Introduction

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INTEGRINS comprise a large family of heterodimeric receptors that mediate the adhesion of cells to extracellular matrices and other cells (Buck and Horwitz, 1987; Hynes, 1987, 1992; Ruoslahti and Pierschbacher, 1987; Ginsberg et al., 1988; Hemler, 1990; Springer, 1990; Watt et al., 1993). In addition, they are involved in transducing extracellular signals into the cell (Hynes, 1992; Juliano and Haskill, 1993; Giancotti and Mainiero, 1994). Both the  and  subunits of integrins have a large extracellular portion, a transmembrane segment, and generally a short cytoplasmic domain. The cytoplasmic domains of integrins interact with the cytoskeleton and possibly with signaling molecules, but the molecular mechanisms of these interactions are not well understood.

The ₆₄ integrin is a basement membrane receptor for laminins (Kajiji et al., 1989; De Luca et al., 1990; Sonnenberg et al., 1990a,b; Lee et al., 1992) that engages in cytoplasmic interactions distinct from those of all other known integrins. Instead of being localized at adhesion plaques that serve as anchoring structures of actin filament networks, this receptor is part of hemidesmosomes (Carter et al., 1990; Stepp et al., 1990; Jones et al., 1991; Sonnenberg et al., 1991), junctional complexes that anchor cytokeratin intermediate filament (IF)¹ networks and mediate adhesion of epithelial cells to the underlying basement membrane. The intracellular interactions of integrin ₆₄ are mediated by the ₄ subunit, the intracellular portion of which is much larger (~1,000 amino acids) than that of all the other known  subunits (~50 amino acids) and bears no apparent sequence homology to them (Hogervorst et al., 1990; Suzuki and Naitoh, 1990; Tamura et al., 1990). It contains four regions with homology to fibronectin type III (FNIII) repeats, arranged in two pairs separated by a 143-amino acid-long connecting segment. This large cytoplasmic tail of integrin ₄ is required, and probably sufficient, for incorporation of the integrin into hemidesmosomes. Specifically, a minimal region on the integrin ₄ subunit located in the linking segment between the second and third repeat has been reported to be critical to its localization in hemidesmosomes (Niessen et al., 1997a). The targeted inactivation of the integrin ₄ gene in mice convincingly demonstrated that hemidesmosome formation and proper skin attachment to the basal lamina are crucially dependent on the expression of this integrin subunit (Dowling et al., 1996; van der Neut et al., 1996).

Cytoplasmic components of the hemidesmosome that have been implicated in the attachment of IFs to this adhesion structure include the bullous pemphigoid antigen 1 (Stanley et al., 1981), also referred to as BP230 or BPAG1e (Yang et al., 1996), plectin (Wiche et al., 1984), the 200-kD 6A5 antigen (P200; Kurpakus and Jones, 1991), HD1 (Owaribe et al., 1991; Hieda et al., 1992), and the IF-associated protein 300 (Skalli et al., 1994). Among these, plectin, IF-associated protein 300, and HD1 have been shown to bind to IFs in vitro (Foisner et al., 1988; Skalli et al., 1994; Fontao et al., 1997), and the IF-binding site of plectin has recently been mapped to the fifth repeat domain within the carboxy-terminal globular region of the molecule (Nikolic et al., 1996). Also, BP230, which shares partial sequence homology with plectin and desmoplakin and is structurally related to these molecules (Tanaka et al., 1991; Green et al., 1992; Klatte and Jones, 1994), is likely to bind to IFs (Yang et al., 1996). A direct interaction of the intracellular domain of the integrin ₄ subunit with any of the cytoplasmic hemidesmosome-associated proteins has not been demonstrated to date. There is evidence that integrin ₄ interacts with HD1, based on coimmunoprecipitation of purified recombinant integrin ₄ polypeptides expressed in bacteria with HD1 present in COS-7 cell lysates, and the observation that overexpression of integrin ₄ leads to a redistribution of HD1 in transfected cells (Sánchez-Aparicio et al., 1997; Niessen et al., 1997a,b), but the question whether this interaction was of direct or indirect nature remains unsolved.

Plectin, the most versatile cytoskeletal linker protein characterized to date, remains a strong candidate for bridging cytokeratin filament networks to hemidesmosomes. In fact, due to the very large size (>500 kD) of plectin molecules predicted on the basis of cDNA sequencing (Wiche et al., 1991; Liu et al., 1996; Elliott et al., 1997), and their extended (~200-nm-long) multi-domain structure, as visualized by electron microscopy (Foisner and Wiche, 1987), plectin molecules would have the dimensions to span the entire tripartite structure characteristic of hemidesmosomes. In this way, they could extend from the plasma membrane, the location of integrin ₄, to the filament anchorage site at the hemidesmosome inner plate structure. A role of plectin in hemidesmosome stabilization by physical linkage of proteins constituting this multi-component complex appears likely also in light of recent studies showing that defects in plectin expression lead to epidermolysis bullosa simplex (EBS)-MD, a severe hereditary skin blistering disease combined with muscular dystrophy (Chavanas et al., 1996; Gache et al., 1996; McLean et al., 1996; Pulkkinen et al., 1996; Smith et al., 1996). Moreover, plectin-deficient mice generated by targeted gene inactivation showed severe skin blistering and degeneration of keratinocytes, apparently caused by a significant reduction in number and mechanical stability of epidermal hemidesmosomes (Andrä et al., 1997).

The aim of the work presented here was to extend the human disease and plectin gene-knockout studies to the molecular level, and to investigate whether plectin directly interacts with integrin ₄, and if so, to define the molecular domains of both proteins involved in this interaction. In addition, we were interested in the consequences that overexpression of integrin ₄ mutant proteins containing putative plectin-binding sites might have on the integrity of cytokeratin filament networks and on the subcellular distribution of plectin. We show here that partially truncated versions of the integrin ₄ cytoplasmic domain expressed in bacteria bind to recombinant carboxy- and amino-terminal domains of plectin in two independent in vitro binding assays, and that protein species shown to interact in vitro colocalized when ectopically coexpressed in living cells. In addition, we observed self-association of integrin ₄ mutant proteins in vitro, and show that plectin proteins can modulate this effect. Furthermore, carboxy-terminal integrin ₄ mutant proteins lacking plasma membrane anchoring sequences, but comprising plectin-binding region(s) in their tail domain, are shown to cause bundling and collapse of IF network arrays upon overexpression in PtK2 and 804G cells, and similar fragments dislocated endogenous plectin from hemidesmosomes in 804G cells.

