Original Article

Binding of Integrin 6ß4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding

Correspondence to: Arnoud Sonnenberg, Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel:(31) 20-5121942 Fax:(31) 20-5121944 E-mail:asonn{at}nki.nl.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the 6ß4 integrin and have shown that the cytoplasmic domain of the ß4 subunit associates with an NH₂-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with 6ß4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that ß4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the ß4 cytoplasmic domain. Mapping of the ß4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that ß4 can compete out the plectin ABD fragment from its association with F-actin. The ability of ß4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament–anchoring hemidesmosomes when ß4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

Key Words: actin, epidermolysis bullosa, hemidesmosome, 6ß4 integrin, plectin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

HEMIDESMOSOMES are junctional protein complexes that stably bind basal epithelial cells to the basement membrane in (pseudo)stratified and some complex epithelia. They are essential for a tight link between the intracellular intermediate filament system and proteins of the extracellular matrix. Hemidesmosomes consist of at least four distinct proteins. Two of these, the integrin 6ß4 and the bullous pemphigoid antigen 180 (BP180)¹, are transmembrane proteins that are involved in anchoring the basal epithelial cells to the underlying basement membrane. The hemidesmosomal plaque proteins, BP230 and plectin, are localized in the cytoplasm, and play a major role in the binding of intermediate filaments to hemidesmosomes (Borradori and Sonnenberg 1996 ; Green and Jones 1996 ; Burgeson and Christiano 1997 ).

The 6ß4 integrin binds to several laminins, particularly the major basement membrane component laminin-5 (Niessen et al. 1994 ). It is prominently expressed in stratified and complex epithelia, in which it is present only in hemidesmosomes (Kajiji et al. 1989 ; Stepp et al. 1990 ; Sonnenberg et al. 1991 ). The 6ß4 integrin has a central role in the assembly and maintenance of hemidesmosomes, as demonstrated by the severe nature of the disease pyloric atresia associated with junctional epidermolysis bullosa (EB) (PA-JEB), which is caused by the absence of 6 or ß4. PA-JEB patients suffer from extensive blistering of the skin due to the absence of functional hemidesmosomes (Vidal et al. 1995 ; Ruzzi et al. 1997 ). A similar skin phenotype occurs in mice in which the gene encoding 6 or ß4 had been disrupted by homologous recombination (Dowling et al. 1996 ; Georges-Labouesse et al. 1996 ; van der Neut et al. 1996 ).

Essential for the role of 6ß4 in hemidesmosome assembly is the unique cytoplasmic domain of the ß4 subunit. It is over 1,000 amino acid residues in length, and contains two pairs of fibronectin type III (FNIII) repeats separated by a connecting segment (CS) (Hogervorst et al. 1990 ; Suzuki and Naitoh 1990 ). Specifically, sequences within the first and second FNIII repeats and in the CS are important for the formation of a complex with plectin and its recruitment into hemidesmosomes at the basal surface of the cell (Spinardi et al. 1993 ; Niessen et al. 1997a ; Nievers et al. 1998 ). Fusion proteins containing the first two FNIII repeats and part of the CS of ß4 form a complex with plectin in vitro (Niessen et al. 1997b ), and recombinant ß4 protein fragments containing these sequences were found to bind plectin in in vitro assays (Rezniczek et al. 1998 ). The CS and third FNIII repeat are essential for association with the BP180 protein (Borradori et al. 1997 ; Aho and Uitto 1998 ; Schaapveld et al. 1998 ). Therefore, the 6ß4 integrin clearly has a structural role, but it is also involved in the transduction of cell adhesion–modulating signals (for reviews see Giancotti 1996 ; Borradori and Sonnenberg 1999 ; Nievers et al. 1999 ).

The BP180 protein (BPAG2), a type II transmembrane protein, is only expressed in epithelial tissues (Nishizawa et al. 1993 ; Aho and Uitto 1999 ), and is probably also a receptor for laminin-5 (Reddy et al. 1998 ). BP180 is recruited into hemidesmosomes by direct binding to the ß4 cytoplasmic domain (Borradori et al. 1997 , Borradori et al. 1998 ; Schaapveld et al. 1998 ; see also Hopkinson et al. 1995 , Hopkinson et al. 1998 ), and there is data suggesting that it is also involved in the recruitment of BP230 to hemidesmosomes (Borradori et al. 1998 ).

The two hemidesmosomal plaque proteins, BP230 and plectin, belong to the plakin family, which is widely expressed cytoskeleton-associated proteins (Ruhrberg and Watt 1997 ). BP230 (BPAG1) is predominantly expressed in stratified and some complex epithelia. The COOH terminus of BP230 was found to decorate intermediate filament networks in vivo and to bind intermediate filament proteins in vitro (Yang et al. 1996 ; Leung et al. 1999 ), but the function of its NH₂ terminus remains unknown. Variant BPAG1 proteins (dystonins) exist that are only expressed in neuronal tissue. These contain an NH₂-terminal actin-binding domain (ABD) as a result of alternative mRNA splicing (Brown et al. 1995 ; Guo et al. 1995 ; Yang et al. 1996 ).

The large plectin protein (~500 kD) is the most widely expressed member of the plakin family (for review see Wiche 1998 ). It belongs to the spectrin superfamily of actin-binding proteins (like dystonin) that share a conserved ABD, itself a tandem duplication of the calponin homology domain (Hartwig 1994 ; Van Troys et al. 1999 ). Three conserved actin-binding sequences (ABS 1–3) have been identified within the ABD (Hemmings et al. 1992 ; Fabbrizio et al. 1993 ; Corrado et al. 1994 ). The PLEC1 gene contains alternative first coding exons, and the resulting plectin splice variants show a characteristic tissue expression. The sequence encoding the ABD is present in all plectin mRNAs (Liu et al. 1996 ; McLean et al. 1996 ; Elliott et al. 1997 ). Indeed, plectin is found in association with actin stress fibers and focal contacts (Seifert et al. 1992 ; Sanchez-Aparicio et al. 1997 ). Recently, it was shown that NH₂-terminal plectin fragments containing the ABD associate with F-actin stress fibers in vivo in transfected plectin-deficient fibroblasts and bind monomeric (G-)actin in vitro. Furthermore, plectin might be involved in the regulation of actin filament dynamics via phosphatidylinositol 4,5-bisphosphate (PIP2) (Andra et al. 1998 ). The COOH-terminal part of plectin contains six tandem repeat domains (R1–R6) that bind to, or can codistribute with, several types of intermediate filament proteins; epidermal keratins, vimentin, desmin and GFAP (glial fibrillary acidic protein), the neurofilament triplet proteins, and lamin B (for review see Wiche 1998 ). The intermediate filament binding site (IFBD) on plectin was mapped to a region of ~50 amino acids at the end of the fifth repeat, R5 (Nikolic et al. 1996 ). In addition to actin and intermediate filaments, there is evidence for an interaction of plectin with microtubules (Herrmann and Wiche 1987 ; Svitkina et al. 1996 ), suggesting that it can interlink all three major protein cytoskeletal systems (Wiche 1998 ). The functional importance of plectin is demonstrated by the fact that mutations in the PLEC1 gene cause muscular dystrophy associated with EB simplex (MD-EBS), a disease characterized by skin blistering and skeletal muscle myopathies (Gache et al. 1996 ; McLean et al. 1996 ; Smith et al. 1996 ). A similar phenotype was seen in null-mutant mice in which the PLEC1 gene had been disrupted (Andra et al. 1997 ). There is ample evidence for an association of plectin with the integrin ß4 subunit within the hemidesmosome (see references above), and there are some indications that plectin can associate with BP180 and thereby influence BP180 localization (Gache et al. 1996 ; Aho and Uitto 1997 ; Schaapveld et al. 1998 ).

