Journal of Biochemistry Advance Access published August 6, 2015 doi:10.1093/jb/mvv048

J. Biochem. 2015;1—15

Desmocollin-2 alone forms functional desmosomal plaques, with the plaque formation requiring the juxtamembrane region and plakophilins Received March 1, 2015; accepted April 9, 2015; published online May 13, 2015

Miwako Fujiwara1, Azusa Nagatomo1, Megumi Tsuda1, Shuichi Obata2,3, Tetsushi Sakuma4, Takashi Yamamoto4 and Shintaro T. Suzuki1,*

*Shintaro T. Suzuki, Kwansei Gakuin University, 2-1 Gakuen, Sanda-shi, Hyogo-ken 669-1337, Japan. Tel: +81-79-565-7672, Fax: +81-79-565-9077, email: [email protected]

The role of the juxtamembrane region of the desmocollin-2 cytoplasmic domain in desmosome formation was investigated by using gene knockout and reconstitution experiments. When a deletion construct of the desmocollin-2 juxtamembrane region was expressed in HaCaT cells, the mutant protein became localized linearly at the cell—cell boundary, suggesting the involvement of this region in desmosomal plaque formation. Then, desmocollin-2 and desmoglein-2 genes of epithelial DLD-1 cells were ablated by using the CRISPR/Cas9 system. The resultant knockout cells did not form desmosomes, but re-expression of desmocollin-2 in the cells formed desmosomal plaques in the absence of desmoglein-2 and the transfectants showed significant cell adhesion activity. Intriguingly, expression of desmocollin-2 lacking its juxtamembrane region did not form the plaques. The results of an immunoprecipitation and GST-fusion protein pulldown assay suggested the binding of plakophilin-2 and -3 to the region. Ablation of plakophilin-2 and -3 genes resulted in disruption of the plaque-like accumulation and linear localization of desmocollin-2 at intercellular contact sites. These results suggest that the juxtamembrane region of desmocollin-2 and plakophilins are involved in the desmosomal plaque formation, possibly through the interaction between this region and plakophilins. Keywords: Cas9/CRISPR/desmocollin-2/desmosome/ DLD-1 cells/juxtamembrane domain/knockout/ plakophilin. Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats; DSC, desmocollin; DSG, desmoglein; DSP, desmoplakin; PG, plakoglobin; PKP, plakophilin; RT-PCR, reverse

Desmosomes form unique disk-like structures and mediate strong cell—cell adhesion. The adhesion proteins of desmosomes are desmosomal cadherins named desmocollins (DSCs) and desmogleins (DSGs), both of which exist as multiple isoforms. Desmosomes also include various scaffold proteins such as desmoplakins (DSP), plakoglobin (PG) and plakophilins (PKPs). It is generally thought that desmosomal cadherins are connected via PG to DSP, which in turn is linked to intermediate filaments and that these structures support the strong cell adhesion (1—3). On the other hand, PKPs were reported to participate in the desmosome formation, but the mechanism involved as remained elusive (4—9). The failure of the desmosome function results in serious disorders such as skin blisters and cardiac failure (10, 11), indicating the important function of the desmosome. Because of the unique structure and vital functions of desmosomes, many investigators have been interested in these structures and have carried out a large number of studies on them. As the results, a variety of data have been accumulated and the basic structure and function have been mostly elucidated. However, several fundamental properties of desmosomes have not been fully elucidated yet (1—3): Various evidences support the notion that desmosomes mediate strong cell—cell adhesion (12), but cell adhesion assays for desmosomes are very difficult to perform and no one has directly shown the strong activity in vitro. It is also unclear why desmosomes construct their unique and complex disk-like structure to fulfil their functions in contrast to other intercellular junctions such as adherens junction and tight junction. One interesting unanswered question is the role of the juxtamembrane region in the cytoplasmic domain of desmosomal cadherins. The overall structures of classical cadherins and desmosomal cadherins are almost the same and their amino acid sequences are highly homologous to each other except for the additional C-terminal region of DSGs. Especially, the juxtamembrane regions are highly conserved (13): the C-terminal region of classical cadherins binds to b-catenin (14), whereas the corresponding region of DSCs and DSGs binds to PG or g—catenin (15), a homologue of b-catenin. The linkage of these proteins

ß The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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1 Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Sanda-shi, Hyogo-ken 669-1337, Japan; 2Department of Liberal Arts, Kitasato University, Sagamihara-shi, Kanagawa-ken, 252-0373, Japan; 3Department of Histology and Cell Biology, Yokohama City University School of Medicine, Yokohama-shi, Kanagawa-ken, 236-0004, Japan; and 4 Department of Mathematics and Life Science, Graduate School of Science, Hiroshima University, Higashihiroshima-shi, Hiroshimaken, 739-8511, Japan

transcription-polymerase chain reaction; TALEN, transcription activator-like effector nuclease.

M. Fujiwara et al.

Materials and Methods Reagents Primary antibodies used were the following: mouse anti-DSG1/2 (Progen, Heiderberg, Germany), mouse anti-PKP2 (Progen), mouse anti-DSC2/3 (Invitrogen, Carlsbad, CA), mouse anti-DSP (Progen), mouse anti-PKP3 (Invitrogen), rabbit anti-DSP (Sant

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Cruz, Sant Cruz, CA), mouse anti-PG (BD Transduction Laboratories, San Jose. CA), rabbit anti-ZO1 (Invitrogen), mouse anti-E-cadherin (Takara, Otsu, Japan), rabbit anti-a-catenin (SigmaAldrich, St. Louis, MO), rabbit anti-b-catenin (Sigma-Aldrich), mouse anti-HA (Nakarai, Kyoto, Japan), mouse anti-FLAG (Invitrogen, Carlsbad, CA) and rabbit anti-HA (MBL, Nagoya, Japan). Secondary antibodies labelled with Alexa Fluor were the products of Invitrogen and alkaline phosphatase labelled secondary antibodies against mouse and rabbit IgGs were obtained from Promega (Madison, WI). Rabbit anti-b-actin antibody was a gift from Dr S. Imaoka (Kwanseigakuin University). Vector pX330 was obtained from Addgene (Cambridge, MA). pGEX-5X-1 was a product of Amersham Pharmacia Biotech Inc. (Piscataway, NJ).