	Materials and Methods

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cDNA Constructs

Integrin ₄. The integrin ₄ subunit protein and cDNA sequences were numbered according to Suzuki and Naitoh (1990; Genbank/EMBL/DDBJ accession number X51841). Nucleotide and primer numbers refer to the nucleotide position (3') relative to the first bp of the start codon (position 127 in X51841). For cloning, a part of the cytoplasmic tail region of integrin ₄ was amplified by PCR from human placenta cDNA (Quick-Clone; CLONTECH Laboratories, Inc., Palo Alto, CA) using primers U3330 (5'-GAG CTT CAC GAG TCA GAT GTT GTC-3') and L5250 (5'-GGG GCA GGG TGC GGT CAA GTT TGG-3'). A mixture of Klen-Taq (AB Peptides, St. Louis, MO) and PfuI (Stratagene, Heidelberg, Germany) polymerases was used under high fidelity conditions described by Barnes (1994). The 1944 bp PCR product obtained was used as template for nested PCR with EcoRI-tailed primers and the amplified fragments were subcloned into the unique EcoRI site of the bacterial expression vector pBN120 (Nikolic et al., 1996), a derivative of pET-15b (Novagen Inc., Madison, WI). Clones generated encoded the following domains of the integrin ₄ subunit: ₄-F_1,2 (amino acid residues 1,126-1,315; plasmid construct pGR1), ₄-L (1,316-1,457; pGR2), ₄-F_3,4C'(1,486-1,752; pGR3), ₄-F₂L (1,219-1,457; pGR4), ₄-F_1,2L'(1,126-1,485; pGR5), and ₄-F_1,2LF_3,4C (1,126-1,752; pGR6). The clone encoding ₄-F_3,4 (1,457-1,662; pJP5) was generated by PCR from pGR6 (₄-F_1,2LF_3,4C; see Fig. 1 A for an overview). The correctness of all PCR-generated clones was verified by DNA sequencing. The clone encoding ₄-F_1,2L (1,126-1,457; pGR36) was obtained by exchanging the SmaI/PstI fragment from pGR5 with that of pGR4. Plasmids encoding ₄-LF_3,4C (1,316-1,752; pGR37) and ₄-F_3,4C (1,457-1,752; pGR44) were constructed by exchanging the SmaI/HindIII fragment of pGR2 with that of pGR6 and the XbaI/NotI fragment of pGR6 with that of pJP5, respectively. For expression of carboxy terminally c-myc-tagged proteins in mammalian cells, EcoRI fragments were subcloned into pAD29 (Nikolic et al., 1996), and excised XbaI/HindIII fragments were subcloned into the eukaryotic expression vector pRc/ CMV (Invitrogen Corp., San Diego, CA). This yielded the mammalian expression constructs encoding ₄-F_3,4C'^myc (pGR13, derived from pGR3), ₄-F_1,2^myc (pGR16, derived from pGR1), ₄-F_1,2L'^myc (pGR19, derived from pGR5), and ₄-F_1,2LF_3,4C^myc (pGR20, derived from pGR6). In addition, the EcoRI fragment from pGR6 was also subcloned into the eukaryotic expression vector pGR29, a modified version of pEGFP-N3 (CLONTECH Laboratories, Inc.). The resulting protein encoded by this construct (₄-F_1,2LF_3,4C^GFP; pGR30) carried an enhanced version of the Aequorea victoria green fluorescent protein (GFP) at its carboxy terminus. Correct expression of proteins encoded by mammalian vector constructs was verified by Western blot analysis of lysates of transfected 804G cells.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   (A) Schematic representation of human integrin 4 mutant proteins and summary of their plectin-binding phenotype in vitro and in vivo. Drawing on top shows major structural motifs contained in the integrin 4 subunit. EC and IC, extracellular part (not fully shown) and intracellular part, respectively; TM, transmembrane domain; F1-F4, fibronectin type III-like repeat domains; L, linking segment between the first and second repeat pair; C, carboxy-terminal domain after the second repeat pair. Details about generation and description of cDNA constructs and expressed proteins are given in the text. Table on right hand side indicates whether (+) or not () a given integrin mutant protein bound to the carboxy- (C = Ple-R3-6T) or amino-terminal (N = Ple-N1) plectin mutant protein in the Eu3+-overlay (in vitro) or transfection assays (in vivo). (B) Schematic representation of rat plectin mutant proteins and summary of their in vitro binding ability to the 4-F1,2LF3,4C polypeptide. Drawings include the major molecular domains of plectin: ABD, actin-binding domain; Rod, plectin rod domain; IFBD, IF binding domain; R1-R6, carboxy-terminal repeats domains 1-6; T, carboxy-terminal tail after repeat 6. Expression plasmids were generated as outlined in the text. Summary table at right shows semiquantitative assessment (strongest reaction: ++++, no binding: ) of the in vitro binding data shown in Fig. 3 (blot overlay of various plectin mutant proteins with the integrin 4 polypeptide 4-F1,2LF3,4C).

View larger version (72K): [in this window] [in a new window]   Fig. 3.   Blot overlay of recombinant plectin proteins with 125I-labeled 4-F1,2LF3,4C. Purified his-tagged recombinant plectin proteins Ple-N1, Ple-N2, Ple-R1, Ple-R3-6T, Ple-R4,5, Ple-R4, Ple-R5, and Ple-R6 (A and C), and whole lysates of induced bacterial cultures (B and D) expressing his-tagged plectin recombinant proteins Ple-R1-3, Ple-R3, Ple-R4-6T, Ple-R6T, and a non-induced bacterial culture, were subjected to SDS 10% PAGE in duplicate. Proteins on one set of gels were stained with Servablue-G (A and B), those on the other set were blotted onto nitrocellulose membranes, overlaid with 125I-4-F1,2LF3,4C (his-tagged), and bound integrin 4 protein detected by autoradiography (C and D). Size markers of 200, 97, 68, 43, and 29 kD are indicated.

