Plectin is a linker of intermediate filaments to Z-discs in skeletal muscle fibers.
Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments, and its deficiency causes the disruption of myofibrils, or muscular dystrophy. To better understand the functional role of plectin in skeletal muscle fibers, we have examined the topological and structural relationships of plectin to intermediate filaments and Z-discs in rat diaphragm muscles by confocal and immunoelectron microscopy. Immunofluorescence analysis revealed that plectin was colocalized with desmin at the periphery of Z-discs. This plectin localization around Z-discs was constantly maintained irrespective of the contracted or extended state of the muscle fibers, suggesting either direct or indirect association of plectin with Z-discs. Immunogold labeling in skinned muscle fibers clearly demonstrated that plectin-labeled fine threads linked desmin intermediate filaments to Z-discs and connected intermediate filaments to each other. These results indicate that through plectin threads desmin intermediate filaments form lateral linkages among adjacent Z-discs, preventing individual myofibrils from disruptive contraction and ensuring effective force generation. (+info)
Plectin is concentrated at intercellular junctions and at the nuclear surface in morphologically differentiated rat Sertoli cells.
Intermediate filaments in Sertoli cells have a well-defined pattern of distribution. They form a basally situated perinuclear network from which filaments extend peripherally to adhesion plaques at the plasma membrane and to sites of codistribution with other major elements of the cytoskeleton, particularly with microtubules. Although the general pattern of intermediate filament distribution is known, the molecular components involved with linking the filaments to organelles and attachment plaques in these cells have not been identified. One candidate for such a linking element is plectin. In this study we test for the presence of, and determine the distribution of, plectin in Sertoli cells of the rat testis. Fixed frozen sections and fixed epithelial fragments of rat testis were probed for plectin and vimentin using antibodies. Tissue was evaluated using standard fluorescence microscopy and confocal microscopy. Plectin in Sertoli cells was concentrated in a narrow zone surrounding the nucleus, and at focal sites, presumably desmosome-like plaques, at interfaces with adjacent cells. Plectin was also concentrated at sites where intermediate filament bundles project into specialized actin-filament containing plaques at sites of attachment to elongate spermatids. Plectin in Sertoli cells is concentrated at the nuclear surface and in junction plaques associated with the plasma membrane. The pattern of distribution is consistent with plectin being involved with linking intermediate filaments centrally (basally) to the nucleus and peripherally to intercellular attachment sites. (+info)
Structure and function of hemidesmosomes: more than simple adhesion complexes.
The attachment of cells to the extracellular matrix is of crucial importance in the maintenance of tissue structure and integrity. In stratified epithelia such as in skin as well as in other complex epithelia multiprotein complexes called hemidesmosomes are involved in promoting the adhesion of epithelial cells to the underlying basement membrane. In the past few years our understanding of the role of hemidesmosomes has improved considerably. Their importance has become apparent in clinical conditions, in which absence or defects of hemidesmosomal proteins result in devastating blistering diseases of the skin. Molecular genetic studies have increased our knowledge of the function of the various components of hemidesmosomes and enabled the characterization of protein-protein interactions involved in their assembly. It has become clear that the alpha6beta4 integrin, a major component of hemidesmosomes, is able to transduce signals from the extracellular matrix to the interior of the cell, that critically modulate the organization of the cytoskeleton, proliferation, apoptosis, and differentiation. Nevertheless, our knowledge of the mechanisms regulating the functional state of hemidesmosomes and, hence, the dynamics of cell adhesion, a process of crucial importance in development, wound healing or tumor invasion, remains limited. The aims of this review are to highlight the recent progresses of our knowledge on the organization and assembly of hemidesmosomes, their involvement in signaling pathways as well as their participation in clinical pathologic conditions. (+info)
Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.
