmoplakin () and other desmosomal plaque proteins (data not shown) to the cell membrane. The ΔC2 fragment did not recruit endogenous desmosomal proteins, although it associated with desmosomes. A major portion of the ΔN2 and ΔC2 fragments remained cytoplasmic (). All fragments were still able to enter the nucleus, but nuclear targeting was more efficient with the ΔN2 and ΔC2 constructs. These experiments show that at least one region mediating plasma membrane targeting of plakophilin 1 as well as a signal directing nuclear localization is retained in all head deletion constructs.
Figure 5 Expression of plakophilin 1 head domain fragments in HaCaT cells. Plasmid DNAs encoding the GFP-tagged plakophilin 1 head domain fragments were transfected into HaCaT cells, and their ability to recruit desmoplakin to the cell membrane was analyzed by (more ...)
Wild-Type Plakophilin 1 and Its Head Domain Accumulate in the Nucleus of HeLa Cells
When overexpressed in simple epithelial HeLa cells, plakophilin 1 accumulated in the nucleus, but was not recruited to the plasma membrane ( a), suggesting that the nuclear function of plakophilin 1 is conserved among all cells, whereas its function in stabilizing intercellular junctions is restricted to certain cell types. The lack of desmosome association of plakophilin 1 in HeLa cells may be due either to the lack of an appropriate binding partner such as cell type–specific Dsg and/or Dsc isoforms, or to different regulatory mechanisms that control modification and/or assembly of desmosomal proteins in HeLa cells. In addition to its nuclear localization, plakophilin 1 was found along actin filaments, as demonstrated by double labeling with phalloidin (, a and a′).
Figure 6 Plakophilin 1 associates with the actin cytoskeleton in HeLa (a–c) and HaCaT (d) cells. Plasmids encoding wild-type plakophilin 1 (a), the head domain (b), and the ΔC1-GFP fusion construct (c) were transfected into HeLa cells, and the (more ...)
Transfection studies with the plakophilin 1 head domain in HeLa cells showed almost exclusive nuclear localization of the fragment ( b). Decoration of actin filaments was not observed, suggesting that the binding site for direct or indirect actin filament association is located in the arm repeat domain (see below). Desmoplakin staining was strong in the transfected cells, but it appeared in a punctate pattern in the cytoplasm rather than in membranes ( b′). A similar distribution of desmoplakin was seen in mitotic cells ( b′, arrowheads), where desmosomal proteins have been internalized in vesicles. Nontransfected, nonmitotic cells revealed the punctate staining pattern along the plasma membrane, which is typical of desmosomes ( b′, arrows). The extent of cytoplasmic staining of desmoplakin seemed to correlate with plakophilin 1 expression levels. The cytoplasmic staining could be due to internalization of desmosomes and/or enhanced synthesis and assembly of desmosomal proteins in the cytoplasm (Demlehner et al. 1995
). The ΔC1 ( c), ΔC2, ΔN1, and ΔN2 (not shown) constructs showed almost exclusive nuclear localization with the same effect on desmoplakin distribution as described above.
Since wild-type plakophilin 1 decorated actin filaments in transfected HeLa cells, we analyzed plakophilin 1 localization more carefully in nontransfected cells to distinguish whether this was an artifact due to heavy overexpression that disturbed the intracellular sorting mechanisms, or whether it was connected to a novel function of plakophilin 1. In a wound healing experiment with HaCaT cells, colocalization of actin filaments and plakophilin 1 was observed at the tips of cellular protrusions ( and ′), suggesting a role for plakophilin 1 in regulating actin filament organization. Association with stress fibers was not observed.
The Plakophilin 1 Head Domain Binds to Desmoglein1, Desmoplakin, and Keratins in the Yeast Two-hybrid Assay
Plakophilin 1 has been shown to bind to Dsg1, Dsc1a, and desmoplakin in in vitro overlay assays (Smith and Fuchs 1998
), and to Dsg1 and desmoplakin in the two-hybrid system (Kowalczyk et al. 1999
). To localize the binding sites of these proteins in plakophilin 1, the cytoplasmic domains of Dsgs1-3 and Dscs1a,b-3a,b and the NH2
terminus of desmoplakin were tested in the yeast two-hybrid system. From all the desmosomal cadherins, only Dsg1 interacted with the plakophilin 1 head domain ( a) and with all head domain deletion constructs ( and ), although the ΔN2 and ΔC2 constructs appeared somewhat less efficient in reporter gene activation, suggesting that the Dsg1 binding site was not completely retained in these constructs. Desmoplakin binding was retained in the ΔC1 and ΔC2 fragments ( b), but not in the ΔN1 and ΔN2 fragments ( c), demonstrating that desmoplakin binds close to the NH2
terminus of plakophilin 1. These results suggest that desmoplakin and Dsg1 do not compete for the same binding site in the plakophilin 1 head.
