Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture

Abstract

Previous studies suggest that plectin, a versatile cytoskeletal linker protein, has an important role in maintaining the structural integrity of diverse cells and tissues. To establish plectin's function in a living organism, we have disrupted its gene in mice. Plectin (-/-) mice died 2-3 days after birth exhibiting skin blistering caused by degeneration of keratinocytes. Ultrastructurally, hemidesmosomes and desmosomes appeared unaffected. In plectin-deficient mice, however, hemidesmosomes were found to be significantly reduced in number and apparently their mechanical stability was altered. The skin phenotype of these mice was similar to that of patients suffering from epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the plectin gene. In addition, plectin (-/-) mice revealed abnormalities reminiscent of minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart. Our results clearly demonstrate a general role of plectin in the reinforcement of mechanically stressed cells. Plectin (-/-) mice will provide a useful tool for the study of EBS-MD, and possibly other types of plectin-related myopathies involving skeletal and cardiac muscle, in an organism amenable to genetic manipulation.

Figure 1

Targeted inactivation of the plectin gene by disruption of rod-encoding exon 31. (A) Schematic representation of the plectin gene locus, the targeting vector, and the disrupted allele. Thick lines in targeting vector diagram represent homology regions. Important restriction sites, exon 31 (solid box), and part of gene replaced by neo^r-cassette (broken lines) are indicated. Digestion of the genomic DNA with restriction enzyme NcoI released a 15-kb fragment from the wild-type (wt) allele and a 2.8-kb fragment from the mutated allele (ma). Both alleles are detectable by a 3′ external probe generated by NcoI digestion, as indicated. (B) Southern blot analysis of genomic DNA prepared from parental ES cell clone (R1), targeted clone (p12-88), and from tail biopsies of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. Presence of wild-type and mutant alleles are indicated by 15- and 2.8-kb fragments, respectively. Note, nonspecific hybridization of probe with a 5-kb fragment (arrowhead) present in all samples. (C) RT–PCR analysis of skin and muscle tissues of plectin wild-type (+/+) and mutant (−/−) mice using plectin-specific or vimentin-specific primer pairs as indicated. (Lanes C) RT–PCRs performed without reverse transcriptase. Size markers (in bp) are indicated. (D) Immunoblotting of skin and muscle tissue homogenates from wild-type (+/+) and plectin (−/−) mice using rabbit antiserum to plectin (21) and mouse antiserum 135-A to the carboxy-terminal domain of plectin (right). Note that no immunoreactive protein bands were found below the ∼300-kD species on the blot portion shown (lower end of lanes, ∼90 kD) or below (data not shown). Comparable amounts of protein were loaded onto gels as confirmed by Ponceau S staining of membranes (data not shown). Molecular mass marker, 200 kD.

Figure 2

Targeted disruption of the plectin gene by replacement of exons 2–4 with a neo^r-cassette. (A) Schematic representation of the plectin gene locus, the targeting vector, and the disrupted allele. For specifications, see Fig. 1. Exons 2–4, disrupted by replacement with neo^r-cassette, are shown as solid boxes. EcoRI–DNA fragments of 13 and 9 kb (both detectable with a 5′ external probe generated by KpnI–HindIII digestion) were indicative of wild-type (wt) and mutant alleles (ma), respectively. (B) Southern blot analysis of genomic DNA prepared from the parental ES cell clone (R1), the targeted clone (p2-86), and tail biopsies of wild-type mice (+/+), and mice heterozygous (+/−), or homozygous (−/−) for the mutation. (C) RT–PCR analysis of skin and muscle tissues of plectin (+/+) and (−/−) mice using plectin-specific (h22/h6; h2/h6) or vimentin-specific (vim1/vim2) primer pairs. Size markers (in bp) are indicated. (D) Immunoblotting of skin and muscle tissue homogenates from wild-type (+/+) and plectin (−/−) mice.

Figure 3

Phenotypic analysis of plectin-deficient mice. (A) Comparison of 2-day-old wild-type (top) and plectin (−/−) (bottom) offsprings. Note large blister on forelimb (arrowhead), bleedings at extremities (arrow) and smaller size of mutant mouse. (B–E) Epoxy resin-embedded and Toluidine blue-stained sections (0.5 μm) of skin (B,C) or muscle biopsies (D,E) taken from 2-day-old animals. Note regular arrangement of epidermal cell layers [(cl) cornified layers; (gl) granular layers; (sl) spinal layers; (bk) basal keratinocytes] in wild-type littermate mouse (B), and blister formation between the dermis and the upper epidermal layers of a plectin (−/−) mouse with EBS (C, asterisk); at the blister margin basal keratinocytes are pale and swollen (arrow). (D) Cross section of skeletal muscle of a heterozygous control littermate animal with normal appearance and arrangement of muscle fibers. (E) Skeletal muscle of a plectin (−/−) mouse, showing scattered degenerating muscle fibers (asterisks). Bars, A, 0.5 cm; B–E, 10 μm.