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 alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-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 alpha6beta4 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 beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 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 beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 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 beta4 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. (+info)
Plectin, the most versatile cytolinker identified to date, has essential functions in maintaining the mechanical integrity of skin, skeletal muscle and heart, as indicated by analyses of plectin-deficient mice and humans. Expression of plectin in a vast variety of tissues and cell types, combined with a large number of different binding partners identified at the molecular level, calls for complex mechanisms regulating gene transcription and expression of the protein. To investigate these mechanisms, we analyzed the transcript diversity and genomic organization of the murine plectin gene and found a remarkable complexity of its 5'-end structure. An unusually high number of 14 alternatively spliced exons, 11 of them directly splicing into plectin exon 2, were identified. Analysis of their tissue distribution revealed that expression of a few of them is restricted to tissues such as brain, or skeletal muscle and heart. In addition, we found two short exons tissue-specifically spliced into a highly conserved set of exons encoding the N-terminal actin binding domain (ABD), common to plectin and the superfamily of spectrin/dystrophin-type actin binding proteins. Using recombinant proteins we show that a novel ABD version contained in the muscle-specific isoform of plectin exhibits significantly higher actin binding activity than other splice forms. This fine tuning mechanism based on alternative splicing is likely to optimize the proposed biological role of plectin as a cytolinker opposing intense mechanical forces in tissues like striated muscle. (+info)
Cytoskeletal linkers: new MAPs for old destinations.
A new isoform of the actin-neurofilament linker protein BPAG has been found that binds to and stabilizes axonal microtubules. This and other newly identified microtubule-associated proteins are likely to be just the tip of an iceberg of multifunctional proteins that stabilize and crosslink cytoskeletal filament networks. (+info)
Identification of the hemidesmosomal 500 kDa protein (HD1) as plectin.
HD1 is a 500 kDa hemidesmosomal plaque protein recognized by monoclonal antibody mAb-121. Recent research on inherited skin disease has suggested that it might be identical to plectin or an isoform. To cast light on this question, we have prepared several monoclonal antibodies that recognize a 500 kDa protein in the hemidesmosome fraction. Unexpectedly, some staining pattern heterogeneity was observed on immunofluorescence microscopy. Attention was focused on two monoclonal antibodies which gave different localization in bovine skin and retinal pigment epithelial cells. Determination of the amino-terminal sequence of an antigenic 100 kDa polypeptide fragment derived from the 500 kDa component of an insoluble fraction of bovine hepatocytes revealed it was identical to that of plectin. Using the two antibodies, we screened a cDNA library derived from BMGE+H, a bovine mammary gland epithelial cell line. The isolated cDNA clones corresponded to the rod domain of bovine plectin, with two separate epitope regions for each of the antibodies. From these results we conclude that the hemidesmosomal 500 kDa component HD1 is identical to plectin. As judged on rough estimation of molar ratios on this basis, hemidesmosomes are composed of plectin, BP230, the integrin beta4 subunit, and alpha6 in a 1:1:1:1 ratio. (+info)
Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin's intermediate filament-binding domain.
Plectin, the largest and most versatile member of the cytolinker/plakin family of proteins characterized to date, has a tripartite structure comprising a central 200 nm-long (&agr;)-helical rod domain flanked by large globular domains. The C-terminal domain comprises a short tail region preceded by six highly conserved repeats (each 28-39 kDa), one of which (repeat 5) contains plectin's intermediate filament (IF)-binding site. We used recombinant and native proteins to assess the effects of plectin repeat 5-binding to IF proteins of different types. Quantitative Eu(3+)-based overlay assays showed that plectin's repeat 5 domain bound to type III IF proteins (vimentin) with preference over type I and II cytokeratins 5 and 14. The ability of both types of IF proteins to self-assemble into filaments in vitro was impaired by plectin's repeat 5 domain in a concentration-dependent manner, as revealed by negative staining and rotary shadowing electron microscopy. This effect was much more pronounced in the case of vimentin compared to cytokeratins 5/14. Preassembled filaments of both types became more and more crosslinked upon incubation with increasing concentrations of plectin repeat 5. However, at high proportions of plectin to IF proteins, disassembly of filaments occurred. Again, vimentin filaments proved considerably more sensitive towards disassembly than those composed of cytokeratins 5 and 14. In general, IFs formed from recombinant proteins were found to be slightly more responsive towards plectin influences than their native counterparts. A dose-dependent plectin-inflicted collapse and putative disruption of IFs was also observed in vivo after ectopic expression of vimentin and plectin's repeat 5 domain in cotransfected vimentin-deficient SW13 (vim(-)) cells. Our results suggest an involvement of plectin not only in crosslinking and stabilization of cytoskeletal IF networks, but also in regulation of their dynamics. (+info)