Two-hybrid analysis of protein–protein interactions. (a) YRG2 yeast cells were double transformed with the plakophilin 1 head in pAS2-1 and desmoplakin-NH2 terminus (DP-NTP, 1), Dsg1 (2), Dsg2 (3), Dsg3 (4), Dsc1a (5), Dsc1b (6), Dsc2a (7), Dsc2b (8), Dsc3a (9), and Dsc3b (10) intracellular domains. All cells grew on selection plates lacking tryptophan and leucine (−TL), indicating that they contain both plasmids. Histidine reporter gene activation was analyzed on plates lacking histidine (−TLH) and LacZ reporter gene activation in a filter lift assay. DP-NTP and Dsg1 activated both reporter genes. (b) Double transformations of the ΔC1 construct in pAS2-1 and Dsg1 (1), Dsc1a (2), Dsc1b (3), and DP-NTP (4), and of ΔC2 with DP-NTP (5), Dsc1b (6), Dsc1a (7), and Dsg1 (8). DP-NTP and Dsg1 interacted with the ΔC1 and ΔC2 constructs, although LacZ reporter gene activation seemed weaker with Dsg1 + ΔC2. (c) Double transformations of the ΔN1 construct in pAS2-1 with Dsg1 (1), Dsc1a (2), Dsc1b (3), and DP-NTP (4) and of ΔN2 with DP-NTP (5), Dsc1b, (6), Dsc1a (7), and Dsg1 (8). ΔΝ1 and ΔN2 reacted with Dsg1, whereas DP-NTP did not interact with the ΔN1 and ΔN2 constructs. Dsc1a and b did not interact with any of the head domain fragments. (d) Double transformants of the head domain with Dsg1 deletion constructs containing the complete cytoplasmic domain (1), the IA domain (2), the CS domain (3), the IA and the CS domain (4), the Dsg domain (5), and the Dsg and CS domains (6). The head domain interacted strongly with the complete Dsg cytoplasmic domain and with the Dsg+CS domain. The interaction with the Dsg domain alone was weaker. (e) Double transformations of the head domain with K8 (1), K18 (2), K6 (3) and K17 (4) and of the arm domain with K8 (5), K18 (6), K6 (7) and K17 (8). The plakophilin 1 head domain interacted weakly with K8 and more strongly with K17 and K18. The arm repeats did not interact with any of the keratins tested. (f) Double transformations of headless plakophilin 1 with the following intracellular domains: DP-NTP (1), Dsg1 (2), Dsg2 (3), Dsg3 (4), Dsc1a (5), Dsc1b (6), Dsc2a (7), Dsc2b (8), Dsc3a (9), and Dsc3b (10). Although the His reporter gene was weakly activated by some constructs, the LacZ reporter gene was not activated. (g) Interactions between the cytoplasmic domain of Dsg1 and DP-NTP and the plakophilin 1 head domain fragments were quantitated using a β-galactosidase assay and the ONPG substrate. The bars represent three independent experiments each performed in triplet. None of the plakophilin 1 constructs activated the LacZ reporter gene on its own. Dsg1 interacted with all constructs tested. However, the ΔC2 and ΔN2 constructs showed a strong decrease in reporter gene activation suggesting that these constructs do not contain the entire Dsg1 binding site. DP-NTP interacted most strongly with the ΔC2 construct and revealed no interaction with the ΔN1 and ΔN2 constructs.
Since plakophilin 1 and plakoglobin (Troyanovsky et al. 1993
; Chitaev and Troyanovsky 1997
) both bind to desmoplakin and Dsg1, we wanted to analyze if these two proteins provide alternative links between the cadherins and the cytoskeleton, or if plakophilin 1 stabilizes the Dsg-plakoglobin-desmoplakin interaction through additional protein interactions. Therefore, we determined the plakophilin 1 binding site in the Dsg1 cytoplasmic domain. The plakophilin 1 head domain interacted with the intact Dsg1 cytoplasmic domain, the Dsg + CS domain and, although to a somewhat lesser extent, with the Dsg domain alone ( d), indicating that the plakophilin 1 binding site differs from the plakoglobin binding site in the CS domain. The requirement of the CS domain for strong binding suggests that the plakophilin 1 binding site is close to the plakoglobin binding site, and that simultaneous binding might be prevented because of steric hindrance.
Since plakophilin 1 has been shown to bind keratins in vitro (Kapprell et al. 1988
; Hatzfeld et al. 1994
; Smith and Fuchs 1998
), we also examined several keratin constructs for their interaction with the plakophilin 1 head domain. As shown in e, the type I keratins K17 and K18 strongly interacted with the plakophilin 1 head, whereas of the type II keratins tested, only K8 showed a weak interaction. Interaction studies with the head fragments revealed binding of K17 and K18 to the ΔC1 and ΔC2 constructs, but not to ΔN1 and ΔN2. The K8 binding site appeared to differ since binding was retained in the ΔN1, ΔC1, and ΔC2 constructs, but was lost in the ΔN2 fragment (not shown).