In this study, we have investigated the role of plectin in the formation of hemidesmosomes. Firstly, we wished to understand why F-actin has not been found in hemidesmosomes, even though plectin contains a spectrin-like ABD, and why ß4, which can form a complex with plectin, is never found in association with the actin cytoskeleton. Secondly, we endeavored to show that the recruitment of plectin into hemidesmosomes by ß4 is the result of a direct interaction between plectin and the ß4 cytoplasmic domain, and if this could be established, the location of the binding sites on these proteins could be determined. Our third aim was to investigate the relation between the binding of plectin to ß4 and intermediate filaments. The recruitment of plectin into hemidesmosomes, and the function of its NH₂-terminal ABD and COOH-terminal IFBD sequences was, for the first time, studied in the absence of endogenous plectin in MD-EBS keratinocytes. We demonstrate that plectin molecules associate in a mutually exclusive manner with either actin filaments or ß4 in hemidesmosomes as a result of competitive binding. In addition, we show which intermediate filament proteins are capable of directly binding to the plectin-IFBD and thereby present further evidence for the versatility of plectin as a cytoskeletal linker protein.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Generation of Immortalized MD-EBS Keratinocytes
Skin punch biopsies (4 mm in diameter) were removed from the trunk of two unrelated patients diagnosed with MD-EBS and transported to the laboratory in transport medium (DMEM containing the following antibiotics: 0.01% (wt/vol) streptomycin, 0.01% (wt/vol) penicillin, and 2.5 U/ml fungisone). The tissue was cut into several pieces and incubated in 0.5% (wt/vol) trypsin, 0.1% (wt/vol) EDTA for 90 min at 37°C to separate the epidermis from the dermis. The epidermal sheets were reincubated in fresh trypsin/EDTA at 37°C for 5 min, pipetted vigorously to encourage basal cell detachment, after which trypsinization was arrested by the addition of DMEM containing 10% FCS, and the resulting cell suspensions were centrifuged. The keratinocyte suspension was plated into two T25 flasks (Greiner) together with 5 x 10⁵ irradiated 3T3 feeder cells and keratinocyte medium (Rheinwald and Green 1975 ) without EGF. Cells were checked and medium (keratinocyte medium with EGF) changed twice weekly. At 80% confluence, cells were trypsinized, split at 1:5, and replated with fresh 3T3 feeder cells.

At passage two, cells were plated in T25 flasks with irradiated 3T3 feeder cells and allowed to grow to 40–50% confluence. The medium was changed to keratinocyte growth medium, a low calcium medium (0.15 mM Ca²⁺), with 10 ng/ml EGF, 5 µg/ml insulin, 0.5 U/ml hydrocortisone, and 0.4% (vol/vol) bovine pituitary extract (Clonetics Corp.) for 48 h, thus allowing cells to spread out as a monolayer. The human papillomavirus 16 expression plasmid pJ416 (Storey et al. 1988 ) used for the immortalization was a gift from Dr. A. Storey (Molecular Virology Laboratory, ICRF, London, United Kingdom). For transfection, keratinocytes were washed once with OPTI-MEM (GIBCO BRL) and 4 ml OPTI-MEM was added to each flask and incubated at 37°C for 4 h. For each T25 flask, the following solutions were prepared and incubated for 30 min at room temperature before mixing: solution A, 10 µg DNA (human papillomavirus 16 plasmid) in 0.5 ml OPTI-MEM; and solution B, 30 µl Lipofectin (18292-011; GIBCO BRL) in 1.5 ml OPTI-MEM. The two solutions were combined, mixed gently, and incubated for a further 10–15 min. All medium was removed from the flasks and 2 ml of transfection mixture was added. The cells were incubated overnight at 37°C, and the next day 4 ml keratinocyte medium and fresh irradiated 3T3 feeder cells were added. Medium was changed twice a week, and cultures were split at 80% confluence.

After transfection, keratinocytes grew for one or two passages and then entered a growth crisis for a period of up to 12 wk. After this crisis, colonies growing from single cells were cloned using cloning rings (C-1059; Sigma Chemical Co.), and then passaged using the same medium. Two MD-EBS cell lines (PEB-1 and 2) were used in this study.

Cell Lines and Antisera
Immortalized normal human keratinocytes (NHK) from foreskin and the immortalized ß4-deficient PA-JEB keratinocyte cell line were described previously (Steenbergen et al. 1996 ; Schaapveld et al. 1998 ). Immortalized NHK, PA-JEB, and MD-EBS keratinocytes were grown in keratinocyte serum-free medium (GIBCO BRL) supplemented with 50 µg/ml bovine pituitary extract, 5 ng/ml EGF, 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were grown at 37°C in a humidified, 5% CO₂ atmosphere.

The MD-EBS and PA-JEB keratinocytes were transiently transfected with cDNA constructs using Lipofectin (GIBCO BRL) according to the manufacturer's procedure.

Rabbit polyclonal antisera against 6 and ß4 have been described previously (Hogervorst et al. 1993 ; Niessen et al. 1994 ). Mouse mAb 58XB4 against ß4 was prepared by Dr. A.M. Martínez de Velasco and Mr. D. Kramer in our laboratory. Mouse mAbs 450-11A (Kennel et al. 1990 ) and 4.3E1 (Hessle et al. 1984 ) against ß4 were kind gifts of Dr. S.J. Kennel (Oak Ridge National Laboratory, Oak Ridge, TN) and Dr. E. Engvall (The Burnham Institute, La Jolla, CA), respectively. Mouse mAb 233 (Nishizawa et al. 1993 ) against BP180 and mouse mAb 121 (Hieda et al. 1992 ) against plectin/HD1 were kind gifts of Dr. K. Owaribe (University of Nagoya, Nagoya, Japan). Rabbit polyclonal antiserum against BP180 was kindly provided by Dr. L. Bruckner-Tuderman (University of Münster, Münster, Germany). The rabbit polyclonal antiserum recognizing BP230 (Tanaka et al. 1990 ) was a kind gift from Dr. J.R. Stanley (University of Pennsylvania, Philadelphia, PA). Polyclonal antibodies against plectin were generated by immunizing rabbits with a glutathinone S-transferase (GST) fusion protein containing the NH₂-terminal domain of plectin (residues 1–339). The antibodies were purified by affinity chromatography on a plectin (1–339)/maltose-binding protein (MBP) Sepharose column. Mouse mAb 6F12, also known as BM-140 (Marinkovich et al. 1992 ), against laminin-5, was a kind gift from Dr. R.E. Burgeson (Cutaneous Biology Research Center, Charlestown, MA). Mouse mAb 7A8 against plectin was purchased from Sigma Chemical Co. Mouse mAbs V9 against vimentin and KL1, recognizing a broad spectrum of keratins, were purchased from Coulter/Immunotech. Mouse mAb SPK-14 against keratin 14 was a kind gift from Dr. D. Ivanyi (Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands). Mouse mAb 12C05 and rabbit polyclonal antiserum HA-11 against the hemagglutinin (HA) epitope (YPYDVPDYA) were purchased from Santa Cruz Biotechnology. Sheep anti–mouse and donkey anti–rabbit HRP-coupled secondary antisera, and donkey anti–rabbit Texas Red conjugated antisera were purchased from Amersham Pharmacia Biotech, and FITC-conjugated goat anti–mouse antiserum from Rockland.

