Plectin regulates Wnt signaling mediated-skeletal muscle development by interacting with Dishevelled-2 and antagonizing autophagy.

Skeletal muscle is the most abundant tissue in the human and animal body, loss of its function can lead to muscle aging and various myogenic diseases. The skeletal muscle development is a complex and tightly regulated process, which is driven by a variety of many factors, signaling pathways and regulatory mechanisms. Plectin (Plec), a cytolinker protein, is ubiquitously expressed in various tissues such as skin, muscle, plasma membrane, and most types of cells. Although known isoforms of Plec is well-characterized in muscle dystrophy, very little is known on the function of Plec in the skeletal muscle development. Here, we found that Plec plays a vital role in promoting C2C12 myoblasts differentiation and proliferation, but inhibits their apoptosis. Also, Plec regulates the expression of atrophy-related genes (atrogin-1 and muRF-1) to rescue muscle atrophy. Furthermore, we have demonstrated that Plec binds to Dishevelled-2 (Dvl-2) and forms a protein complex, which is then activate the canonical Wnt signaling. We also observed that Plec resists ubiquitination by stabilizing Dvl-2 and reduces the level of LC3-labeled Dvl-2 and antagonizes the autophagy system. In conclusion, our findings suggest that Plec regulates canonical Wnt signaling mediated skeletal development by stabilizing Dvl-2 and downregulating the cellular autophagic degradation system.

On the role of tubulin, plectin, desmin, and vimentin in the regulation of mitochondrial energy fluxes in muscle cells.

Mitochondria perform a central role in life and death of the eukaryotic cell. They are major players in the generation of macroergic compounds and function as integrated signaling pathways, including the regulation of Ca2+ signals and apoptosis. A growing amount of evidence is demonstrating that mitochondria of muscle cells use cytoskeletal proteins (both microtubules and intermediate filaments) not only for their movement and proper cellular positioning, but also to maintain their biogenesis, morphology, function, and regulation of energy fluxes through the outer mitochondrial membrane (MOM). Here we consider the known literature data concerning the role of tubulin, plectin, desmin and vimentin in bioenergetic function of mitochondria in striated muscle cells, as well as in controlling the permeability of MOM for adenine nucleotides (ADNs). This is of great interest since dysfunctionality of these cytoskeletal proteins has been shown to result in severe myopathy associated with pronounced mitochondrial dysfunction. Further efforts are needed to uncover the pathways by which the cytoskeleton supports the functional capacity of mitochondria and transport of ADN(s) across the MOM (through voltage-dependent anion channel).

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through ß4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating ß4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells.

Epidermolysis bullosa simplex in sibling Eurasier dogs is caused by a PLEC non-sense variant.

BACKGROUND: Plectin, a large linker protein found in many tissues, acts to connect components of the cytoskeleton to each other. In the epidermis, plectin binds keratin intermediate filaments to hemidesmosomes. A deficiency of plectin in the skin leads to blister formation in the basal layer and the disease epidermolysis bullosa simplex (EBS). HYPOTHESIS/OBJECTIVES: To describe a novel blistering disease that arose spontaneously in a litter of puppies. ANIMALS: Two female and one male 20-day-old Eurasier puppies, from a litter of six, were presented for evaluation of failure to thrive and then euthanized due to poor prognosis. The puppies had ulcers on the lips, tongue, nasal planum, paw pads and abdomen. RESULTS: Immunolabelling on frozen skin for basement membrane proteins revealed patchy and weak to absent staining for plectin as compared with strong linear staining in normal dogs. Ultrastructurally, hemidesmosomes were irregularly shaped and had loss of distinction between inner and outer plaques. Pedigree analysis supported an autosomal recessive mode of inheritance. A premature stop codon was discovered in exon 27 of PLEC that resulted in the production of a severely truncated protein. CONCLUSION: The study describes the first documented spontaneous EBS associated with a PLEC variant in domestic animals.

Determining the mechanical properties of plectin in mouse myoblasts and keratinocytes.

Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated paramagnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between plectin-deficient and wild-type keratinocytes, indicating that plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in plectin-deficient and wild-type myoblasts, but was significantly increased in plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of plectin has divergent implications on biomechanical properties depending on the respective cell type.

Stabilization of the dystroglycan complex in Cajal bands of myelinating Schwann cells through plectin-mediated anchorage to vimentin filaments.