cDNA constructs Human cDNAs for DSC2a and DSG2 were obtained by polymerase chain reaction (PCR) by using nucleotide sequences published in the GenBank. In this study, we used DSC2a isoform and its derivatives and DSC2 indicates DSC2a unless otherwise specified. To make DSC2 deletion mutants, we amplified DNAs by PCR using human DSC2 cDNA as a template and appropriate primers (Supplementary Table SI, DSC2-s, DSC2-as, DSC2(CS1)-s, DSC2(CS1)-as, DSC2(CS2)-s, DSC2(CS2)-as, DSC2(CS3)-as). The resultant cDNAs were cloned into the expression vector pEF1 (Invitrogen, Carlsbad, CA) by using an appropriate site. A HA-tag was introduced at the C-termini of all constructs by using the NotI-PmeI site. To construct the substitution mutant of the DSC2 CS1 region with the corresponding region of human E-cadherin (DSC2CS1-eCS1), we performed PCR amplifications by using DSC2 cDNA and E-cadherin cDNA as templates and appropriate primers (Supplementary Table SI, DSC2(CS1-eCS1)-1 s, DSC2(CS1eCS1)-1as, DSC2(CS1-eCS1)-2 s, DSC2(CS1-eCS1)-2as, DSC2as, Ecad(CS1-eCS1)-s, Ecad(CS1-eCS1)-as). The resultant fragments were fused by PCR and replaced with the CS1 region of DSC2 of pEF1-DSC2. The expression constructs of PKP-2 and PKP-3 were made by PCR using appropriate primers (Supplementary Table SI, PKP2-s, PKP2-as, PKP3-s, PKP3-as). The resultant cDNAs were cloned into the expression vector pCAG-Flag-IRESpuro. These constructs had a FLAG-tag at their N-termini. Constructs for GST fusion proteins with the cytoplasmic domain of DSC2 (GST-DSC2CP and GST-DSC2CP(CS1)) were generated by incorporating PCR-amplified cDNA into the pGEX5X-1 vector by using an appropriate site. PCR was carried out by using pEF1-DSC2 (GST-DSC2CP) or pEF1-DSC2CS1 (GST-DSC2CP(CS1)) as a template and appropriate primers (Supplementary Table SI, GST-DSC2-s, GST-DSC2-as, GST-DSC2(CS1)-s, GST-DSC2(CS1)-as).

Knockout by use of the CRISPR/Cas9 system Disruption of genes was carried out by the method of CRISPR/Cas9 system. For the CRISPR/Cas9 system, the pX330 vector was used according to the method described by Cong et al. (23). Briefly, to obtain knockout cells, we co-transfected DLD-1 cells with the CRISPR/Cas9 construct and a pCAG-Flag-IRESpuro vector by using the Lipofectamine LTX protocol or the calcium phosphate precipitation method. After screening with puromycin, resultant colonies were picked up and cultured; and the expression of the target protein was analysed by immunoblotting. To confirm the deletion or addition at the target site region of the genomic DNA, by PCR we amplified the DNA fragment containing the region from the genomic DNA purified from the cells. PCR products were subcloned into a vector and sequenced by using an ABI PRISM 3100 Genetic Analyzer.

Cell culture and transfection Cells of the human adenocarcinoma cell line DLD-1, human keratinocyte cell line HaCaT and human embryonic kidney cell line HEK293T were cultured in Dulbecco’s modified Eagle’s mediumF12 (1:1) containing 10% fetal bovine serum in 5% CO2 atmosphere at 37 C. For transient expression, HEK293T cells grown at 40% confluence were transfected with a construct DNA by the calcium phosphate precipitation method. After 16 h of culture, the medium was changed and analysis was performed at 40—48 h after transfection. To obtain stable clones, we transfected cells at 40% confluence with a construct DNA by using the Lipofectamine LTX

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plays a central role in cadherin functions. On the other hand, the juxtamembrane region of classical cadherins associates with p120-catenin and regulates the endocytosis of the cadherins (16, 17); whereas the corresponding regions of DSCs and most of DSGs are not known to associate with any specific protein(s), although an important role for these regions in desmosome assembly and/or other function have been suggested (18). These questions are very interesting to address, but desmosome studies have several intrinsic problems. One difficulty is that desmosomes show relatively complex and variable structures depending on the cells expressing them (2, 3, 19). For example, keratinocytes usually express multiple DSCs and DSGs in the same cell. Each desmosomal cadherin seems to have its unique role and each combination of cadherins may show specific properties; but the details have not been clarified yet (2, 3, 20). Another difficulty is that desmosomes are highly insoluble; especially DSP is quite difficult to dissolve by the standard solubilization method, which hinders biochemical studies on desmosomes (21). Moreover, cell adhesion assays for desmosomes are notoriously difficult to carry out, because classical cadherins are required for desmosome formation; and their strong cell adhesion activity disturbs the cell adhesion assay for desmosomes. In this study, we focused on the juxtamembrane region of the DSC-2 (DSC2) cytoplasmic domain and characterized its role. We initially used HaCaT cells, a keratinocyte cell line, for the study and obtained evidence for an important role of the juxtamembrane region in desmosome assembly. However, we became confronted with various difficulties such as those described above. Hence, we took two approaches to circumvent or reduce these problems in the latter part of this study. First, we chose DLD-1 cells, a human cell line derived from an adenocarcinoma, because the cells are epithelial and construct relatively simple desmosomes consisting of only DSC2 and DSG-2 (DSG2) as desmosomal cadherins. Second, we tried to establish a reconstitution system for desmosomes, because it is well known that reconstitution experiments are a very powerful tool to analyse the function of a given protein. To achieve reconstitution experiments, however, we needed to obtain appropriate cells that lacked the expression of the target protein, which cells had been very difficult to find in most cases. Recently, however, new gene editing methods, such as TALEN and CRISPR/Cas9 system, have become available (22, 23). Herein, we applied this method to desmosomal cadherin genes of DLD-1 cells and established a reconstitution system for desmosomes. Base on the results obtained, we discussed the assembly of desmosomes.