Plectin. Rat plectin cDNA constructs were generated by PCR and/or other cloning techniques on the basis of the complete rat cDNA sequence (Genbank/EMBL/DDBJ database entry X59601). All constructs used were subcloned into the bacterial expression vector pBN120 (Nikolic et al., 1996), which enabled expression of amino terminally his-tagged proteins, except for the ones encoding Ple-N₁ (pGR48) and Ple-R_3-6T (pGR49), which were inserted into the unique EcoRI site of pFS23, a pET23a derivative driving the expression of carboxy terminally his-tagged proteins. Plectin constructs encoded the following segments of the protein (see Fig. 1 B for an overview): Ple-N₁ (amino acids 1-1,128; construct pGR48), Ple-N₂ (546-1,128; pMZ4), Ple-R₁ (2,777-3,161; pJD11), Ple-R_1-3 (2,777-3,851; pJD22), Ple-R_3-6T (3,346-4,687; pGR49), Ple-R₃ (3,346-3,851; pJD21), Ple-R_4-6T (3,850-4,687; pJD23), Ple-R_4,5 (3,780-4,367; pMZ3), Ple-R₄ (3,780-4,024; pBN135), Ple-R₅ (4,025-4,367; pBN132), Ple-R₆T (4,262- 4,687; pMZ5), and Ple-R₆ (4,277-4,620; pBN144). For expression in mammalian cells, EcoRI fragments of pGR48 (Ple-N₁) and pGR49 (Ple-R_3-6T) were subcloned into the eukaryotic expression vector pGR29, resulting in constructs encoding GFP-tagged Ple-N₁^GFP (pGR31) and Ple-R_3-6T^GFP (pGR33). The latter plectin fragment was also expressed as a c-myc-tagged version (Ple-R_3-6T^myc), encoded by pBN72, a pRc/CMV derived plasmid.

Cell Culture, DNA Transfection, and Immunofluorescence Microscopy

Rat kangaroo PtK2 (CCL 56; American Type Culture Collection, Rockville, MD) and rat bladder carcinoma 804G cells (Izumi et al., 1981) were cultured in DME, supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% of heat-inactivated fetal calf serum, at 37°C and 5% CO₂. For transfection, cells were grown on glass coverslips and, while still subconfluent, transiently transfected with either 30 µg plasmid DNA per 10-cm plate using the calcium phosphate precipitation method (Graham and van der Eb, 1973) or 0.6 µg plasmid DNA per 13-mm coverslip in wells of 24-well tissue culture plates (Falcon Plastics, Cockeysville, MD) using the LipofectAMINE (GIBCO BRL, Paisley, Scotland) transfection method. In the latter case, the transfection medium (0.6 ml/coverslip) contained DNA and LipofectAMINE at final concentrations of 1 µg/ml and 8 µg/ml, respectively, in serum-free medium (Opti-MEM, GIBCO BRL); it was replaced with DME/10% FCS after 4.5 h of incubation at 37°C and 5% CO₂. Cells were fixed 24-48 (PtK2) or 8-24 h (804G) after transfection using chilled (20°C) methanol and processed for immunofluorescence microscopy as previously described (Wiche et al., 1993). The following immunoreagents were used: rabbit anti-human ₄-antiserum (Giancotti et al., 1992; kindly provided by F.G. Giancotti), mouse monoclonal anti-rat plectin antibody (mAb) 5B3 (Foisner et al., 1994), guinea pig anti-mouse liver cytokeratin antibody (Denk et al., 1981; kindly provided by H. Denk), mouse monoclonal anti-vimentin antibody (clone V9; DAKOPATTS, Copenhagen, Denmark), and anti-myc monoclonal antibody 1-9E10.2 (American Type Culture Collection) as primary antibodies; and AMCA-, fluorescein (FITC)- or Texas red-conjugated AffiniPure donkey anti-mouse IgG (H+L), anti-rabbit IgG (H+L), and anti-guinea pig IgG (H+L; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as secondary antibodies. Specimens were viewed in a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Oberkochen, Germany) or using the Bio-Rad MRC600 confocal scanning laser microscope (Richmond, CA). Photographs were taken using Ilford ASA 400 black and white film, and digital images were processed using the NIH Image 1.59 and Adobe Photoshop 4.01 software packages.

Expression of Recombinant Proteins in Bacteria

His-tagged recombinant proteins encoded by pBN120 or pFS23 vector constructs were expressed in Escherichia coli BL21(DE3) and purified from inclusion bodies by solubilization in 6 M urea, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9 (binding buffer) containing 5 mM imidazole, followed by affinity binding to His-Bind metal chelation resin, according to the manufacturer's (Novagen Inc.) protocol. Bound proteins were eluted from affinity columns using 250 mM imidazole in binding buffer and stored frozen at 20°C. Samples were dialyzed against desired buffers before use to remove urea.

Gel Electrophoresis, Immunoblotting, and Preparation of 804G Cell Lysates

SDS-polyacrylamide gel electrophoresis (PAGE) was carried out under reducing conditions (Laemmli, 1970). Proteins on gels were visualized by staining with Servablue-G (Serva, Heidelberg, Germany). For immunoblotting, proteins were transferred to nitrocellulose sheets (Schleicher & Schuell, Dassel, Germany) for 4 h at 35 V in 25 mM Tris, 191 mM glycine, 0.01% SDS. After blocking with 3% BSA in TBS-T (TBS, containing 0.1% Tween-20), membranes were incubated with mAb 5B3 (1 h, room temperature), washed with TBS-T, and incubated with alkaline phosphatase-conjugated secondary goat anti-mouse antibody (Promega, Heidelberg, Germany). After washing, antibody binding was visualized with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl₂.

Lysates of 804G cells were prepared by boiling cells in Laemmli's sample buffer (400 µl per confluent 10-cm plate) for 5 min.

Eu³⁺ Labeling of Recombinant Proteins and Microtiter Plate Overlay Assay

Recombinant mutant proteins were labeled with Eu³⁺ following the recommendations provided by the manufacturer (Wallac, Turku, Finland). 50-200 µg of recombinant integrin (₄-F_1,2, ₄-F_1,2LF_3,4C, ₄-F_1,2L, ₄-LF_3,4C, ₄-F_3,4C, ₄-F_3,4) and plectin (Ple-N₁, Ple-R_3-6T) mutant proteins were dialyzed against labeling buffer (50 mM sodium carbonate, pH 8.5) overnight at 4°C, and subsequently incubated with the labeling reagent (Eu³⁺-chelate of N¹-(p-isothiocyanatobenzyl)-DTTA) at room temperature for ~30 h. Unreacted labeling reagent was removed by gel filtration through a P6 column (Bio-Rad) equilibrated with 50 mM Tris-HCl, pH 7.5, 0.9% NaCl, 0.01% NaN₃. Proteins were stored at 4°C until use. Microtiter plates were coated with recombinant proteins expressed in bacteria (100 µl of a 100 nM solution in 25 mM sodium borate buffer, pH 9.2) overnight at 4°C. Blocking was carried out with 4% BSA in TBS for 1 h and then binding with Eu³⁺-labeled proteins in 100 µl overlay buffer (TBS, pH 7.5, containing 1 mM EGTA, 2 mM MgCl₂, 1 mM DTT, and 0.1% Tween 20) for 90 min at room temperature. After extensive washing with overlay buffer, the amount of bound proteins was determined by releasing complexed Eu³⁺ with enhancement solution and measuring the fluorescence with a Delfia time-resolved fluorometer (Wallac). The fluorescence values were converted to concentrations by comparison with an Eu³⁺ standard.