Western Blotting
MD-EBS keratinocytes were plated in 6-well tissue culture plates and lysed in 80 µl sample buffer. Lysates were boiled for 5 min at 95°C, and 40 µl samples analyzed by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore) and blots were blocked in 2% (wt/vol) baby milk powder in TBST (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.01% [vol/vol] Tween 20) for 1 h at 37°C. Subsequently, blots were incubated with primary antisera in 0.2% (wt/vol) baby milk powder in TBST for 1 h at room temperature. After extensive washing, blots were incubated for 1 h at room temperature with secondary HRP-coupled antisera in the same buffer as used for the primary antiserum incubation. Proteins were detected using the chemiluminescence procedure (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Immunofluorescence
MD-EBS and PA-JEB keratinocytes were switched to HAMF12/DMEM (1:3) containing 10% (vol/vol) FCS, 2 mM L-glutamine, 0.4 µg/ml hydrocortisone (Sigma Chemical Co.), and 1 µM isoproterenol (Sigma Chemical Co.) 24 h before the immunolabeling procedure was started. Keratinocytes grown on glass coverslips were washed twice with PBS (pH 7.2), and fixed for 10 min at room temperature in freshly prepared 1% (wt/vol) paraformaldehyde in PBS. Fixed cells were washed twice with PBS and permeabilized in 0.5% (vol/vol) Triton X-100 in PBS for 5 min at room temperature. Cells were rinsed and incubated in 2% (wt/vol) BSA in PBS for 30 min at room temperature, washed with PBS, and incubated with primary antisera or TRITC-conjugated phalloidin in PBS containing 2% BSA for 30 min at 37°C. After washing with PBS, cells were incubated with the secondary antiserum, goat anti–mouse–FITC or donkey anti–rabbit–Texas red diluted 1:100 in PBS containing 2% BSA. After rinsing in PBS, the preparations were mounted in Vectashield (Vector Laboratories Inc.) and viewed under a BioRad MRC-600 confocal laser scanning microscope.

cDNA Constructs
All nucleotide and amino acid positions are numbered with the ATG initiation codon at position one. Plasmid inserts were generated by PCR using the proofreading Pwo DNA polymerase (Boehringer Mannheim) and gene-specific sense and antisense primers containing restriction site tags. All plasmid inserts were confirmed by sequence analysis using the ^T7Sequencing kit (Amersham Pharmacia Biotech).

Plectin-ABD, a clone containing alternative exon 1c, exons 2–8, and almost the complete exon 9 of human epithelial plectin cDNA (position 1–1018), and plectin-IFBD (see below) were inserted into pcDNA3HA (a kind gift from Dr. E. Sander, Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands), a derivative of the eukaryotic expression vector pcDNA3 (Invitrogen Corp.) that contains an extra sequence 5' of the multiple cloning site encoding the HA tag.

The yeast galactose metabolism regulatory gene 4 (GAL4) plasmids containing human ß4 (sequence data available from EMBL/GenBank/DDBJ under accession nos. X51841 and X52186), human epithelial plectin (U53204), and mouse plectin (rat accession number X59601) cDNA subclones and full-length cDNA encoding human skeletal muscle actin (J00068), human ß cytoplasmic actin (AB004047), human cytoplasmic actin (M19283), human keratin 5 (M28496), human keratin 8 (M34225), human keratin 14 (J00124), human keratin 18 (M26326), human GFAP (S40719), and mouse vimentin (M26251) are described in Figure 5, Figure 7, and Figure 9. Numbers in superscript correspond to the amino acid residues of subclones encoded within the GAL4 activation domain (AD) or binding domain (BD) fusion proteins. Vectors used were the yeast GAL4 AD or BD expression vectors pACT2 or pAS2-1 (Clontech). The template DNAs used for PCR were full-length cDNAs encoding human ß4, human skeletal muscle actin, mouse vimentin, human plectin (kind gifts from Dr. T. Magin, Institut für Genetik, University of Bonn, Bonn, Germany), human keratins 5, 8, 14, and 18, and cDNA subclones containing bps 1–1018, 2377–3156, and 13054–13725 of human epithelial plectin. Other templates used were EST clones obtained from the IMAGE cDNA clone collection at the Resource Center/Primary Database of the German Human Genome Project (RZPD, Berlin, Germany): 975524 and 1064756 for mouse and 52195 for human plectin 3' clones; 361338, 364065, and 381924 for human GFAP; 611141 and 613287 for human ß cytoplasmic actin; and 41909 and 362511 for human cytoplasmic actin.

View larger version (45K): [in this window] [in a new window]   Figure 1. Expression of hemidesmosomal proteins in immortalized MD-EBS keratinocytes. Western blot of lysates of normal (NHK; lanes 1) and plectin-deficient (MD-EBS; lanes 2) human keratinocytes, probed with mAb 450-11A against integrin ß4 (200 kD), mAb 7A8 against plectin (>500 kD), polyclonal antiserum against BP230 (230 kD), or polyclonal antiserum against BP180 (180 kD). All hemidesmosome proteins tested, except plectin, were present in MD-EBS keratinocytes.

View larger version (63K): [in this window] [in a new window]   Figure 2. Localization of hemidesmosome proteins in immortalized MD-EBS keratinocytes. Cells were fixed, immunolabeled, and subjected to confocal immunofluorescence microscopy using polyclonal antiserum against 6 (A) or ß4 (B), mAb 233 against BP180 (C), mAb 6F12 against laminin-5 (D), mAb 121 against plectin/HD1 (E), or polyclonal antiserum against BP230 (F). Results obtained for cell lines from two different, unrelated human patients were similar; shown here are the results for one patient. All hemidesmosome proteins tested, as well as the extracellular matrix protein laminin-5 (a ligand for the integrin 6ß4), are concentrated at sites of cell–substrate contact in patches characteristic for hemidesmosome-like clusters, except plectin, which is not expressed. Sections were focused at the cell–substrate interface. Bar, 10 µm.

View larger version (148K): [in this window] [in a new window]   Figure 3. Subcellular distribution of HA-tagged plectin-ABD fragment in transfected MD-EBS keratinocytes. 48 h after transfection, cells were fixed, double-immunolabeled using mAb 12C05 against the HA epitope (green; A, D, and G) and polyclonal antiserum against ß4 (red; H), and processed for immunofluorescence. TRITC-labeled phalloidin was used to stain F-actin (red; B and E). Composite images (C, F, and I) show the superimposition of the green and red signals, with areas of overlap appearing yellow. Cells were visualized by confocal microscopy. The plectin protein fragment is colocalized with actin stress fibers, or with 6ß4-containing hemidesmosome-like basal clusters. Sections were focused at the cell–substrate interface. Bar, 10 µm.