Role of CS1 and plakophilins protocol or calcium phosphate precipitation method. The cells were screened in the presence of G418 (400 mg/ml) for about 2 weeks and resultant clones were examined by immunoblot.

Light microscopy Cells for microscopy were grown on cover glasses. For examination of cell shape, cultured cells were fixed and examined with a light microscope equipped with a differential interference contrast apparatus. Immunofluorescence staining was performed as described previously (24). Photographs were taken mainly with a Nikon A1 laser confocal microscope (Nikon) in this study, unless otherwise specified. For proximity ligation assay (PLA) staining, cells were fixed with cold-methanol at 20 C for 5 min. After having been washed with tris-buffered saline (TBS), the fixed cells were incubated with 1% BSA in TBS for 30 min at room temperature for blocking and then reacted with primary antibody for 3 h at room temperature or overnight at 4 C. Then, ligation and amplification reactions were performed according to the manufacturer’s manual (Olink Bioscience, Uppsala, Sweden).

The cell dissociation assay was basically carried out according to the method described by Chen et al. (25) with some modifications. In brief, confluent cells were washed with HEPES-buffered saline (HBS) and then incubated with 200 U/ml dispase in HBS until the cell sheets had been released from the culture dishes. The cell sheets were then rinsed gently with HBS and transferred into a conical tube. The tube was inverted 10 times and the fragments were subsequently transferred into a dish and counted.

Immunoprecipitation and pull-down assay Cells for immunoprecipitation were solubilized for 20 min with 1% NP-40 in RIPA buffer (10 mM Tris-HCl [pH 8], containing 150 mM NaCl, 1 mM EDTA and 1 mM phenymethysulfony fluoride). The cell lysates were centrifuged at 4 C for 15 min at 15,000  g and the resultant solution was incubated for 90 min at 4 C with 0.5 mg antibody bound to anti-mouse IgG conjugated Dynabeads. After washing three times with RIPA buffer, the beads were mixed with sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS—PAGE) sample buffer, and boiled. The resultant samples were separated on 7% SDS-PAGE gels and examined by Immunoblot analysis. Pull-down assay by using GST fusion proteins was carried out as described previously (24).

Other methods Electron microscopy was carried out as described previously (26). Electron micrographs were taken by using an H7500 electron microscope (Hitachi High-Technologies, Tokyo, Japan). Reverse transcription-PCR (RT-PCR) was performed as described previously (24).

Results Role of juxtamembrane CS1 region of DSC2

To investigate the function of the juxtamembrane region of the DSC2 cytoplasmic domain, we constructed three deletion mutants of the highly conserved regions in the cytoplasmic domain (Fig. 1A) and expressed these mutants in HaCaT cells (Fig. 1B). We named the conserved regions CS1 (KVIPDDLA QQNLIVSNTEAPGDD), CS2 (DNCRYTYSEWHS FTQPRLGEK) and CS3 (SVAGSVGCCSERQEED GLEFLDNLEPKFRTLAEACMKR); and the present CS1 and CS2 regions included the respective CSI and CSII segments defined by Troyanovsky et al. (18). The DSC2 mutant lacking the juxtamembrane CS1 region (DSC2(CS1)) as well as the one lacking the C-terminal CS3 region (DSC2(CS3)) did not show a linear dotted localization; and these

Knockout of desmosomal cadherin genes

Because the above results were obtained by using a keratinocyte cell line, HaCaT cells, we were interested in knowing whether other types of cells would give similar results. Another important question is what kind of ultrastructure the DSC2 constructs formed. It was very difficult, however, to examine the ultrastructure made by the DSC2 constructs, because we could not differentiate well the structures formed by the mutants from the endogenous desmosomes at the electron microscopy level. To avoid these problems, therefore, we chose another human epithelial cell line, DLD-1, the cells of which only express DSC2 and DSG2 as desmosomal cadherins and generated knockout cells for these desmosomal cadherin genes by use of the CRISPR/Cas9 system. Then, we used the resultant cells for desmosome reconstitution experiments. Before carrying out the knockout experiments, we expressed the deletion constructs of DSC2 in DLD-1 cells and examined their localization. The results were essentially the same as those for the HaCaT cells (data not shown). Then, we prepared knockout cells for these desmosomal cadherin genes. The target sites are shown in Fig. 3A. In this study, we generated and used double knockout cells for DSC2 and DSG2 genes (DLD-1(DSC2DSG2)), because the single knockout cells for DSG2 or DSC2 formed desmosomal plaques (data not shown). After screening, multiple clones that did not show the expression of DSC2 or DSG2 were identified by immunoblot analysis and the genomic sequencing revealed that the DSC2 and DSG2 genes had deletions at 3