¹²⁵I Labeling of Proteins and Blot Overlay Assay

The recombinant integrin ₄ mutant protein ₄-F_1,2LF_3,4C (~0.8 mg) was dialyzed against 20 mM Tris-HCl, pH 7.5, and incubated with one Iodo-Bead (Pierce, Rockford, IL) and 0.5 mCi of Na¹²⁵I (DuPont-NEN, Boston, MA) at room temperature for 15 min. Free iodine was removed by gel filtration using a Sephadex G-25 (Pharmacia Biotech Sverige, Uppsala, Sweden) column (0.9 × 14 cm) equilibrated with 20 mM Tris-HCl, pH 7.5, 1 mM DTT. Labeled proteins were stored at 4°C until use.

Purified recombinant integrin and plectin mutant proteins and lysates of induced bacterial cultures expressing plectin protein fragments were analyzed by SDS-10% PAGE. Proteins were blotted onto 0.2-µm nitrocellulose membranes (Schleicher & Schuell), overnight in 25 mM Tris, pH 8.8, and 192 mM glycine, at 25 V using a wet mini-blot apparatus (Bio-Rad). Membranes were incubated with 0.1% gelatin (Merck, Darmstadt, Germany) in PBS for 4 h at room temperature, and rinsed once with TSDT (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT, 1% (vol/vol) Triton X-100). After incubation with 10 µg/ml of ¹²⁵I- ₄-F_1,2LF_3,4C in TSDT containing 0.5% BSA for 4 h and washing with TSDT, sheets were dried, sealed in plastic bags, and X-Omat film (Eastman Kodak, Rochester, NY) was exposed to them.

Immunoelectron Microscopy

Vascular perfusion of Wistar rats was performed with 4% paraformaldehyde in PBS, pH 7.4. Skin samples were immersed in fresh paraformaldehyde solution for 30 min and embedded in lowicryl-HM20 (Agar Scientific Ltd., Stansted, UK) as described in detail by Villinger (1991). Small pieces (~1-2 mm²) of fixed rat skin were rinsed in PBS and dehydrated in a series of ethanol dilutions at 20°C and ethanol finally replaced by overnight infiltration of pure lowicryl. Samples were transferred into transparent tubes (Eppendorf, Hamburg, Germany) that were filled with freshly prepared lowicryl. Polymerization was complete after 24 h of exposure to UV light 28°C.

Lowicryl thin sections (60-80 nm) cut with an ultramicrotome (Ultracut S; Reichert, Vienna, Austria) were mounted on formvar-coated nickel slot grids. Postembedding immunolabeling was performed as previously described (Polak and Varndell, 1984). Free aldehyde groups were reduced by incubating sections in 0.1 M glycine in PBS, three times for 5 min, and blocking was performed in normal goat serum (BioCell Res. Lab, Cardiff, UK) for 30 min. For immunolabeling, sections were incubated for 1 h at room temperature, or overnight at 4°C with one of the following domain-specific anti-plectin antibodies: a) anti-rod mAb 7A8 (Foisner et al., 1996), b) anti-amino terminus rabbit serum E1A raised against a synthetic peptide corresponding to a 12-amino acid residue-long sequence of human exon 1a, and c) anti-carboxy terminus mouse serum 135C raised against a recombinant rat plectin mutant protein corresponding to the carboxy-terminal repeat 4 domain (see Andrä et al., 1997), or with non- immune serum or PBS as controls. Secondary labeling using 5 nm gold-conjugated goat-anti-mouse IgG and 1 nm gold-conjugated goat-anti- rabbit IgG (BioCell Res. Lab) was performed for 1 h at room temperature. For visualization in the electron microscope, 1 nm or 5 nm colloidal gold particles were silver enhanced (Stierhof et al., 1991). Thin sections were counterstained with uranyl acetate and lead citrate and were viewed at 80 kV in a JEOL JEM-1210 electron microscope.

	Results

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Expression and Purification of Recombinant Forms of Integrin ₄ and Plectin

Localization of plectin at hemidesmosomes (Wiche et al., 1984) and its function as an IF (cytokeratin)-binding protein make it a likely candidate to act as a linker between the cytokeratin filament network and the hemidesmosomal integrin ₆₄. To test whether this proposed function of plectin involves its direct interaction with the uniquely long cytoplasmic tail of the ₄ subunit of integrin ₆₄, we generated a series of human integrin ₄ and rat plectin truncation mutants (Fig. 1) for use in biochemical in vitro binding, as well as in vivo cell transfection assays.

The original design of our integrin ₄ expression constructs was based on a report by Spinardi et al. (1993), who showed that a 303-amino acid-long region of the cytoplasmic tail comprising the first of its two pairs of FNIII repeats and the following segment between the first and second pair was necessary for the recruitment of the integrin ₄ subunit into hemidesmosomes. Integrin ₄ subdomain-specific clones generated in the pET expression system are shown in Fig. 1 A. Encoded proteins contained the cytoplasmic tail starting at the beginning of the first FNIII repeat (₄-F_1,2LF_3,4C), the 303-amino acid region described above (₄-F_1,2L'), and the rest of the tail, starting in the third FNIII repeat, close to its beginning (₄-F_3,4C'). ₄-F_1,2L and ₄-F_3,4C were similar to ₄-F_1,2L' and ₄-F_3,4C', respectively, except for excluding (₄-F_1,2L) and including (₄-F_3,4C) the third FNIII repeat in its entirety. ₄-F_1,2 corresponded to the first FNIII repeat pair, ₄-F_3,4 to the second; ₄-LF_3,4C lacked the first FNIII repeat pair. SDS-PAGE analysis of purified recombinant integrin ₄ polypeptides expressed in E. coli confirmed that they were of the sizes predicted on the basis of the corresponding cDNA sequences, including the 2.6-kD his-tags (Fig. 2).

View larger version (41K): [in this window] [in a new window]   Fig. 2.   SDS-PAGE of integrin 4 mutant proteins expressed in bacteria. His-tagged recombinant proteins were purified from bacterial inclusion bodies as described in the text and analyzed on a 12% polyacrylamide gel. Proteins were visualized by staining the gel with Servablue-G. The predicted masses of recombinant proteins were: 71.4 kD (4-F1,2LF3,4C), 39.4 kD (4-F1,2L), 24.0 kD (4-F1,2), 50.4 kD (4-LF3,4C), 35.0 kD (4-F3,4C), 25.4 kD (4-F3,4), 42.6 kD (4-F1,2L'), and 31.8 kD (4-F3,4C'). Molecular weight markers are indicated (×103).

