In skeletal muscle, the cytolinker plectin is prominently expressed at Z-disks and the sarcolemma. Alternative splicing of plectin transcripts gives rise to more than eight protein isoforms differing only in small N-terminal sequences (5–180 residues), four of which (plectins 1, 1b, 1d, and 1f) are found at substantial levels in muscle tissue. Using plectin isoform–specific antibodies and isoform expression constructs, we show the differential regulation of plectin isoforms during myotube differentiation and their localization to different compartments of muscle fibers, identifying plectins 1 and 1f as sarcolemma-associated isoforms, whereas plectin 1d localizes exclusively to Z-disks. Coimmunoprecipitation and in vitro binding assays using recombinant protein fragments revealed the direct binding of plectin to dystrophin (utrophin) and β-dystroglycan, the key components of the dystrophin–glycoprotein complex. We propose a model in which plectin acts as a universal mediator of desmin intermediate filament anchorage at the sarcolemma and Z-disks. It also explains the plectin phenotype observed in dystrophic skeletal muscle of mdx mice and Duchenne muscular dystrophy patients.

Plectin, a major linker and scaffolding protein of the cytoskeleton, has been shown to be essential for the mechanical integrity of skin, skeletal muscle, and heart. Studying fibroblast and astroglial cell cultures derived from plectin (-/-) mice, we found that their actin cytoskeleton, including focal adhesion contacts, was developed more extensively than in wild-type cells. Also it failed to show characteristic short-term rearrangments in response to extracellular stimuli activating the Rho/Rac/Cdc42 signaling cascades. As a consequence, cell motility, adherence, and shear stress resistance were altered, and morphogenic processes were delayed. Furthermore, we show that plectin interacts with G-actin in vitro in a phosphatidylinositol-4,5-biphosphate-dependent manner and associates with actin stress fibers in living cells. The actin stress fiber phenotype of plectin-deficient fibroblasts could be reversed to a large degree by transient transfection of full-length plectin or plectin fragments containing the amino-terminal actin-binding domain (ABD). These results reveal a novel role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its proposed role in scaffolding and mechanical stabilization of cells.

Abstract. We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of ~50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IFbinding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg 428°) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.