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Cell dissociation assay

mutant proteins were diffusely localized at cell-cell contact sites in the transfectants (Fig. 1C), whereas the DSC2 mutant with the deleted CS2 region (DSC2(CS2)) showed almost the same localization as control. These results suggest that the CS1 region in addition to the CS3 one played a role in a linear dotted localization of DSC2. When the deletion mutant proteins were immunoprecipitated, DSC2(CS1) and DSC2(CS2) co-precipitated with PG, whereas DSC2(CS3) did not (Fig. 1D), suggesting that the CS1 and CS3 regions had different functions in a linear dotted localization. The above results also suggest that the DSC2(CS1) was not incorporated into desmosome plaques. For further examination, we treated the transfectants with NP-40, a non-ionic detergent, and characterized the solubility. As shown in Fig. 2A, wild type DSC2 was resistant to the solubilization with NP-40; whereas DSC2(CS1) was easily extracted with the detergent. After solubilization, positive staining for DSC2(CS1) almost disappeared from the cell-cell boundaries (Fig. 2B). Moreover, DSC2(CS1) did not co-localize with endogenous DSP (Fig. 2C). The results of the PLA assay showed that DSC2 and DSP were localized within a proximate distance of 540 nm, whereas DSC2(CS1) and DSP were not (Fig. 2D and Supplementary Fig. S1). These results suggest that the CS1 region of DSC2 was involved in the plaque formation of desmosomes.

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Fig. 1 Effect of deletion of well conserved regions in the cytoplasmic domain of DSC2 on the assembly of desmosomes. (A) Schematic drawing of wild type DSC2 and three deletion constructs of DSC2. PG binds to CS3 region. (B) Immunoblot analysis of expression of wild type and deletion constructs in HaCaT cells. b-actin was used as an internal standard. (C) Immunofluorescence staining of DSC2 and deletion constructs expressed in HaCaT cells. DSC2 and DSC2(CS2) showed dot-like localization at intercellular contact sites, whereas DSC2(CS1) and DSC2(CS3) showed continuous staining at cell—cell contact sites. Monoclonal anti-HA antibody (HA) and anti-DSC2 (DSC) antibody were used for the staining. Scale bar, 10 mm. (D) Wild type DSC2, DSC2(CS1), and DSC2(CS2) co-precipitated with PG by immunoprecipitation (IP), but DSC2(CS3) did not.

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Re-expression of DSC2 constructs in double knockout cells

Fig. 2 Characterization of DSC2 and DSC2("CS1) expressed in HaCaT cells. (A) Immunoblot analysis showed that DSC2 expressed in HaCaT cells was resistant to NP-40 solubilization but that DSC2(CS1) was not. (B) Immunostaining of HaCaT cells treated with 0.5% NP-40. After solubilization, significant fluorescence of DSC2 remained at intercellular contact sites of the cells, but that for DSC2(CS1) was almost undetectable at these sites. Monoclonal anti-HA antibody (HA) and anti-DSC2 (DSC) were used for the staining. (C) Immunofluorescence staining for DSC2 and DSC2(CS1) expressed in HaCaT cells. DSC2 co-localized with DSP in the transfectants, whereas DSC2(CS1) did not well co-localize with DSP in them. (D) PLA assay using anti-HA (HA) and anti-DSP (DSP) antibodies revealed that DSC2 and DSP were present within a close distance in the DSC2-expressing cells but that DSC2(CS1) and DSP were not in such close proximity in the DSC2(CS1)-expressing ones. Photographs were taken with a Nikon Eclipse TE2000-U fluorescence microscope. Scale bar, 10 mm.

Then, DSC2 was re-expressed in the double knockout DLD-1(DSC2DSG2) cells (Fig. 4A). When immunostained, the resultant transfectants showed a desmosome-like accumulation of DSC2 without the DSG2 expression; and the expressed DSC2 was co-localized with DSP (Fig. 4B). In addition, the re-expressed DSC2 co-precipitated with PG (Fig. 4C) and was resistant to solubilization with NP-40 (Fig. 4D). Next, DSC2(CS1), DSC2(CS2) and DSC2(CS3) were expressed in the double knockout DLD-1(DSC2DSG2) cells. In the resultant transfectants, DSC2(CS1) did not show a patch-like accumulation and was localized diffusely at cell—cell contact sites (Fig. 4B). Furthermore, the expressed DSC2(CS1) did not co-localize with DSP. On the other hand, DSC2(CS3) showed a localization similar to that of DSC2(CS1) in the transfectants, whereas DSC2(CS2) showed a localization similar to that of DSC2 in parental DLD-1 cells (Supplementary Fig. S2). When the transfectant cells were solubilized with NP-40, the DSC2(CS1) was easily solubilized by the treatment, suggesting that the DSC2(CS1) had not become firmly integrated into some structure (Fig. 4D). These results support the notion that the CS1 region of DSC2 was involved in the plaque formation. Next, the cell adhesion activities of the DSC2 transfectants were examined by performing a cell dissociation assay. As shown in Fig. 4E, the DSC2 transfectants of the double knockout DLD-1 cells showed significant cell adhesion activity similar to that of the wild type DLD-1 cells. In contrast, the double knockout DLD-1 cells transfected with DSC2(CS1) showed only weak activity. Substitution experiments on the CS1 region of DSC2

The above results suggest that the CS1 region played an important role in the assembly of desmosomal plaques. However, another explanation is possible, i.e. that deletion of the CS1 region might have 5

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the target regions in which the initiation codon had been destroyed or new stop codons had been generated (Fig. 3B). Immunoblot analysis did not detect the expression of DSC2 and DSG2 in the cells, but did show that of other desmosomal scaffold proteins at levels similar to those of the wild-type cells (Fig. 3C). The knockout cells showed normal epithelial morphology that was indistinguishable from that of wild type DLD-1 cells. Immunofluorescence staining for DSC2 or DSG2 showed no signals, but staining for DSP revealed a weak distribution (Fig. 3D). Furthermore, expression of other desmosomal cadherins was not detected in the knockout cells by RT-PCR (Fig. 3E). E-cadherin, afadin and ZO-1 were localized at cell—cell contact sites of knockout cells (data not shown). In this study, we used at least two target sites for each gene and obtained multiple knockout cells. The above results were obtained by using clone 1, but the other clones showed essentially the same properties. Clone 1 was used in the subsequent experiments.