Plectin mutant proteins corresponding to different molecular regions of the amino- and carboxy-terminal globular domains of the molecule (Fig. 1 B) were generated by subcloning rat plectin cDNAs into the pET expression system. Two amino-terminal protein fragments used covered the amino acid sequence encoded by exons 1-24 (Ple-N₁) and exons 12-24 (PleN₂). The other fragments covered, partly overlapping, the complete carboxy-terminal globular domain with its repeat structure. Ple-R₁ started within the hinge region after the rod domain and included the first and part of the second repeat domain; Ple-R_3-6T contained the rest of plectin's carboxy terminus. Two constructs, Ple-R₃ and Ple-R_4-6T, split construct Ple-R_3-6T into two shorter ones, one corresponding to repeat 3 and parts of the flanking repeats 2 and 4, the other to the carboxy terminus starting in repeat 4. Ple-R_1-3 was a combination of Ple-R₁ and Ple-R₃, ranging from plectin's hinge region into repeat 4. Ple-R_4,5 contained repeat 4 and 5, Ple-R₄ repeat 4 alone, and Ple-R₅ repeat 5 alone. Ple-R₆T contained a small portion of repeat 5, repeat 6 and the carboxy-terminal tail. Ple-R₆ was equivalent to Ple-R₆T but lacked the tail. Plectin mutant proteins expressed from these plasmids were all of the expected sizes, as determined by SDS-PAGE (Fig. 3, A and B). The double band observed for Ple-N₁ was likely due to an additional translation initiation event at an in-frame ATG codon, resulting in a ~15-kD shorter his-tagged protein version.

Different Subdomains of the Integrin ₄ Cytoplasmic Tail Region Each Bind to Amino- and Carboxy-terminal Domains of Plectin

To detect direct interaction between the candidate integrin ₄ cytoplasmic tail region and various subdomains of plectin, we first used a qualitative blot overlay assay in which recombinant plectin fragments immobilized on nitrocellulose membranes were overlaid with ¹²⁵I-labeled purified recombinant integrin ₄-F_1,2LF_3,4C (Fig. 3, C and D). The plectin fragments used in these experiments covered the complete cDNA sequence (exons 1-32; Liu et al., 1996) with the exception of the predicted rod region (exon 31), a small part of the preceding amino-terminal globular domain (exons 25-30), and the three additional alternative start exons (1a, 1b, and 1c) identified recently (Elliott et al., 1997). Among nearly a dozen recombinant plectin polypeptides tested, the one displaying highest binding activity to ₄-F_1,2LF_3,4C was Ple-R_3-6T, which contained most of plectin's carboxy-terminal globular domain. Plectin protein fragments corresponding to individual repeats (Ple-R₃, Ple-R₄, Ple-R₅) showed no or hardly any binding activity, neither did fragments containing repeat 1 and part of the hinge region (Ple-R₁), repeats 1-3 (Ple-R_1-3), or repeats 4 and 5 combined (Ple-R_4,5). Aside from Ple-R_3-6T, the ₄-F_1,2LF_3,4C polypeptide bound to all other plectin mutant proteins that contained repeat 6, such as Ple-R_4-6T, Ple-R₆T, and Ple-R₆. ₄-F_1,2LF_3,4C also reacted with the amino-terminal polypeptide fragments Ple-N₁ and Ple-N₂, suggesting that one or more additional interaction site(s) resided in this region of the plectin molecule.

To verify the blot overlay, and in order to assess plectin-integrin ₄ interaction in a more quantitative way, we used an alternative, nonradioactive microtiter plate-binding assay involving Eu³⁺-labeled proteins, which had successfully been used in the molecular mapping of plectin's IF-binding site (Nikolic et al., 1996). The two plectin protein fragments representing amino- and carboxy-terminal domains and exhibiting highest binding efficiency in the blot overlay assay (Ple-N₁ and Ple-R_3-6T, Fig. 3) were used for this assay. ₄-F_1,2LF_3,4C was coated onto 96-well microtiter plates and overlaid with increasing concentrations of the Eu³⁺-labeled plectin proteins (Fig. 4 A). The amounts of proteins bound to the coated integrin fragment were determined by measuring released Eu³⁺ by time-resolved fluorometry (Soini and Lövgren, 1987). Non-specific binding to coated BSA was also determined and subtracted to give results for specific binding to ₄-F_1,2LF_3,4C. Binding of both plectin proteins was similar up to 500 nM, where Ple-N₁ reached saturation, contrary to Ple-R_3-6T, which exhibited approximately twofold higher binding at 1,000 nM, consistent with the apparently stronger binding of ¹²⁵I-₄-F_1,2LF_3,4C to this protein in the blot overlay assay (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Concentration dependent (A) and competitive binding (B) of Eu3+- labeled recombinant plectin polypeptides to immobilized recombinant integrin 4 cytoplasmic tail polypeptide. (A) 4-F1,2LF3,4C (100 nM) was coated onto microtiter plates and overlaid with increasing concentrations (10-1,000 nM) of Eu3+-labeled Ple-N1 () or Ple-R3-6T (). Data shown have been corrected for nonspecific binding to BSA. Note, for concentrations up to 500 nM error bars do not extend outside of symbols. (B) Table summarizing competition experiments: 4-F1,2L or 4-F3,4C were coated onto microtiter plates (100 nM) and overlaid with either Eu3+-labeled Ple-N1 (100 nM) in the absence and presence of a 10-fold molar excess (1 µM) of unlabeled Ple-R3-6T (column 2) or Eu3+-labeled Ple-R3-6T (100 nM) in the absence and presence of a 10-fold molar excess (1 µM) of unlabeled Ple-N1 (column 3). Extent of inhibition of binding was calculated as 100  100 × (binding with competitor/binding without competitor). Amounts of plectin proteins bound were determined by measuring released Eu3+ by time-resolved fluorometry after addition of enhancement solution as described in the text. All data (A and B) are presented as the mean ± SD of duplicate determinations.