View larger version (114K): [in this window] [in a new window]   Figure 4. Distribution of plectin compared with F-actin in PA-JEB cells. PA-JEB keratinocytes and PA-JEB cells stably expressing the integrin 6ß4 were fixed, double-immunolabeled using mAb 121 against plectin (green; A, D, and G) or an affinity-purified polyclonal antiserum against the plectin-ABD (green; H and J) and F-actin (red; B, E, and K), and processed for immunofluorescence. Composite images (C, F, I, and L) were generated by superimposition of the green and red signals, with areas of overlap appearing yellow. Cells were visualized by confocal microscopy. In PA-JEB cells (A–C), the staining patterns of plectin and F-actin partly overlapped, whereas in the 6ß4-positive cells (D–F) plectin was exclusively localized in hemidesmosome-like basal clusters that did not stain for F-actin. These sections (A–F) were taken at the optical plane corresponding to the cell–substrate interface. In another plane of focus (G–I), it is shown that the two anti-plectin antibodies produce a similar pattern of staining of the cytoskeleton in PA-JEB keratinocytes. (J–L) Higher magnification shows plectin associated with F-actin filaments in PA-JEB keratinocytes (arrows). Bars: 10 µm (A, D, and G); and 5 µm (J).

View larger version (30K): [in this window] [in a new window]   Figure 5. Mapping of the binding site between the NH2-terminal part of plectin and the cytoplasmic domain of ß4 and of the internal folding of the ß4 cytoplasmic domain by yeast two-hybrid analysis. (A) Cotransformation of yeast host strain PJ69-4A with pAS2-plectin1–339 and one each of the listed pACT2-ß4 constructs or empty pACT2 vector to determine the plectin-ß4 interaction site. (B) Cotransformation with pAS2-plectin1–339 or pAS2-ß41457–1752, and pACT2-ß41115–1457 (wild-type) or -ß41115–1457* (R1281W) to characterize plectin-ß4 binding and the internal folding of the ß4 cytoplasmic domain. Transformation mixtures were spread on SC-LT and SC-LTHA plates and grown for 12 d at 30°C. Plating efficiency on selective SC-LTHA plates is expressed as a percentage of plating efficiency on nonselective SC-LT plates of the same transformation. ++, >50%; +, >50% (slow-growing colonies); ±, 5–25%; -, 0%. Plates were scored after 6 and 12 d of growth. Slow-growing colonies could only be scored after 12 d of growth. Plating efficiencies of <25% always represented slow-growing colonies. All efficiencies listed represent an average of multiple independent transformations on at least three separate occasions.

View larger version (43K): [in this window] [in a new window]   Figure 6. Localization of hemidesmosomal components in PA-JEB keratinocytes transiently transfected with cDNAs encoding full-length wild-type ß4 or ß4R1281W. Cells were fixed, double-immunolabeled using polyclonal antiserum against 6 (red; A–F) and mAb 121 against plectin/HD1 (green; A and D), mAb 233 against BP180 (green; B and E), or polyclonal antiserum against BP230 (green; C and F), and processed for immunofluorescence. Cells were visualized by confocal microscopy. Expression of ß4 results in the formation of hemidesmosome-like structures at sites of cell–substrate contact, in which 6ß4 is concentrated, as shown by staining for 6. Similar results were obtained when instead of the anti-6 antiserum, a polyclonal antiserum against ß4 was used. The wild-type integrin 6ß4 (A–C) is colocalized with plectin, BP180, and BP230. The mutant integrin 6ß4R1281W is not colocalized with plectin (D), but is colocalized with BP180 (E) and BP230 (F). Sections were focused at the cell–substrate interface. Bar, 10 µm.

View larger version (36K): [in this window] [in a new window]   Figure 7. Mapping of the interaction site for ß4 and for actin on the NH2 terminus of plectin by yeast two-hybrid analysis. (A) Cotransformation of yeast host strain PJ69-4A with one each of the pAS2-plectin subclones listed together with pACT2-ß41115–1457 to determine the interaction site for ß4 on plectin (A) and with one each of the pAS2-plectin ABD subclones listed together with pACT2-ß41115–1457 or actin for a comparison of the ß4- and actin-binding sites on plectin (B). The results shown for plectin36–302, plectin36–236, plectin36–181, and plectin36–142 are identical for those obtained with two sets of four similar subclones starting at residues 1 or 5, respectively. The results shown are for ß cytoplasmic actin. No binding was found between pACT2-ß41115–1457 and pAS2-actin (data not shown). Details are as for Figure 5.

View larger version (50K): [in this window] [in a new window]   Figure 8. Dot blot overlay and actin sedimentation assay of recombinant purified ß4 protein fragments with radiolabeled plectin-ABD fragment. (A) SDS-PAGE of 4 ng in vitro translated [35S]-methionine/cysteine-labeled HA-tagged plectin-ABD protein (40 kD). Autoradiogram. (B) Recombinant purified GST fusion proteins encoding ß41115–1328, ß41115–1382 (wild-type sequence), ß41115–1382* (R1281W), ß41457–1752 (3 µg each), or GST alone (2 µg), and purified F-actin (1, 2, or 3 µg) were immobilized on nitrocellulose by dot blotting and incubated with 100 ng [35S]methionine/cysteine-labeled plectin-ABD in 3 ml APB or AGB (upper and middle lanes, respectively). Protein loading was verified by amido-black staining (bottom lanes). The GST protein alone does not bind to plectin-ABD. (C) Actin filaments polymerized in the presence of and complexed with MBP-plectin ABD fusion protein were incubated with a 10-fold molar excess of GST fusion proteins encoding ß41115–1382 (lanes 1 and 2), ß41115–1382* (lanes 3 and 4), ß41115–1328 (lanes 5 and 6), or no GST fusion protein (lanes 7 and 8). The F-actin complexes were then isolated by centrifugation, after which supernatant (S; odd-numbered lanes) and pellet (P; even-numbered lanes) were isolated and analyzed using SDS-PAGE. Proteins were visualized using Coomassie brilliant blue staining.

View larger version (20K): [in this window] [in a new window]   Figure 9. Mapping of the interaction site between the COOH terminus of plectin and the cytoplasmic domain of ß4 and of full-length intermediate filament proteins by yeast two-hybrid analysis. Yeast host strain PJ69-4A was cotransformed with pAS2-plectin and one each of the listed pACT2-IF or pACT2-ß4 subclone constructs, or with empty pACT2 vector to determine the interaction sites between plectin and ß4 or intermediate filament proteins. The plectin R5-R6 clone encodes amino acid residues 4068–4687 of mouse plectin (containing most of repeat R5, repeat R6, and the COOH tail), the plectin R6 clone encodes residues 4351–4574 of human plectin (most of repeat R6 and the COOH tail). Similar results as for the human plectin R6 clone were obtained with a mouse plectin clone encoding residues 4206–4687 (part of repeat R5, repeat R6, and the COOH tail; data not shown). Details are as for Figure 5.

The full-length ß4 cDNA construct in the eukaryotic expression vector pRc/CMV (Invitrogen Corp.) has been described previously (Niessen et al. 1997a , Niessen et al. 1997b ). The CGG to TGG mutation in codon 1281 of ß4, a human patient mutation that results in an R to W amino acid substitution at position 1281 (Pulkkinen et al. 1998 ), was introduced into human ß4 cDNA by sequence overhang extension PCR. The mutagenesis primers 5'-GACAACCCTAAGAACTGGATGCTGCTTATTG-3' (sense) and 5'-CAATAAGCAGCATCCAGTTCTTAGGGTTGTC-3' (antisense), representing positions 3826–3856 of the human ß4 coding sequence, were used with normal human ß4 cDNA as a template. The resulting DNA fragments were first cloned into the pACT2 vector, verified by DNA sequencing, and used in yeast two-hybrid analysis (see Figure 5 B). For cell transfection experiments, the mutation was subsequently introduced into full-length ß4 cDNA in pRc/CMV by exchanging of the appropriate DNA restriction fragments (see Figure 6).