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Fig. 3 Knockout of DSC2 and DSG2 genes in DLD-1 cells. (A) Target sites of DSC2 and DSG2 knockout. (B) Genomic sequencing revealed deletions in the target regions that destroyed the initiation codon of DSC2 and generated new stop codons in the downstream region of DSG2. (C) Immunoblot analysis of the double knockout cells demonstrated that the cells did not express DSC2 or DSG2, but did expressed other junctional proteins at normal levels. b-actin was used as an internal standard. (D) In contrast to the wild type DLD-1 cells, the double knockout ones did not show positive immunofluorescence staining for DSC2 or DSG2, but did show faint staining for DSP. Scale bar, 10 mm. (E) RT-PCR for DSC1, DSC3, DSG1, DSG3 and DSG4 detected no signal of these cadherins in DLD-1 cells. (C). HaCaT cells were used as positive control for the expression of desmosomal cadherins and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard. DSC2DSG2, DSC2 and DSG2 double knockout DLD-1 cells.

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Fig. 4 Expression of DSC2 and DSC2("CS1) in the DSC2 and DSG2 double-knockout DLD-1 cells. (A) Immunoblot analysis of expression of DSC2 and DSC2(CS1). b-actin was used as an internal standard. (B) Immunofluorescence staining of DLD-1(DSC2DSG2) cells, DSC2expressing double knockout cells and DSC2(CS1)-expressing double-knockout cells with anti HA antibody and anti-DSP antibody. DSC2 accumulated in a patch-like fashion and co-localized with DSP in DSC2-expressing double-knockout cells, whereas DSC2(CS1) was localized linearly at intercellular contact sites in the DSC2(CS1)-expressing double-knockout cells and did not co-localized with DSP. The doubleknockout cells showed weak DSP staining. Scale bar, 10 mm. (C) In the immunoprecipitation (IP) experiment, DSC2 and DSC2(CS1) coprecipitated with PG. b-actin was used as an internal standard. (D) DSC2 and DSC2(CS1) expressed in the double-knockout cells were incubated with 1% NP-40 and then analysed by immunoblotting using anti-HA antibody (HA). DSP was resistant to the solubilization with NP40, whereas DSC2(CS1) was not. (E) Cell dissociation assay (a) and the quantified results (b). Wild type DLD-1 and DSC2-expressing doubleknockout cells showed strong cell adhesion activity, whereas the double-knockout ones and DSC2(CS1)-expressing double-knockout cells showed only weak activity. DSC2DSG2, DSC2 and DSG2 double-knockout DLD-1 cells.

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Fig. 5 Substitution of CS1 region of DSC2 and highly conserved amino acid residues in the region. (A) Amino acid sequences of CS1 region of DSC2 and the corresponding region of E-cadherin are shown. The two amino acid residues E743 and D747 in the region were changed to alanine and are indicated in red. (B) Immunoblot analysis of the effect of swapping with the corresponding E-cadherin region and of substitution constructs in the DSC2 DSG2 double knockout DLD-1 cells. b-actin was used as an internal standard. (C) Immunofluorescence staining of the DSC2 mutants and DSP in the transfectants. Swapping construct transfectants revealed linear localization of the swapping construct and patchlike localization of DSP at the intercellular contact sites. Similarly, DSC2(E743A) transfectants showed a linear localization of DSC2(E743A) and weak patch-like accumulation of DSP at the intercellular contact sites, whereas DSC2(D747A) transfectants showed the localization of DSC2(D747) and DSP similar to that of wild type DLD-1 cells. Scale bar, 10 mm.DSC2DSG2, DSC2 and DSG2 double knockout DLD-1 cells.

disrupted the higher structure of the region or the cytoplasmic domain, which in turn could have inhibited the assembly of desmosomal plaques. To clarify this problem, we substituted the CS1 region of DSC2 with the corresponding region of E-cadherin, which region is

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highly homologous to that of DSC2 in terms of amino acid sequence (Fig. 5A). The resultant transfectants showed that the construct was localized at cell—cell contact sites but formed no clear plaques (Fig. 5C), supporting the notion that the CS1 region

Role of CS1 and plakophilins

Interaction of DSC2 with PKP2 and PKP3

The above results suggest that some protein had bound to the juxtamembrane CS1 region and regulated the formation of desmosomal plaques of DSC2. We first examined the interaction between DSC2 and p120-catenin, because E-cadherin associates with p120-catenin at the region corresponding to the CS1 region of DSC2; and Dsg3 was reported to bind to p120-catenin at this region (27). However, punctate staining of p120-catenin was not obtained in DLD-1 cells; and immunoprecipitation experiments did not show any evidence that p120-catenin bound to this region (data not shown). Next, we postulated that PKP2 and/or PKP3 bound to the CS1 region of DSC2, because PKPs are homologues of p120-catenin and have been thought to associate with the desmosomal cadherins (5, 28). Furthermore, Bonne´ et al. (6) suggested the interaction between PKP3 and the juxtamembrane domains of some of the desmosomal cadherins. Because PKP1 was not expressed in DLD-1 cells, FLAG-tagged PKP2 or PKP3 and HA-tagged DSC2 were expressed in HEK293T cells and immunoprecipitation experiments were then carried out (Fig. 6). When PKP2FLAG was precipitated with anti-FLAG antibody, DSC2-HA co-precipitated with it (Fig. 6A). Similarly, PKP3-FLAG co-precipitated with DSC2HA (Fig. 6B), but neither PKP2-FLAG nor PKP3FLAG co-precipitated with DSC2(CS1)-HA. A faint band of PKP3 occasionally co-precipitated with DSC2(CS1)-HA in the experiments, which may indicate weak interaction between PKP3 and CS3 region. In contrast, immunoprecipitation of DSC2-HA with anti-HA antibody did not bring down PKP2-FLAG or PKP3-FLAG (data not shown). The reason for this finding is unclear. To examine this association further, we carried out pull-down experiments by using fusion proteins of GST with the cytoplasmic domain of DSC2 (GST-DSC2CP), which constructs are schematically depicted in Fig. 6C. Extracts of DLD-1 cells were