To map the plectin-binding site(s) in the integrin ₄ cytoplasmic domain more precisely, Ple-R_3-6T and Ple-N₁ were separately coated and overlaid with samples of Eu³⁺-labeled integrin ₄ mutant proteins (Fig. 5, A and B). Consistent with the qualitative blot overlay assay (Fig. 3), strongest binding was observed between ₄-F_1,2LF_3,4C and the carboxy-terminal plectin fragment Ple-R_3-6T (Fig. 5 A, first bar). The same integrin ₄ fragment bound to the amino-terminal plectin fragment Ple-N₁ with just ~30% lower efficiency (Fig. 5 B, first bar). Among a panel of truncated versions of ₄-F_1,2LF_3,4C tested, only those containing either the region between the second and third FNIII repeat (₄-F_1,2L and ₄-LF_3,4C) and/or the carboxy-terminal tail region following the last FNIII repeat (₄- LF_3,4C and ₄-F_3,4C) showed significant binding to either one of the plectin fragments (Fig. 5, A and B). ₄-F_1,2 and ₄-F_3,4 hardly showed any detectable binding. These results suggested that at least two different plectin-binding domains were present in the cytoplasmic domain of the integrin ₄ subunit, one between the two pairs of FNIII repeats, the other in the ultimate tail region trailing the second pair of FNIII repeats. Moreover, both binding domains apparently comprised interaction sites for amino- as well as carboxy-terminal domains of plectin.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Binding of Eu3+- labeled recombinant integrin 4 polypeptides to plectin carboxy- (A) and amino-terminal (B) protein domains immobilized on microtiter plates. 100 nM purified his-tagged recombinant plectin mutant proteins Ple-R3-6T and Ple-N1 were coated onto microtiter plates and separately overlaid with Eu3+- labeled integrin 4 proteins 4-F1,2LF3,4C, 4-F1,2L, 4-F1,2, 4-LF3,4C, 4-F3,4C, and 4-F3,4 (2.5 µM each). Amounts of bound integrin 4 proteins were determined as described in Fig. 4. Data are presented as the mean ± SD of duplicate determinations.

To examine whether the different binding sites on integrin and plectin molecules have differential affinities for each other, integrin fragments ₄-F_1,2L and ₄-F_3,4C, each containing just one of the two putative plectin-binding regions, were coated onto microtiter plates and overlaid with either Eu³⁺-labeled Ple-N₁ with or without a 10-fold molar excess of unlabeled Ple-R_3-6T (Fig. 4 B, first column) or, vice versa, with Eu³⁺-labeled Ple-R_3-6T with or without a 10-fold molar excess of unlabeled Ple-N₁ (Fig. 4 B, second column). The results of these competition experiments, documented as the relative percentage of inhibition of binding in the presence of the competitor, are summarized in Fig. 4 B. Ple-R_3-6T was able to efficiently displace Ple-N₁ from both, ₄-F_1,2L and ₄-F_3,4C, whereas reversely, Ple-N₁ hardly could displace Ple-R_3-6T from ₄-F_1,2L and inhibited its binding to ₄-F_3,4C by only ~50%. The ability of the plectin mutant proteins to displace each other from the more carboxy-terminal integrin ₄ plectin-binding domain (contained in ₄-F_3,4C) indicated that both exhibited affinities for the same binding site. Apparently, however, Ple-R_3-6T bound to this site with higher affinity compared to Ple-N₁, as indicated by its higher competition efficiency. This was consistent with the more efficient binding of ₄-F_3,4C to immobilized Ple-R_3-6T, as compared to Ple-N₁ in the microtiter plate assay (compare bars ₄-F_3,4C in Fig. 5, A and B). The inability of Ple-N₁ to displace Ple-R_3-6T from ₄-F_1,2L indicated also a significantly higher affinity of Ple-R_3-6T, compared to Ple-N₁, for the second, more amino-terminal plectin-binding site contained in this integrin ₄ fragment (₄-F_1,2L).

Integrin ₄ Mutant Proteins Exhibit Self-association upon Immobilization

Competition experiments similar to those shown above (Fig. 4 B) using immobilized plectin proteins and Eu³⁺- labeled integrin fragments in solution failed due to excessive self-interaction of integrin ₄ mutant proteins upon immobilization, a situation which also prevented the determination of dissociation constants. The ability of integrin ₄ cytoplasmic tail domains to self-associate potentially could have implications for certain functions of the protein, such as integrin clustering in hemidesmosomal complex formation and/or hemidesmosomal plaque-cytoskeleton anchorage. To obtain some information on possible molecular mechanisms and to identify the domain(s) mediating self-association, purified recombinant integrin ₄ polypeptides representing various molecular domains were resolved by SDS-PAGE, blotted onto nitrocellulose, and overlaid with ¹²⁵I-₄-F_1,2LF_3,4C (Fig. 6, A and B). ₄-F_1,2LF_3,4C reacted strongly with itself, ₄-F_1,2L, and ₄-F_3,4C, but not detectably with ₄-F_1,2 or ₄-F_3,4. This indicated that the domains involved in the aggregation of integrin ₄ were similar to those mediating integrin ₄-plectin interactions. In solution, no such aggregation of integrin ₄ proteins was found (data not shown). When ₄-F_1,2LF_3,4C was coated and overlaid with Eu³⁺-₄-F_1,2LF_3,4C (Fig. 6 C, filled bars) in either the standard overlay assay buffer (150 mM NaCl), buffer without NaCl, or buffer with increased NaCl concentration (500 mM), binding under high-salt conditions was fivefold increased over standard condition and about 10-fold reduced at low-ionic strength. Non-specific binding to BSA (Fig. 6 C, open bars) was low and even decreased with increasing ionic strength. This experiment indicated that self-interaction of ₄-F_1,2LF_3,4C was of hydrophobic nature. In a further experiment, increasing concentrations of Ple-R_3-6T were added to a constant amount of Eu³⁺- ₄-F_1,2LF_3,4C. As seen in Fig. 6 D, plectin fragments decreased the extent of integrin ₄ self-association in a concentration dependent manner. Presumably by binding to coated integrin ₄ molecules, plectin fragments blocked integrin ₄ self-association sites, which reside near or are even overlapping with the plectin-binding sites, as indicated by the blot overlay assay (Fig. 6, A and B).

View larger version (27K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Fig. 6.   Self-association of recombinant integrin 4 cytoplasmic tail fragments. (A and B) Blot overlay: purified his-tagged recombinant integrin 4 proteins 4-F1,2LF3,4C, 4-F1,2L, 4-F1,2, 4-F3,4C, and 4-F3,4 were subjected to SDS 10% PAGE in duplicate. Proteins on one gel were stained with Servablue-G (A), those on the other were blotted onto nitrocellulose membrane, overlaid with 125I-4-F1,2LF3,4C, and bound integrin 4 protein detected by autoradiography (B). Size markers of 97, 68, 43, 29, 18.4, and 14.3 kD are indicated. (C) Ionic strength dependence: 4-F1,2LF3,4C (filled bars) and BSA (open bars) were coated (both at 100 nM) and overlaid with Eu3+-labeled 4-F1,2LF3,4C (50 nM) in solutions of different ionic strengths (0, 150, and 500 mM NaCl). Scale is normalized for extent of binding under standard conditions (150 nM NaCl). Data are presented as the mean ± SD of duplicate determinations. (D) Competition experiment: 4-F1,2LF3,4C was coated (100 nM) and overlaid with 100 nM Eu3+-labeled 4-F1,2LF3,4C and different concentrations (0-2 µM) of Ple-R3-6T. Scale is normalized for extent of binding in the absence (0 µM) of Ple-R3-6T (100%). Data are presented as the mean ± SD of duplicate determinations.