DNA fragments encoding different fragments of the ß4 cytoplasmic domain were isolated from pACT2-ß4 plasmids (described above) and cloned into the bacterial GST fusion protein expression vector pRP261, a derivative of the pGEX-3X vector (Amrad Corp. Ltd.) that contains a slightly modified multiple cloning site, for the production of recombinant GST fusion proteins (see Figure 8).

Plectin-ABD was isolated from pcDNA3HA-plectin ABD (described above) and inserted into the bacterial MBP fusion protein expression vector pMAL-c2X (New England Biolabs Inc.), for the production of MBP fusion proteins (see Figure 8).

The ß4 cDNA expression constructs used for the experiments in Figure 11 have been described previously (Niessen et al. 1997a , Niessen et al. 1997b ).

View larger version (128K): [in this window] [in a new window]   Figure 10. Subcellular distribution of HA-tagged plectin-IFBD fragment in MD-EBS keratinocytes after transient transfection. Cells were transiently transfected with an HA-tagged plectin R5-R6 cDNA construct. Cells were fixed, double-immunolabeled using polyclonal antiserum HA-11 against the HA tag (green; A, D, and G) and a mixture of mAbs 58XB4 and 4.3E1 against ß4 (red; B), a mixture of mAbs SPK-14 and KL1 against keratins (red; E), or mAb V9 against vimentin (red; H), and processed for immunofluorescence. Composite images (C, F, and I) were generated by superimposition of the green and red signals, with areas of overlap appearing yellow. Cells were visualized by confocal microscopy. The plectin-IFBD protein fragment is exclusively found in cytoplasmic filament structures (A, D, and G). 6ß4 is present in basally located hemidesmosome-like clusters (B), together with the plectin-IFBD (C). Keratins are observed in a dense cytoplasmic filament network (E), which is decorated by the plectin-IFBD (F). Expression of the IFBD-fragment of plectin causes a collapse of the vimentin filament network (H and I). Bar, 10 µm.

View larger version (83K): [in this window] [in a new window]   Figure 11. Subcellular distribution of wild-type and mutant ß4 and study of the effect of ß4 expression on the vimentin intermediate filament network in transfected PA-JEB keratinocytes. PA-JEB cells were transfected with cDNA encoding full-length ß4 (A–C), COOH-terminal deletion mutants ß41355 (D–F), and ß41328 (G–I). Cells were fixed, double-immunolabeled, and processed for confocal immunofluorescence microscopy using polyclonal antiserum against the 6 subunit (green; A, D, and G), or mAb V9 against vimentin (red; B, E, and H). Composite images (C, F, and I) were generated by superimposition of the green and red signals, with areas of overlap appearing yellow. Expression of wild-type full-length ß4 results in the formation of hemidesmosome-like structures, in which 6ß4 is concentrated at sites of cell–substrate contact, as is shown by staining for 6, and codistributes with the vimentin network, most probably dependent upon its binding to plectin (A–C). The ß41355 mutant protein, which efficiently binds to plectin, is found colocalized with the vimentin network (D–F). The ß41328 mutant protein, which can only bind plectin with low efficiency in vitro, was only very occasionally found to be colocalized with vimentin (G–I). Bar, 10 µm.

Yeast Two-Hybrid Assay
Yeast strain Saccharomyces cerevisiae PJ69-4A (a gift from Dr. P. James, Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI), which contains the genetic markers trp1-901, leu2-3, his3-200, gal4, gal80, LYS2::GAL1-HIS3, GAL2-ADE2 (James et al. 1996 ), was used as the host for the two-hybrid assay. It contains two tightly regulated reporter genes, His and Ade, which makes it suitable for the sensitive detection of protein interactions. The use of PJ69-4A was essentially as described in Schaapveld et al. 1998 . In brief, PJ69-4A cells were cotransformed with a pACT2(-derived) as well as a pAS2-1(-derived) plasmid, and aliquots of the same transformation mixture were spread on plates containing SC-LT medium, yeast synthetic complete medium (SC) lacking only the vector markers Leu (for pACT2 and derivatives) and Trp (for pAS2-1 and derivatives), as well as on SC-LTHA plates, lacking Leu, Trp, as well as the interaction markers His and Ade. Plates were scored after 6 and 12 d of growth, and the number of colonies on the SC-LTHA plate compared with that on the SC-LT plate. Positive and negative controls used were as in Schaapveld et al. 1998 . Cotransformation efficiencies (on nonselective SC-LT plates) for all plasmid combinations were always at least 10⁴ cfu/µg plasmid DNA, and the difference between the various plasmid combinations tested was never greater than twofold. Cotransformation of yeast PJ69-4A with an empty pAS2-1 and an empty pACT2 vector, with a derived pAS2 plasmid and an empty pACT2 vector, or with an empty pAS2 vector and a derived pACT2 plasmid never resulted in the growth of colonies on selective SC-LTHA plates, showing that none of the GAL4 fusion proteins encoded by the recombinant plasmids used could cause activation of the His and Ade reporter genes by themselves. For pACT2-ß4^1457–1752, –keratin 5, and –keratin 8 constructs, a slight autonomous activation of the reporter genes was found, but this could be repressed by the addition of 2 mM 3-amino-1,2,4-triazole (a His antagonist) (A8506; Sigma Chemical Co.) to the medium.

Purification of Recombinant Fusion Proteins
Escherichia coli strain BL21(DE3), genotype F^- ompT gal [dcm] [lon] hsdS_B carrying DE3 prophage with the T7 RNA polymerase gene (Novagen), was transformed with recombinant pRP261 plasmids. Colonies obtained were used to inoculate Luria-Bertani medium containing 100 µg/ml ampicillin, and cultures were grown overnight at 37°C and 250 rpm. Cultures were then diluted 1:20 in fresh medium, grown to an OD₆₀₀ of 0.7 at 30°C and 200 rpm, and induced by the addition of isopropyl ß-D-thiogalactopyranoside (IPTG) to 0.4 mM for an additional 3 h. Bacteria were harvested by centrifugation at 4,000 g, resuspended in PBS containing 1 mM EDTA and 1% (vol/vol) Triton X-100, and lysed by sonication. Lysates were cleared by centrifugation for 10 min at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione agarose beads (G 4510; Sigma Chemical Co.). Beads with affinity-bound proteins were washed three times with PBS containing 1% (vol/vol) Triton X-100, and equilibrated in 50 mM Tris-HCl (pH 8.0). Bound proteins were eluted in 50 mM Tris-HCl (pH 8.0), containing 10 mM reduced glutathione.

Recombinant MBP-plectin ABD fusion protein was expressed and purified as described above, except that amylose resin (800-21; New England Biolabs) was used for the affinity purification, and that equilibration and elution of the resin was in 20 mM Tris-HCl (pH 7.4), 1 mM ß-mercaptoethanol without or with 10 mM maltose, respectively. Buffers containing the eluted fusion proteins were exchanged by ultrafiltration using Centricon 10 filters (Amicon; Millipore).