Fig. 6 Interaction between DSC2 and PKP2 or PKP3. (A) Immunoprecipitation of PKP2. When FLAG-tagged PKP2 and HAtagged DSC2 or HA-tagged DSC2(CP) were ectopically expressed in HEK293T cells and the PKP2 was immunoprecipitated with antiFLAG antibody, PKP2 and DSC2 were co-precipitated, but PKP2 and DSC2(CP) did not. DSC2(CP), a DSC2 mutant lacking the entire cytoplasmic domain; *DSC2, Empty IRES vector was used instead of the PKP expression IRES vector. (B) Immunoprecipitation of PKP3. When FLAG-tagged PKP3 and HA-tagged DSC2 or HA-tagged DSC2(CP) were ectopically expressed in HEK293T cells and the PKP3 was immunoprecipitated with anti-FLAG antibody, PKP3 and DSC2 were co-precipitated, but PKP3 and DSC2(CP) did not. (C) Schematic drawing of GST-fusion constructs. (D) GSTDSC2CP pulled down PKP2 and PKP3, but GST-DSC2CP(CS1) did not. b-actin was used as an internal standard (A and B). Control, DSC2 and DSG2 double knockout DLD-1 cells; IP, immunoprecipitation.

prepared and examined. The GST-DSC2CP fusion protein pulled down PKP2 and PKP3, but the GST-DSC2CP(CS1) fusion protein lacking the CS1 region did not (Fig. 6D). Taken together, our data suggest that the CS1 region of DSC2 interacted with PKP2 and PKP3. Knockout of PKP genes

The above results suggest a vital role of PKP2 and PKP3 in the assembly of desmosomes. Hence, knockout experiments of PKP2 and PKP3 genes were carried out in the DSC2-expressing double knockout DLD-1(DSC2DSG2) cells to characterize further the biological role of PKP2 and PKP3 in desmosome

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had an indispensable function in the formation of desmosomal plaques. Interestingly, immunofluorescence staining showed the formation of a small number of DSP plaques in the absence of plaque-forming desmosomal cadherins (Fig. 5C). Furthermore, we carried out amino acid substitution experiments: We postulated that highly conserved polar amino acid residues in the CS1 region might play a key role in the formation of desmosomal plaques. The substitution of the glutamic acid residue at position 743 in the region with alanine (DSC2(E473A)) also resulted in a linear localization (Fig. 5C) similar to that of DSC2(CS1) (Fig. 4B). In contrast, another substitution mutant of the nearby aspartic acid residue at position 747 with alanine (DSC2(D747A)) did not have appreciable effects on the localization (Fig. 5C), indicating the specificity of amino acid substitution. These results also suggest that the juxtamembrane CS1 region was involved in the assembly of desmosomes.

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Fig. 7 Knockout of PKP2 and PKP3 genes in DLD-1 cells by use of the CRISPR/Cas9 system. (A) Target sites of PKP2 and PKP3 knockout by CRISPR/Cas9. (B) In the immunoblot analysis, the expression of PKP2 and PKP3 was not detected in the PKP2 and PKP3 double knockout cells (PKP2PKP3) derived from the DSC2 and DSG2 double knockout DLD-1 cells, but the expression of the other junctional proteins did not change much. b-actin was used as an internal standard. (C) Genomic sequences of target regions of the knockout cells contained an addition or a deletion that generated new stop codons in the downstream regions or disrupted the initiation site of translation. (D) Immunofluorescence staining of the knockout cells showed that PKP2, PKP3 or DSP was not stained but that DSC2 was stained linearly at intercellular sites. Positive staining for E-cadherin and PG did not change much. DSC2DSG2, DSC2 and DSG2 double knockout DLD-1 cells. Scale bar, 10 mm.

assembly. The target sites are shown in Fig. 7A. The results demonstrated that single knockout cells for PKP2 or PKP3 only very mildly affected the formation of desmosomal plaques. Therefore, we performed double knockout of PKP2 and PKP3 genes next. Immunoblot analysis did not detect the expression of 10

PKP2 or PKP3 in the resultant cells (Fig. 7B) and genomic sequencing showed that the target regions had deletions (Fig. 7C). Immunofluorescence staining of the DSC2 transfectants of the knockout cells (DLD-1(DSC2DSG2PKP2PKP3)) revealed that DSC2 had become localized diffusely at cell—cell

Role of CS1 and plakophilins

contact sites and did not show any linear dotted pattern (Fig. 7D, HA). Interestingly, patch-like accumulation of DSP was not observed well in the cells. E-cadherin (Fig. 7D), afadin (data not shown) and ZO-1 (data not shown) localized at cell-cell contact sites of the cells, a distribution very similar to that found for the wild-type cells. Examination of desmosome formation in mutant cells by electron microscopy

Discussion In this study, we successfully used the CRISPR/Cas9 system to obtain knockout cells for desmosomal cadherins and used the resultant cells for reconstitution experiments. We used at least two target sites for each gene to exclude off-target effects and examined multiple knockout clones, which gave similar results. Moreover, when knockout of a gene(s) caused some defects, we carried out rescue experiments and confirmed the results. Hence, we think that the present results are not simple artefacts, but reflect the actual properties of DSC2. One may wonder if knockout of an important gene in differentiated cells would alter the expression of