Colocalization of Integrin ₄ and Plectin Mutant Proteins Ectopically Expressed in Transfected PtK2 Cells

To test whether integrin ₄ and plectin mutant proteins shown to interact in vitro would also interact when ectopically expressed in living cells, we transiently cotransfected PtK2 cells using various plectin and integrin ₄ cDNA vector constructs driving the expression of c-myc-tagged and GFP-tagged proteins. Based on the in vitro binding data, we chose to express two plectin fragments, Ple-N₁^GFP, containing the amino-terminal integrin ₄-binding site(s) identified above, and Ple-R_3-6T^myc (Ple-R_3-6T^GFP), the carboxy-terminal polypeptide exhibiting the strongest binding to integrin ₄ among all the recombinant plectin mutant proteins tested. In a first series, PtK2 cells were cotransfected with plasmids encoding the carboxy-terminal plectin fragment and one of the plasmids encoding correspondingly tagged integrin ₄ fragments ₄-F_1,2LF_3,4C^GFP, ₄-F_1,2LF_3,4C^myc, ₄-F_1,2L'^myc, ₄-F_1,2^myc, and ₄-F_3,4C'^myc (Fig. 7). Most transfected cells were double-transfected, expressing both the plectin and integrin proteins. A few cells bearing only one plasmid served as controls for single-transfected cells.

View larger version (99K): [in this window] [in a new window]   Fig. 7.   Coexpression of the carboxy-terminal plectin protein Ple-R3-6T and various integrin 4 mutant proteins in PtK2 cells. Cells plated on glass coverslips were cotransfected with a plectin construct encoding Ple-R3-6T (with either c-myc-tag or GFP linked to the carboxy terminus) and one of the integrin 4 constructs encoding 4-F1,2LF3,4CGFP (A-C), 4-F1,2L'myc (D-F), 4-F3,4C'myc (G and H), or 4-F1,2myc (I and J). After ~48 h, cells were fixed and double- or triple-stained. (A, D, G, and I) Ectopically expressed integrin 4 proteins, tagged with GFP (A; FITC optics) or c-myc (D, G, and I; Texas red optics). (B, E, H, and J) Ectopically expressed plectin protein tagged with c-myc (B; Texas red optics) or GFP (E, H, and J; FITC optics). (C and F), cytokeratins and vimentin, respectively, visualized by UV optics (AMCA). Note colocalization of 4-F1,2LF3,4CGFP/myc, 4-F1,2L'myc, and 4-F3,4C'myc, but not 4-F1,2myc, with the carboxy-terminal plectin polypeptide (Ple-R3-6TGPF/myc). Bar, 15 µm.

In cells coexpressing Ple-R_3-6T^myc and ₄-F_1,2LF_3,4C^GFP, the bulk of both ectopically expressed proteins showed codistribution and association with dense structures surrounding the nucleus; in addition, both were associated with small dot- or patch-like structures of unknown nature throughout the cytosol in superimposable patterns (Fig. 7, A and B). In rarely observed single-transfected cells expressing the plectin mutant protein alone, similar dot- and patch-like structures were not observed; such cells rather displayed filament association of mutant proteins (e.g., single-transfected cell in upper right-hand corner of Fig. 7 B), or bundling and collapse of IFs, as previously reported (Nikolic et al., 1996). As revealed by triple-staining, the cytokeratin filaments of such cotransfected cells seemed to have completely collapsed onto the nuclei, rendering the remainder of the cytoplasmic space a cytokeratin-free zone, clearly outlined by adjacent cytokeratin-positive cells (Fig. 7 C).

₄-F_1,2L'^myc, containing the first pair of FNIII repeats plus the region between the second and third FNIII repeat, colocalized with the carboxy-terminal plectin protein (Ple-R_3-6T^GFP) in dense aggregates as well as in more delicate filamentous structures (Fig. 7, D and E). These delicate structures were not seen in cells expressing Ple-R_3-6T^GFP (see e.g., single-transfected cell in upper right-hand part of Fig. 7 E) or ₄-F_1,2L'^myc alone (see e.g., transfected cell in center of Fig. 10 I), suggesting that their appearance was dependent on the coexpression of both the plectin and integrin ₄ mutant proteins. This notion was supported by the observation that in double-transfected cells, expressing comparatively low levels of ₄-F_1,2L'^myc compared to Ple-R_3-6T^GFP, delicate structures were hardly observed, but integrin-specific staining was confined to dense structures typical of collapsed filaments (see e.g., double-transfected cell in lower right-hand part of Fig. 7, D-F).

View larger version (134K): [in this window] [in a new window]   Fig. 10.   Collapse of IF network arrays in PtK2 cells ectopically expressing different integrin 4 cytoplasmic tail domain fragments. Cells plated on glass coverslips were transfected with cDNA constructs encoding mutant integrin 4-F1,2LF3,4Cmyc (A-E), 4-F3,4C'myc (F-H), or 4-F1,2L'myc (I-K). After ~48 h, cells were fixed and double- or triple-stained. (A, C, F, and I), c-myc-tagged integrin 4 proteins. (D, G, and J), vimentin, FITC optics. (B, E, H, and K), cytokeratins, UV optics (AMCA). Focus was set for the Texas red channel. Mutant proteins containing the carboxy-terminal 267 amino acids of the 4 cytoplasmic tail (4-F1,2LF3,4Cmyc and 4-F3,4C'myc) led to the collapse of both IF systems (B, D, E, G, and H), whereas the protein lacking this domain (4-F1,2L'myc) did not (J and K). Bar, 15 µm.

The expression product ₄-F_3,4C'^myc, similar to ₄-F_1,2LF_3,4C^GFP and ₄-F_1,2L'^myc, colocalized with the coexpressed carboxy-terminal plectin polypeptide in dense perinuclear structures (Fig. 7, G and H), different from the dispersed patches observed when this integrin construct was expressed alone (Fig. 7 G). In contrast, ₄-F_1,2^myc, which lacked plectin-binding activity in vitro, was diffusely localized in small dots throughout the cell, regardless of whether or not the cell was cotransfected with the plectin construct (Fig. 7, I and J). As expected, plectin staining was independent of integrin ₄ localization and its characteristics were similar to those of plectin only expression (Fig. 7 J).