Dot Blot Assay
Purified recombinant GST fusion proteins containing different fragments of the ß4 cytoplasmic domain (0.4 mg/ml) dissolved in actin polymerization buffer (APB) (10 mM Tris-HCl, 2 mM MgCl₂, 100 mM KCl, 0.5 mM ATP, 0.1 mM ß-mercaptoethanol, pH 7.5) or actin-G buffer (AGB) (10 mM Tris-HCl, 0.2 mM CaCl₂, 0.1 mM ß-mercaptoethanol, pH 7.5) were precleared by centrifugation at 100,000 g for 30 minutes at 4°C in a TLA 100.3 rotor (Beckman). Subsequently, protein concentration of the cleared lysates was determined using Bradford protein assay. Bovine skeletal muscle actin (A3653; Sigma Chemical Co.) in AGB was polymerized at 4.5 µM for 1 h at room temperature. Samples were diluted to 10–30 µg/ml in the appropriate buffer and spotted, 100 µl per well, on nitrocellulose membrane using a Hoeffer 96 wells dot blot system (Amersham Pharmacia Biotech). Membrane strips were subsequently incubated in blocking buffer (APB or AGB supplemented with 0.2% [wt/vol] heat-inactivated BSA, and 10 µg/ml each of the protease inhibitors aprotinin, leupeptin, and pepstatin). [³⁵S]methionine/cysteine-labeled plectin-ABD protein was obtained by coupled in vitro transcription/translation of pcDNA3HA-plectin ABD DNA using the TNT^® coupled reticulocyte lysate system (Promega). Nonincorporated radiolabeled amino acids were removed from the in vitro translation mixture using Centricon 10 filters. Purified translation mixture (25 µl) was diluted into 3 ml of blocking buffer and incubated for 3 h at room temperature with the nitrocellulose strips. The strips were then washed three times in blocking buffer, air dried, and exposed using Kodak X-Omat AR film.

Actin Cosedimentation Assay
Bovine skeletal muscle actin (5 µM in AGB) was allowed to polymerize in the presence of MBP-plectin ABD fusion protein (1 µM) by the addition of 0.1 vol of 10x initiation mix (20 mM MgCl₂, 1 M KCl, 5 mM ATP) for 1 h at room temperature. Polymerized actin filaments complexed with plectin were pelleted by centrifugation for 1 h at 100,000 g and 20°C, and resuspended in APB. Actin–plectin complexes were then incubated with 10 µM of purified, precleared GST-ß4 fusion proteins (as described above) for 1 h at room temperature. Actin filaments with bound protein were pelleted as described above, and corresponding amounts of pellet and supernatant were analyzed by SDS-PAGE.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Expression and Distribution of Hemidesmosomal Proteins in Immortalized MD-EBS Keratinocytes
Two immortalized keratinocyte cell lines were derived from unrelated MD-EBS patients, and analyzed by Western blot using specific antisera for the different hemidesmosome proteins (Figure 1). Expression of the hemidesmosome proteins was similar in the two MD-EBS cell lines, but different from that in NHK cells because the MD-EBS keratinocytes expressed ß4, BP230, and BP180, but not plectin.

Next, we analyzed the distribution of hemidesmosome proteins in MD-EBS cells using confocal immunofluorescence microscopy (Figure 2). As expected, no reaction was found with plectin antiserum (Figure 2 E). The integrin 6ß4, as well as BP180 and BP230 were concentrated at cell–substrate contact sites in patch-like structures (Figure 2, A–C and F). This staining pattern is typical for hemidesmosome-like structures in cultured keratinocytes (Borradori et al. 1998 ; Schaapveld et al. 1998 ). The extracellular ligand of 6ß4, laminin-5, was concentrated underneath the hemidesmosome-like structures (Figure 2 D). These observations indicate that at least on a laminin-5 matrix, keratinocytes can form hemidesmosomes in the absence of plectin.

Expression of the Plectin-ABD in MD-EBS Keratinocytes Results in Its Association with both Actin Stress Fibers and Hemidesmosomes
Plectin contains a highly conserved NH₂-terminal ABD, and is therefore potentially capable of binding actin; yet F-actin has not been found in association with hemidesmosomes. We speculated that binding of 6ß4 to plectin might prevent it from binding to F-actin, possibly by competing with actin for overlapping binding sites on plectin. To examine the subcellular distribution of the NH₂-terminal ABD of plectin, an expression vector carrying plectin-ABD cDNA, a clone encoding the first 339 amino acid residues of human epithelial plectin encompassing its ABD fused to an HA tag, was transiently transfected in MD-EBS cells (Figure 3).

In the majority of cells, the plectin fragment was efficiently expressed, and its staining pattern overlapped with that of phalloidin-stained F-actin (Figure 3, A–C). However, in a population of ~10–20% of the cells, it was also present in basally located hemidesmosome-like clusters not associated with F-actin (Figure 3, D–F). Only very few cells exclusively express the plectin fragment in hemidesmosome-like clusters (Figure 3, G–I).

These data suggest that the NH₂ terminus of plectin is not only capable of associating with F-actin, but also with 6ß4-containing hemidesmosome-like structures.

Expression of ß4 in PA-JEB Keratinocytes Alters the Distribution of Plectin Compared with that of F-Actin
The above results prompted us to investigate whether the intact plectin molecule is also capable of associating with F-actin. Therefore, we compared its distribution with that of F-actin in PA-JEB keratinocytes that endogenously express full-length plectin. In addition, we studied the distribution of these two proteins in PA-JEB cells that stably express the integrin 6ß4 at their cell surface. In both cell types the staining pattern of plectin with the mAb 121 against plectin/HD1 and an affinity-purified polyclonal antiserum against the plectin-ABD was similar. In the PA-JEB keratinocytes, both antibodies stained plectin diffusely throughout the cytoplasm, with some enrichment of the protein in regions where F-actin was also found to be concentrated (Figure 4, A–C). In general, the staining pattern of plectin seen with the mAb 121 appeared more in spots, whereas the staining produced by the polyclonal plectin antiserum was often continuous and clearly associated with cytoskeletal elements (Figure 4, G–I). Plectin was only occasionally found to be associated with F-actin at the cell periphery, where microtubules and/or intermediate filaments may be running parallel to F-actin and be linked to it by plectin (Figure 4, J–L) (Svitkina et al. 1996 ). Association of plectin with F-actin probably also occurs at other sites in the cell, but it may not be as easily recognized there because the cytoskeletal fibers it links do not run parallel to one another but are intertwined. Furthermore, it should be realized that the distribution of F-actin may be different from that of plectin, because plectin can also cross-link other cytoskeletal systems, i.e., intermediate filament with microtubules (Svitkina et al. 1996 ). In PA-JEB cells that stably express the integrin 6ß4, both antibodies stained hemidesmosome-like structures, which were devoid of F-actin (Figure 4, D–F).

These results demonstrate that plectin is associated with the cytoskeleton, including F-actin, or with hemidesmosome-like structures, and that the localization of plectin with the latter is dependent on the expression of 6ß4.

Direct Interaction between the NH₂ Terminus of Plectin and the Cytoplasmic Domain of ß4
To investigate whether the NH₂-terminal ABD of plectin can interact directly with the cytoplasmic domain of the ß4 integrin subunit, and to determine the binding site on ß4, a yeast two-hybrid assay was performed. The plectin-ABD fragment was expressed as a GAL4 BD fusion, together with one of a set of overlapping fragments of the ß4 cytoplasmic domain (as GAL4 AD fusions) in the yeast strain PJ69-4A. Interactions were detected by the growth of yeast colonies on selective SC-LTHA plates (Figure 5 A).