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In the above experiments, immunofluorescence microscopy was used to examine the formation of desmosomal plaques in the cells. This method, however, has a clear limitation in that it could not clarify what types of ultrastructures the desmosomal cadherins actually made in these cells. Therefore, we examined the ultrastructures that the DSC2 constructs and their associated proteins formed in the cells by electron microscopy. As shown in Fig. 8 panels a and b, wild type DLD-1 cells clearly formed desmosomes. Single knockout cells of DSG2 (Fig. 8, c and d) or DSC2 (data not shown) formed desmosomal structures very similar to those of the wild-type cells, but double knockout cells for DSC2 and DSG2 (DSC2DSG2) did not show any desmosome or desmosome-like structure at al. (Fig. 8, e and f). Re-expression of DSC2 in the double knockout cells, however, resulted in the formation of desmosome-like structures indistinguishable from those of wild-type cells (Fig. 8, g and h). Intriguingly, expression of DSC2(CS1) in the double knockout cells (DSG2(CS1)/(DSC2DSG2)) did not lead to the formation of any desmosome-like structures, although a slightly dense amorphous structure (arrows) was observed at cell—cell contact sites (Fig. 8, i and j). It was unclear whether this structure was related to desmosomes. On the other hand, PKP2 and PKP3 double knockout cells (DSC2/(DSC2DSG2 PKP2PKP3)) did not show the formation of desmosomes or desmosome-like structure (Fig. 8, k and l). These results were mostly consistent with those of immunofluorescence staining and indicated that desmosomes or desmosome-like structures were made with DSC2 without DSG2 or vice versa. Moreover, the results also demonstrated that the CS1 region of DSC2 and PKP2 and PKP3 were required for the formation of the desmosome structure.

other genes, resulting in serious modification of the basic properties of the target cells. As far as we examined, however, the knockout cells retained their original epithelial properties; and, furthermore, re-expression of the knocked out gene by transfection with its cDNA rescued the cells from their defects. Recently, Incontro et al. (29) reported that knockout of synaptic protein genes did not change much the properties of neurons, which finding is consistent with our results. These results suggest that knockout of structural genes such as desmosomal cadherin genes may not affect much the properties of differentiated epithelial cells. If this assumption is held in other cases, the present approach should be a useful tool to characterize the functions of various proteins in differentiated cells in culture, although the CRISPR/Cas9 system is now mainly used for research using undifferentiated stem cells and eggs (30). Similar approach is possible by using knockdown cells, but it is very difficult to perform, especially for complex systems. The role of two types of desmosomal cadherins, DSCs and DSGs, is an interesting question in desmosome research. The question is, however, hard to answer. This study partly answered this question: DSC2 alone in the absence of DSG2 clearly formed desmosome-like structures. Moreover, the structure was resistant to solubilization by a detergent and had significant cell adhesion activity: DSC2 was already reported to have weak cell adhesion activity in L cells (31). On the other hand, Koeser et al. (32) reported that DSG2 also formed desmosome plaques. Burdett et al. (33) and Nekrasova et al. (34) obtained data indicating that DSCs might play an important role in the initial step of desmosome assembly. Indeed, Lowndes et al. (35) recently suggested that DSC2 alone formed desmosome plaques by an in vitro study. Taken together, the present data indicate that a desmosome or desmosome-like structure can be formed with only one type of desmosomal cadherin DSC2 and is functional at least in part and that this structure may be involved in the first step of desmosome plaque formation. More studies are needed to clarify the role of DSCs and DSGs. The importance of the CS1 region of DSCs in desmosome function has been implicated. This study using deletion experiments of the DSC2 CS1 region provided evidence that the region was involved in desmosome assembly. This conclusion was further supported by amino acid substitution of the well conserved amino acids in the CS1 region and swapping of this region with the corresponding region of E-cadherin (Fig. 6). Franke’s group also came to a similar conclusion by using chimeric proteins made of DSC1 and connexin-32 (18). Hence, the CS1 region of DSCs seems to play a vital role in desmosome assembly. For further elucidation of the mechanism of function of the CS1 region, identification of the proteins that interact with the region is essential. Kanno et al. reported that p120-catenin bound to the corresponding region of Dsg3 (27). However, we could not obtain any evidence for the binding of p120-catenin to DSC2 despite the fact that the juxtamembrane region of DSC2 is

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Fig. 8 Examination of desmosome formation in various DLD-1 cells by Electron microscopy. Wild type DLD-1 cells made typical desmosomes (arrowheads) (a and b). Single knockout DLD-1 cells for DSG2 formed desmosome-like plaques (arrowheads) (c and d), whereas double knockout DLD-1 cells for DSC2 and DSG2 failed to form desmosomes (e and f). Re-expression of DSC2 in the double knockout cells, however, reconstructed the desmosome-like structure (arrowheads) (g and h); whereas re-expression of the deletion mutant DSC2(CS1) did not result in the formation of desmosome-like structures, although a slightly dense structure was observed at intercellular contact sites (arrows) (i and j). Double knockout DLD-1 cells for PKP2 and PKP3 did not make desmosomes (k and l). DSC2DSG2, DSC2 and DSG2 double knockout DLD-1 cells; PKP2PKP3, PKP2 and PKP3 double knockout cells derived from DSC2 and DSG2 double knockout DLD-1 cells. Photographs at the right are enlargements of those at the left. Scale bars: 0.5 mm (a, c, e, g, I and k); 0.1 mm (b, d, f, h, j and l).