Interactions between the plakophilin 1 head fragments and desmoplakin and Dsg1 were quantitated by measuring LacZ reporter gene activation with the ONPG substrate. As shown in g, the desmoplakin–plakophilin 1 and the Dsg1–plakophilin 1 interactions were much stronger with the plakophilin 1 head in the pBD vector compared with the pAS2-1 vector, although this vector allows high protein expression levels as verified by Western blotting with Gal4 and plakophilin 1–specific antibodies ( a). The high protein expression could either interfere with correct folding of plakophilin 1, or the desmoplakin and Dsg1 binding sites are masked by inter- or intramolecular interactions after expression in the pAS vector. Dsg1 interacted most strongly with the ΔN1 construct, which lacks the desmoplakin binding site. Interaction with the ΔC1 construct was somewhat weaker. In contrast, the ΔC2 and ΔN2 constructs showed considerably reduced reporter gene activation, suggesting that these constructs did not contain the entire Dsg1 binding site. Desmoplakin interacted most strongly with the ΔC2 construct, which lacks part of the Dsg1 binding site. The interaction was lost with the ΔN1 and ΔN2 constructs, indicating that the binding site is in the NH2-terminal region.
Figure 8 Expression of plakophilin 1 constructs in yeast (a) and HeLa cells (b). (a) Yeast cell extracts were prepared as described in Materials and Methods, and the cell extracts were stained with Coomassie (lanes 1–7) or blotted with anti-GAL4 (lanes (more ...)
Since all previous experiments had shown interactions between cadherins and the arm repeat domains, but not the end domains of arm proteins, we also analyzed whether the headless plakophilin 1 or the repeat domain interacted with any of the desmosomal cadherins. As shown in f, none of the desmosomal cadherins interacted with headless plakophilin 1. The same result was obtained with the arm domain construct. Expression of the headless fragment in yeast cells was verified by Western blotting with anti-Gal4 antibodies ( a, lane 7′). Here, the headless fragment gave the strongest signal, indicating that a lack of protein expression did not account for the lack of binding.
The armadillo Repeat Domain of Plakophilin 1 Associates with Actin and Induces the Formation of Filopodia and Long Cellular Protrusions
The colocalization of wild-type plakophilin 1 with stress fibers as well as actin-rich structures at the tips of filopodia () pointed to a possible role of plakophilin 1 in regulating the actin cytoskeleton. Therefore, we expressed the plakophilin 1 arm repeat and the headless domain in HeLa and HaCaT cells and verified expression by Western blotting ( b). Cells with high levels of these plakophilin 1 fragments displayed a highly unusual morphology with formation of filopodia, lamellipodia, or long protrusions (, a–d), which interfered with normal monolayer formation. The transfected cells often sat on top of the other cells. Cells with lower expression levels still displayed a normal cell morphology (, a–e). However, double labeling with desmoplakin antibodies showed that desmoplakin had been internalized, and disintegration of junctions had already begun ( and ′). The plakophilin 1 arm repeat domain colocalized with actin in lamellipodia (, a, c, and d) and sometimes stress fibers ( and ′), suggesting a role in regulating actin polymerization and filopodia formation. This phenotype was observed in HeLa and HaCaT cells.
Figure 9 Expression of the arm repeat domain in HaCaT (a and b), L6 (c), and HeLa (d) cells. Cells were transfected with the arm repeat domain in pCMV5 (a, b, and d) or the headless construct in pEGFP (c). Transfected cells were visualized with the T7 antibody (more ...)
Figure 10 Expression of the arm repeat domain in HaCaT and HeLa cells. (a) HaCaT cells were transfected with plakophilin 1 arm repeats and processed for immunofluorescence after 20 h. Cells were stained with the plakophilin 1 repeat antibody (a) and FITC-phalloidin (more ...)
The Phenotype Produced by the Plakophilin 1 Arm Domain and Its Capacity to Associate with Actin Filaments Critically Depend on a Conserved Motif
A similar phenotype, the formation of long dendritelike cellular protrusions, had been observed in transfection studies with full-length p120ctn
(Reynolds et al. 1996
) and δ-catenin (Lu et al. 1999
). This suggested that the phenotype is conserved among p120ctn
family members, and might depend on the interaction with a common binding partner that is involved in regulating actin filament organization. To characterize this binding site in plakophilin 1, we constructed a deletion mutant that lacks a central pentapeptide motif conserved among all p120ctn
family members. The motif (ENCM/VC) is specific for this family and not detected in other arm related proteins. Transfection studies with this mutant construct (plakophilin 1 arm ΔENCMC) showed that the mutant had lost its capacity to induce changes in cell morphology and no longer associated with actin filaments in filopodia ( and ′). Instead, it accumulated in the cytoplasm, sometimes in an aggregated form ( f).
To analyze if the interaction between plakophilin 1 and actin is direct, we used the two-hybrid system. These experiments revealed no direct interaction between β-actin and the plakophilin 1 repeat domain (data not shown), suggesting that the interaction either depends on an intact microfilament or is mediated through an actin-associated protein in vivo.