A high plating efficiency, indicating that the reporter genes were efficiently expressed as a result of a strong interaction between the plectin-ABD and ß4, was observed with the ß4^1115–1457, ß4^1115–1382, and ß4^1115–1355 constructs. The site of interaction on the ß4 cytoplasmic domain could be mapped to the first and second FNIII repeat and 27 residues of the CS, as present in the ß4^1115–1355 construct. The ß4^1115–1328 construct with the last 27 amino acids deleted interacted only weakly with the plectin-ABD. The first FNIII repeat itself does not bind plectin-ABD, but is nevertheless essential for this binding, since its deletion in the ß4^1217–1328 construct results in a complete abrogation of binding. No interaction could be found with the third or fourth FNIII repeat, alone or when present as a pair. Therefore, the interaction of the NH₂ terminus of plectin with ß4 is specific for the first pair of FNIII repeats and 27 amino acids of the CS (amino acids 1115–1355).

The R1281W ß4 Mutation Abrogates Binding to Plectin without Interfering with Other Protein Interactions of ß4
Recently, two patients with nonlethal PA-JEB have been described who were either homozygous or heterozygous for a missense mutation, i.e., a single amino acid substitution from R to W at residue 1281 of ß4 (Pulkkinen et al. 1998 ). Since the R to W mutation was within the binding site for plectin mentioned above, we introduced it into wild-type ß4 cDNA to study its effect on the binding of ß4 to plectin. This was first tested in a yeast two-hybrid assay (Figure 5 B). The results show that ß4-plectin binding was completely abrogated by this mutation, confirming that the second FNIII repeat of ß4 is essential for binding to plectin. We then tested whether the intramolecular interaction within the wild-type ß4 cytoplasmic domain, as described previously (Schaapveld et al. 1998 ), was affected in the ß4^R1281W mutant. Wild-type (ß4^1115–1457) and R1281W (ß4^1115–1457*) constructs were expressed in yeast as GAL4 AD fusions together with a GAL4 BD fusion of ß4^1457–1752. Both wild-type (ß4^1115–1457) and mutant (ß4^1115–1457*) bound strongly to the ß4^1457–1752 fusion protein, indicating that intramolecular binding could still occur in the ß4^R1281W mutant protein.

Next, we tested the effect of the R1281W mutation on the function of ß4 in hemidesmosome formation in immortalized keratinocytes isolated from a PA-JEB patient who completely lacked ß4 (Schaapveld et al. 1998 ). PA-JEB keratinocytes were transiently transfected with full-length wild-type ß4 or ß4^R1281W cDNA and analyzed by confocal immunofluorescence microscopy (Figure 6). In transfected PA-JEB cells, both the wild-type ß4 and the ß4^R1281W protein were found at the basal side of the cell in hemidesmosome-like clusters. Whereas wild-type ß4 was colocalized with plectin, BP180, and BP230 in hemidesmosome-like structures (Figure 6A, Figure B, and Figure C, respectively), the ß4^R1281W protein was only colocalized with BP180 and BP230 (Figure 6, D–F).

Thus, the data show that plectin can directly bind ß4. The recruitment of BP180 into hemidesmosomes does not seem to require plectin; BP180 (and BP230) and plectin can be independently recruited by ß4.

Direct Binding of both ß4 and Actin to the Plectin-ABD and Mapping of Overlapping Sites of Interaction
The cell transfection studies described in Figure 3 suggest that both F-actin and ß4 can bind to the plectin-ABD. Binding of ß4 to an NH₂-terminal part of plectin (residues 1–1128) has been shown previously by Rezniczek et al. 1998 using in vitro binding assays, but this interaction appeared not to require the presence of the plectin-ABD, since its deletion from a smaller plectin fragment (residues 546–1128) did not result in the abrogation of binding to ß4. To examine the binding of ß4 to the plectin-ABD and the presence of additional binding sites COOH-terminal of the ABD, a set of overlapping fragments of the entire plectin NH₂ terminus (residues 1–1155) was generated. Each of these was expressed in yeast as a GAL4 BD fusion, together with ß4^1115–1457 as a GAL4 AD fusion (Figure 7 A). Binding of ß4^1115–1457 to the plectin NH₂ terminus could only be observed for the plectin-ABD fragment that contains residues 1–339 used in the experiments described above.

The plectin-ABD clone contains the first 9 exons of the plectin PLEC1 gene, including the variable exon 1c (encoding residues 1–65), which is specific for epithelial plectin and encodes a protein sequence with no known homologies. Exons 2–8 encode the plectin-ABD (amino acid residues 66–302), and exon 9 (residues 303–343) encodes a unique stretch of amino acids. For further mapping of the ß4-binding site and for comparing it with that of actin, plectin-ABD subclones were constructed and tested in a yeast two-hybrid assay with ß4^1115–1457 or with full-length actin. As shown in Figure 7 B, the plectin-ABD binds not only ß4, but also actin. The sequences encoded by exons 1c and 9 are not involved in binding to ß4. Deletion of NH₂-terminal sequences within the ABD, even of only the first four residues, disrupted binding to ß4 completely, suggesting that the first part of the ABD is essential. COOH-terminal deletions showed that deletions extending into the ABD abrogate binding even when all three ABS sequences (residues 72–83, 144–170, and 182–196) were intact, showing that the last part of the ABD is also required. Therefore, the minimal region in plectin required for binding to ß4 comprises residues 65–302, i.e., the complete ABD.

Investigation of the binding site for actin on the same panel of plectin fragments showed that deletion of the first 35 residues in plectin did not affect its interaction with actin, but that removal of all 64 residues encoded by exon 1c abolished it, in contrast to the binding of ß4. This could be due to steric hindrance of the ABD in juxtaposition to the GAL4 BD moiety present in the GAL4-plectin^65–339 fusion protein, since data in the literature do not reveal that amino acids NH₂-terminal of the ABD are required for binding to actin in other actin-binding proteins (Fabbrizio et al. 1993 ; Corrado et al. 1994 ). COOH-terminal deletions showed that no residues COOH-terminal of the plectin-ABD are required for binding of actin either. However, in contrast to ß4, actin could still bind when COOH-terminal parts of the ABD were deleted up to and including ABS3. Only deletion of ABS2 resulted in complete loss of actin binding. Therefore, ABS1 and ABS2 are sufficient to bind actin. Plectin is versatile in its binding of actin; three different actin isoforms were tested for interaction with plectin^1–339: skeletal muscle actin, ß cytoplasmic actin, and cytoplasmic actin, and all effectively bind plectin (data not shown).

In conclusion, the binding sites for ß4 and actin both reside in the plectin-ABD. Both sites start at the beginning of the ABD; actin binding only requires ABS1 and ABS2; the binding of ß4 also requires ABS3 and more COOH-terminal sequences. ß4 and actin do not bind to each other; they do not interact in the yeast two-hybrid assays (data not shown). Together, these results suggest that the binding site for ß4 overlaps with that for actin. Therefore, only one of these proteins can bind to a single plectin molecule, binding thus being mutually exclusive.