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Role of CS1 and plakophilins

integrated into the desmosomal plaques, thereby playing a vital role in desmosome function (2, 42). The present results, however, revealed a new property of DSP: The double-knockout cells for desmosomal cadherins showed weak plaque-like accumulation of DSP without desmosomes and the number was low. We do not have good explanation for the results: DSP may interact with the other structure. Our result indicated that the plaque-like localization of DSP does not necessarily mean its association with desmosomes, hence formation of desmosomes. Taken together, the role of DSP in desmosome assembly needs to be reexamined. The present results clarified some of the problems in desmosome assembly such as the role of CS1 and PKPs, but a part of our present results contradicts or does not fit well with current views of desmosomes. One possible explanation could be that properties of desmosomes vary depending on their composition and the cells that express them. Naturally, more studies are essential to clarify this problem, but this study should provide a basis for such future studies.

Supplementary Data Supplementary Data are available at JB Online.

Acknowledgement We give thanks to Dr K. Ohbo, Dr. M. Ono and Dr. K. Yoshida (Yokohama City University School of Medicine) for their helpful discussion on electron Microscopy. Funding This study was supported in part by grants-in-aid from Ministry of Education and Culture (KAKENHI 24240062 to S.O) and from Kwansei Gakuin University (to S.T.S.). Conflict of Interest None declared.

References 1. Delva, E., Tucker, D.K., and Kowalczyk, A.P. (2009) The desmosome. Cold Spring Harb. Perspect. Biol. 1, a002543 2. Nekrasova, O.E., Amargo, E.V., Smith, W.O., Chen, J., Kreitzer, G.E., and Green, K.J. (2011) Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes. J. Cell Biol. 195, 1185—1203 3. Thomason, H.A, Scothern, A., McHarg, S., and Garrod, D.R. (2010) Desmosomes: adhesive strength and signalling in health and disease. Biochem. J. 429, 419—433 4. Bass-Zubek, A.E., Hobbs, R.P., Amargo, E.V., Garcia, N.J., Hsieh, S.N., Chen, X., Wahl, J.K. III, Denning, M.F., and Green, K.J. (2008) Plakophilin 2: a critical scaffold for PKC alpha that regulates intercellular junction assembly. J. Cell Biol. 181, 605—613 5. Chen, X., Bonne´, S., Hatzfeld, M., van Roy, F., and Green, K.J. (2002) Protein binding and functional characterization of plakophilin 2. Evidence for its diverse roles in desmosomes and beta-catenin signaling. J. Biol. Chem. 277, 10512—10522 6. Bonne´, S., Gilbert, B., Hatzfeld, M., Chen, X., Green, K.J., and van Roy, F. (2003) Defining desmosomal plakophilin-3 interactions. J. Cell Biol. 161, 403—416 13

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more homologous to that of classical cadherins than is that region of DSGs. Instead, we obtained evidence that PKP2 and PKP3 bound to the CS1 region of DSC2: Immunostaining showed partial co-localization of PKPs and DSC2. Immunoprecipitation and pulldown assay also supported the binding (Fig. 6), although the results were slightly inconclusive. Furthermore, the knockout cells of PKP2 and PKP3 revealed a phenotype of desmosomal cadherin localization similar to that of the DSC2(CS1) mutant. These results suggest that PKP2 and PKP3 bound to the CS1 region and functioned in desmosome assembly. Bonne´ et al. (6) provided some evidence that the interaction between PKP3 and the juxtamembrane regions of DSGs and DSC3 but not DSC2, although they did not clarify its function. It is unclear, however, how the interaction between DSC2 and PKP2 or PKP3 works in desmosome assembly. The interaction appears to be weak, implying that it has some regulatory role, but not a structural one. Indeed, various roles of PKPs have been reported (6, 36—39). Recently, Todorovic´ et al. (39) reported that PKP3 is involved in desmosome formation as well as in construction of the adherens junction through activation of Rap1 and that this function is different from that of PKP2. However, our results were slightly different: The functions of PKP2 and PKP3 appeared to overlap, as the function of PKP2 could be compensated by PKP3 and vice versa: single knockout of PKP2 or PKP3 had only very mild effects on desmosome assembly and localization; whereas the double knockout severely affected them (Fig. 7). Furthermore, activation of adenyl cyclase with forskolin, which should activate Rap1, did not rescue the weak accumulation of DSP (data not shown), in contrast to the results of Todorovic´ et al. (39). The reason for this discrepancy between the two studies is unclear. The functions of PKP2 and PKP3 may vary depending on the conditions, because PKP2 and PKP3 are thought to be involved in multiple processes (4, 40), as was reported for the closely related protein p120-catenin (41). Indeed, the localization of PKP2 and PKP3 was similar to but not identical with that of DSC2 in the DSC2 transfecntants of the double knockout cells (Fig. 7D). These results are consistent with the multiple roles of PKPs. Nevertheless, it is noteworthy that the PKP double knockout resulted in poor localization of DSP, despite the fact that the expression level of DSP did not decrease much (Fig. 7B). Furthermore, DSC2 showed abnormal linear localization in the double knockout cells (Fig. 7D). On the other hand, DSC2(CS1), which could not interact with PKPs, did not form desmosomal plaques (Figs 4 and 6) and PKP2 and PKP3 did not show any dotted localization at intercellular contact sites of the DSC2(CS1) transfectants (data not shown). These results indicate that the CS1 region and PKPs are likely to be involved in the proper transport and/or accumulation of desmosomal proteins including DSC2 and DSP, thereby resulting in desmosome formation. DSP has been broadly used as a marker of desmosomes, because it has been thought to be intimately

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Desmocollin-2 alone forms functional desmosomal plaques, with the plaque formation requiring the juxtamembrane region and plakophilins.

The role of the juxtamembrane region of the desmocollin-2 cytoplasmic domain in desmosome formation was investigated by using gene knockout and recons...
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