The plasmid encoding the amino-terminal plectin protein Ple-N₁^GFP was used in a second set of similar cotransfection experiments using PtK2 cells (Fig. 8). Cells, cotransfected with the plasmid encoding ₄-F_1,2LF_3,4C^myc, displayed colocalization of both ectopically expressed polypeptides in brightly stained patches (Fig. 8, A and B). Colocalization was also observed with ₄-F_1,2L'^myc (Fig. 8, C and D), but the dots and patches double-stained in this case were smaller and distributed more uniformly throughout the cell as compared to the structures formed with ₄-F_1,2LF_3,4C^myc. A similar confinement of both mutant proteins within distinct dots and patches was also found in cells coexpressing ₄-F_3,4C'^myc and Ple-N₁^GFP (Fig. 8, E and F). In a single-transfected cell the weakly expressed ₄-F_3,4C'^myc was found diffusely distributed within the nuclear compartment and in form of dots and patches in the cytosol (Fig. 8 E). In contrast, ₄-F_1,2^myc, which lacks both putative plectin-binding sites, remained diffusely distributed throughout the cell (Fig. 8 G), without showing the dotty staining pattern of the plectin protein (Fig. 8 H).

View larger version (55K): [in this window] [in a new window]   Fig. 8.   Coexpression of the amino-terminal plectin protein Ple-N1 and various integrin 4 mutant proteins in PtK2 cells. Cells plated on glass coverslips were cotransfected with a cDNA construct encoding Ple-N1GFP and one of the plasmids encoding the integrin 4 mutants 4-F1,2LF3,4Cmyc (A and B), 4-F1,2L'myc (C and D), 4-F3,4C'myc (E and F), or 4-F1,2myc (G and H). After ~48 h, cells were fixed and double-stained for ectopically expressed c-myc-tagged integrin 4 proteins (A, C, E, and G; Texas red optics) and the GFP-tagged plectin protein (B, D, F, and H; FITC optics). Note colocalization of 4-F1,2LF3,4Cmyc, 4-F1,2L'myc, and 4-F3,4C'myc, but not 4-F1,2myc, with Ple-N1GFP. Bar, 15 µm.

Ultrastructural Localization of Plectin at Hemidesmosomes Using Domain-specific Antibodies

To examine whether plectin molecules found at hemidesmosomal junctions in tissues are located within distances short enough to enable their interaction with plasma membrane-anchored integrin ₄, immunogold electron microscopy of acrylic resin-embedded rat skin samples was performed. Embedding in lowicryl HM20 was found to provide excellent conditions for thin sectioning of rat skin specimens (below 100 nm), and enabled adequate hemidesmosomal fine structure resolution, comparable to other protocols (Rappersberger et al., 1990; Shimizu et al., 1992). Considering that individual plectin molecules have been visualized as structures 200 nm in length, and probably can extend over even longer distances (Foisner and Wiche, 1987; Foisner et al., 1995; Svitkina et al., 1996), we used antibodies raised against epitopes of plectin residing in three different domains of the molecule, the amino terminus (serum E1A), the rod (mAb 7A8), and the carboxy terminus (serum 135C). All three of these domain-specific immunoreagents were found to be reactive with epitopes at the periphery as well as in the cytoplasmic interior of keratinocytes. Labeling was most intense near basolateral plasma membrane domains, in particular at the locations of hemidesmosomes (Fig. 9, A-C) and desmosomes (not shown; see also Eger et al., 1997), and along cytoplasmic cytokeratin filament bundles. In the area of hemidesmosomes, label was found at the plaque, at the inner plate, and in association with cytokeratin filaments (Fig. 9, A-C).

View larger version (117K): [in this window] [in a new window]   Fig. 9.   Postembedding immunogold electron microscopy of plectin in rat skin (A-C) and schematic model highlighting plectin's putative role as a stabilizer of hemidesmosomes (D). (A-C) Indirect immunogold labeling of plectin molecules using antibodies specific to the rod domain (A; mAb 7A8, 5 nm gold), the amino terminus (B; antiserum E1A, 1 nm gold silver enhanced), and the carboxy terminus (C; antiserum 135C, 5 nm gold silver enhanced). An overview over the interface between the basal keratinocyte layer and the basement membrane showing five regularly aligned hemidesmosomes is presented in A, and details of the hemidesmosomal ultrastructure comprising the inner plate linked to keratin filaments, plaque, lamina lucida, lamina densa, and extracellular anchoring filaments and fibrils can be identified in the ~60-nm-thick sections shown in B and C. Note that all three domain-specific immunoreagents were found to be reactive with basolateral components of hemidesmosomes as well as cytoplasmic keratin filaments. Also note that specific immunolabeling using antiserum E1A was weak compared to mAb 7A8 and antiserum 135C and was observed only in combination with 1 nm, but not 5 or 10 nm, colloidal gold-conjugated secondary antibodies, indicating low accessibility of amino-terminal plectin epitopes, probably because of steric hindrance. Bars, 100 nM. (D) In this schematic drawing of a hemidesmosome, plectin molecules are depicted to connect the plaque structure with cytokeratin filaments anchored at the inner plate in a clamp-like fashion via interaction sites located at their opposite ends. Note that plectin molecules in dimeric (two polypeptide chains arranged parallel) as well as tetrameric forms (two dimers arranged anti-parallel) could provide IF- and integrin 4-binding sites at opposite ends and would be able to bridge the entire cytoplasmic domain of the hemidesmosome via their ~200-nm-long rod domains. Hd, hemidesmosome; LD, lamina densa; kf, keratin filaments; Af, anchoring filaments; Fb, anchoring fibrils; IP, inner plate; P, plaque; Ple, plectin; M, cell membrane; N, plectin amino terminus; C, plectin carboxy terminus.

None of the domain-specific anti-plectin antibodies showed noticeable preference in labeling of any hemidesmosomal substructures. Furthermore, based on the resolution of the indirect immunogold labeling used (20-30 nm), it was unlikely that plectin molecules in hemidesmosomal basolateral regions were oriented in the same way and arranged in equidistal positions. These data were consistent with a model, in which randomly oriented plectin molecules can span over the entire distance between plasma membrane-associated hemidesmosomal integrin receptor complexes and hemidesmosomal inner plate-anchored cytokeratin filament bundles (Fig. 9 D).

Overexpression of Integrin