Biochemical Evidence for Competitive Binding of ß4 and F-Actin to the Plectin-ABD
To confirm the yeast two-hybrid results described above, an in vitro binding assay was performed. GST-ß4 fusion proteins or F-actin were spotted on nitrocellulose using a dot blot system and the immobilized proteins were overlaid with in vitro translated, radio-labeled plectin-ABD protein (Figure 8A and Figure B). Two different buffers were used in the overlay assay: APB, a high-salt buffer which maintains F-actin in a polymerized state, and AGB, a low-salt buffer in which F-actin can depolymerize to monomeric G-actin. The plectin-ABD strongly bound F-actin, GST-ß4^1115–1382, but not GST-ß4^1115–1382* (R1281W) or GST-ß4^1457–1752, which is in accordance with the yeast two-hybrid assay results (Figure 5 and Figure 7). No significant binding was found to GST-ß4^1115–1328, although this mutant protein interacted weakly with the plectin-ABD in the yeast two-hybrid assay. These different results may reflect a difference in the sensitivity of the two assays, with the yeast two-hybrid assay being more sensitive than the dot blot assay. Consistent with the absence of binding of the ß4^1115–1328 mutant to plectin in the dot blot assay, the mutant protein was also unable to induce a redistribution of plectin in cell transfection experiments (Niessen et al. 1997b ; Schaapveld et al. 1998 ). Binding of the plectin-ABD to the GST-ß4^1115–1382 fusion protein was stronger in the presence of low salt (AGB). Thus, binding is probably ionic in nature, explaining why charged residues, i.e., R1281, are important for ß4–plectin interactions. In contrast, binding of the plectin-ABD to actin was significantly weaker in the presence of low salt.

To obtain evidence that ß4 and F-actin indeed compete for binding to the plectin-ABD, we performed an in vitro competition assay. F-actin, polymerized in the presence of MBP-plectin-ABD fusion protein, was incubated with a 10-fold molar excess of soluble GST fusion proteins. Then the F-actin complexes were precipitated by centrifugation and the supernatant and pellet were collected separately (Figure 8 C). In the absence of GST-ß4 fusion protein, or in the presence of GST-ß4^1115–1328 or GST-ß4^1115–1382*, all MBP-plectin protein was found in the pellet together with actin. However, in the presence of GST-ß4^1115–1382, about half of the MBP-plectin protein appeared in the supernatant due to binding to the soluble GST-ß4 fusion protein. The results show that ß4 can efficiently compete with F-actin for binding to the plectin-ABD.

The Plectin COOH Terminus Interacts with Intermediate Filament Proteins, but Not with ß4
The results presented so far provide evidence for a role of the NH₂ terminus of plectin in the binding of both F-actin and ß4. To rule out a role for the COOH terminus of plectin more precisely of its globular repeat domains, we also tested cDNA clones encoding this part of the plectin molecule. Since earlier studies (Wiche et al. 1993 ; Nikolic et al. 1996 ) had indicated a role for sequences at the end of the R5 domain in binding to intermediate filament proteins, plectin fragments containing this putative IFBD were also tested for interaction with the keratins 5, 8, 14, and 18, and with vimentin and GFAP. Two plectin cDNA clones were tested in the yeast two-hybrid assay. One encoded most of R5, the complete R6, and the COOH tail, and the other encoded most of R6 and the COOH tail. They were expressed in yeast as GAL4 BD fusion proteins together with GAL4 AD fusion proteins of ß4^1115–1457, ß4^1320–1668, or ß4^1457–1752, or of different intermediate filament proteins (Figure 9). No interaction was found between the plectin fragments and any of the ß4 fragments tested. However, specific interaction of the larger plectin fragment, which contained the IFBD at the end of R5, could be detected with different types of intermediate filament proteins. Binding was observed with keratins 14 and 18, but not with keratins 5 or 8, and with both vimentin and GFAP.

Thus, the results show that the plectin COOH terminus has no function in the binding to the ß4 cytoplasmic domain, but confirm the presence of a distinct intermediate filament protein–binding site in the plectin domain R5, which we have shown to bind the type I and III, but not type II intermediate filament proteins.

The Plectin-IFBD Associates with the Intermediate Filament Cytoskeleton in Plectin-deficient MD-EBS Keratinocytes
To investigate whether the plectin-IFBD fragment that interacts with intermediate filament proteins in a yeast two-hybrid assay also associates with intermediate filaments in keratinocytes, we transiently transfected MD-EBS cells with an expression vector containing the plectin-IFBD fused to an HA tag, and analyzed the results by confocal immunofluorescence microscopy (Figure 10). In contrast to normal keratinocytes in vivo, which do not express vimentin but only keratins, the EBS-MD keratinocytes coexpressed vimentin and keratin intermediate filaments. In cells expressing plectin-IFBD, the HA-tagged protein was found to be distributed together with keratins in delicate filamentous structures (Figure 10, D–F). Although the expression of plectin-IFBD had no apparent effect on the keratin intermediate filament network, in many cells it induced a collapse of the vimentin network into dense clusters around the nucleus (Figure 10, G–I). Colocalization of these two proteins in filamentous structures was only observed in a few cells (data not shown). The dramatic effect of the plectin-IFBD on the integrity of the vimentin intermediate filament network suggests that it interferes with another protein with properties similar to plectin that mediates the anchorage of these filaments at distal or peripheral sites in the cell. It may also indicate that plectin-IFBD binds more strongly to vimentin than to keratins. The 6ß4 integrin was always present in basally located hemidesmosome-like clusters, and showed no obvious colocalization with plectin-IFBD (Figure 10, A–C). These results suggest that in a normal situation, when the intact plectin protein is present, it helps to provide a scaffold for the vimentin cytoskeleton.

The Integrin 6ß4 Organizes the Vimentin Intermediate Filament Network through Plectin upon Expression in PA-JEB Keratinocytes
To study a possible indirect role for the integrin 6ß4 (via plectin) in the organization of the vimentin intermediate filament network, immortalized ß4-deficient PA-JEB keratinocytes were transiently transfected with cDNAs encoding wild-type or mutant ß4 and used in confocal immunofluorescence microscopy (Figure 11). In nontransfected cells, vimentin is present in rather loose filamentous networks throughout the cytoplasm, but in cells expressing ß4 it was also detected in dense basally located clusters colocalized with 6ß4 (Figure 11, A–C). These results show that expression of ß4 leads to a reorganization of the vimentin intermediate filament network. This most likely occurs by binding of ß4 to the ABD in the plectin NH₂ terminus, accompanied by binding of vimentin to the IFBD in its COOH terminus. The effect of ß4 on the vimentin network would then be indirect and dependent on the binding of ß4 to plectin.

Further evidence for this was obtained by transfection of one mutant ß4 protein, ß4¹³⁵⁵, that can efficiently bind plectin, and one ß4 mutant, ß4¹³²⁸, that binds plectin in yeast two-hybrid assays, but much less efficiently. In cells transfected with ß4¹³⁵⁵ cDNA, the ß4 protein fragment was present in basal hemidesmosome-like clusters colocalized with vimentin (Figure 11, D–F). The ß4¹³²⁸ mutant was also found in basal clusters, but it only occasionally colocalized with vimentin (Figure 11, G–I).

These results show that ß4 is capable of organizing the vimentin intermediate filament cytoskeleton. It does so indirectly, via plectin, since only ß4 mutants that contained the plectin binding site (like ß4¹³⁵⁵) were capable of organizing vimentin filaments into hemidesmosome-like basal clusters. Since this ß4 mutant cannot recruit BP180 or BP230 (Schaapveld et al. 1998 ), the BP proteins do not seem to play a role in this process.

	Discussion

Top Abstract Introduction Materials and M