Figure 4

Ultrastructural analysis of plectin (−/−) mouse skin. Specimens shown are from exon 2–4 (A,B,E–H) and exon 31-deficient mice (C,D). (A) Basal keratinocyte at the edge of an epidermal blister with cytoplasmic rupture in perinuclear regions; cytoplasmic components, including keratin filaments, still appear intact (arrows). The epidermal basement membrane is marked with arrowheads. (N) nucleus. (B,C) More advanced degeneration of basal keratinocyte with appearance of empty spaces in the cytoplasm and disorganization of cytoplasmic components; these are replaced by granular, moderate electron-dense material, as particularly evident in C. Arrowheads in B and small arrowheads in C mark epidermal basement membrane; large arrowhead in C, position of insert shown in D. (D) Higher magnification of C; note, inner plate of the hemidesmosome (arrowhead) is still preserved. (E) Complete dissolution of the basal keratinocyte layer, which is replaced by the fluid-filled blister. Arrowheads indicate position of epidermal basement membrane. (F) Higher magnification of the same blister as shown in E; in spite of complete dissolution of the basal keratinocyte, remnants of the cell membrane are still preserved and attached to the epidermal basement membrane (arrow). (G) Hemidesmosomes are present in basal keratinocytes outside blisters. Arrowheads denote intact outer and inner plates as well as insertion of keratin filaments into the inner plate structure. (H) Desmosomes in the upper epidermal layers are regularly structured. Bars, 1 μm.

Figure 5

Electron micrographs of skin, skeletal muscle, and heart muscle biopsies taken from 2-day-old wild-type mice. (A) Skin sections showing normal hemidesmosomes with emanating dense keratin filament bundles (arrow). (B,C) Sections through skeletal muscle showing intact sarcomeric structures (B) and the intact plasma membrane (arrow) of a muscle fiber (C). (D,E) Sections through heart muscle exhibiting intact intercalated discs (D, arrow) and normal sarcomeric structures (E). Bars, 1 μm (A,D); 0.5 μm (B); 0.2 μm (C,E).

Figure 6

Detection of plectin, integrin β4, and BPAG1 in skin of wild-type (+/+) and plectin-deficient (−/−) mice using immunofluorescence microscopy. Frozen sections were prepared from skin biopsies of 2-day-old mice. (A, A′ and B, B′) Double staining using anti-plectin mAb 10F6 (A,B) and anti-integrin β4 cytoplasmic domain antiserum (A′,B′). (C,D) Anti-integrin β4 extracellular domain mAb 346-11A; (E,F) anti-BPAG1 (mAb-5E). Arrowheads in A, A′, B, B′, and E denote basal membrane of basal keratinocytes, in F, blister floor and blister top. (G,H) Optical confocal microscopy sections in a plane (G, arrows) perpendicular to the layer of the basal keratinocytes. Bars, 20 μm (A–F); 6 μm (G,H).

Figure 7

Electron micrographs of skeletal and heart muscle of plectin (−/−) mice. (A,B) Longitudinal sections through skeletal muscle biopsies obtained from 2-day-old plectin (−/−) mice. Corelike focal disruption of myofibrils are indicated by stars, intact Z-bands by arrows, and disrupted Z-bands by arrowheads. (C) Higher magnification of B; intact plasma membrane is indicated by arrows, disrupted membrane regions by arrowheads. (D–F) Heart muscle biopsies of 2-day-old plectin-deficient mice. Note aberrant isolated myofibril bundles (D, arrowheads), dilated intercalated disc (E, arrowhead) alongside normal desmosome exhibiting keratin filament anchorage (E, arrow), and focal loss of Z-bands (F, arrowheads) next to still intact Z-bands (F, arrow). Bars, 0.5 μm (A–C); 1 μm (D); 0.2 μm (E); and 2 μm (F).

Figure 8

Immunofluorescence microscopy of cross sections through skeletal muscle from 2-day-old wild-type (+/+) and plectin-deficient (−/−) mice. (A,B) Anti-plectin mAb 10F6; arrows in A indicate pronounced staining at the periphery of muscle fibers. (C,D) Anti-vinculin; note prominent staining at the periphery of control muscle fibers (C, arrow) and the irregular, patchy staining pattern of muscle fibers in plectin-deficient mice (D, stars). (E,F) anti-spectrin; the peripheral staining observed in wild-type mice (E, arrow) is substantially diminished in plectin-deficient mice (F, star). Bar, 20 μm.

Figure 9

Immunfluorescence microscopy (anti-plectin mAb 10F6) of longitudinal sections through skeletal muscle. Muscle biopsies were obtained from 2-day-old wild type (A) and plectin-deficient (B) mice. Bar, 12 μm.