Previous studies have unmasked plectin, a uniquely versatile intermediate filament-associated cytolinker protein, to be essential for skin and skeletal muscle integrity. Different sets of isoforms of the protein were found to stabilize cells mechanically, regulate cytoskeletal dynamics, and serve as a scaffolding platform for signaling molecules. Here, we investigated whether a similar scenario prevails in myelinating Schwann cells. Using isoform-specific antibodies, the two plectin variants predominantly expressed in the cytoplasmic compartment (Cajal bands) of Schwann cells were identified as plectin (P)1 and P1c. Coimmunoprecipitation and immunolocalization experiments revealed complex formation of Cajal band plectin with ß-dystroglycan, the core component of the dystrophin glycoprotein complex that in Schwann cells is crucial for the compartmentalization and stabilization of the myelin sheath. To study the functional implications of Schwann cell-specific plectin-ß-dystroglycan interaction, we generated conditional (Schwann cell-restricted) plectin knockout mice. Ablation of plectin in myelinating Schwann cells (SCs) was found not to affect myelin sheath formation but to abrogate the tight association of the dystroglycan complex with the intermediate filament cytoskeleton. We show that the disruption of this association leads to the destabilization of the dystroglycan complex combined with increased myelin sheath deformations observed in the peripheral nerve during ageing of the animal.

Keratins mediate localization of hemidesmosomes and repress cell motility.

The keratin (K)-hemidesmosome (HD) interaction is crucial for cell-matrix adhesion and migration in several epithelia, including the epidermis. Mutations in constituent proteins cause severe blistering skin disorders by disrupting the adhesion complex. Despite extensive studies, the role of keratins in HD assembly and maintenance is only partially understood. Here we address this issue in keratinocytes in which all keratins are depleted by genome engineering. Unexpectedly, such keratinocytes maintain many characteristics of their normal counterparts. However, the absence of the entire keratin cytoskeleton leads to loss of plectin from the hemidesmosomal plaque and scattering of the HD transmembrane core along the basement membrane zone. To investigate the functional consequences, we performed migration and adhesion assays. These revealed that, in the absence of keratins, keratinocytes adhere much faster to extracellular matrix substrates and migrate approximately two times faster compared with wild-type cells. Reexpression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a role of keratins, which to our knowledge is previously unreported, in the maintenance of HDs upstream of plectin, with implications for epidermal homeostasis and pathogenesis. They support the view that the downregulation of keratins observed during epithelial-mesenchymal transition supports the migratory and invasive behavior of tumor cells.

Peristomal and generalized bullous pemphigoid in patients with underlying inflammatory bowel disease: is plectin the missing link?

Bullous pemphigoid (BP) is a blistering disorder of the skin and mucosa that may coexist with inflammatory bowel disease (IBD). The authors' experiences with peristomal and generalized BP in five patients (three with ulcerative colitis [UC] post colostomy surgery and two with Crohn's disease [CD] post ileostomy surgery, time since surgery 5 to 20 years) is described. The patients presented with peristomal blisters and erosions, subsequently confirmed as BP by skin biopsy. Treatments for the skin disease included potent alcohol-based topical corticosteroids, oral tetracyclines, and oral corticosteroids. In three patients (two with UC, one with CD), the initially localized peristomal disease later became generalized across the skin; these patients were more likely to require systemic immunosuppressive therapy. Because an involvement of plectin, a cytoskeletal protein that attaches skin and mucosal cells to their extracellular matrix, in IBD has been shown, it is possible that this protein forms the missing link between IBD and BP via epitope spreading. The inflammation of IBD exposes plectin, stimulating a secondary immune response that may, in susceptible individuals, crossreact with the skin, provoking BP. Further research into this area could enable clinical testing for plectin auto-antibodies in patient sera, possibly preempting the development of BP and expediting the initiation of early effective treatment.

Plectin promotes migration and invasion of cancer cells and is a novel prognostic marker for head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 1/2 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate with the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC.

BRCA2 interacts with the cytoskeletal linker protein plectin to form a complex controlling centrosome localization.

The breast cancer susceptibility gene (BRCA2) is localized mainly in the nucleus where it plays an important role in DNA damage repair. Some BRCA2 protein is also present in the centrosome. Here, we demonstrate that BRCA2 interacts with plectin, a cytoskeletal cross-linker protein, and that this interaction controls the position of the centrosome. Phosphorylation of plectin by cyclin-dependent kinase 1/cyclin B (CDK1/CycB) kinase has been reported to abolish its cross-linking function during mitosis. Here, we induced phosphorylation of plectin in prepared fractions of HeLa cells by adding activated CDK1/CycB kinase. Consequently, there was significant dissociation of the centrosome from the nuclear membrane. Plectin has six homologous ankyrin-like repeat domains (termed PLEC M1-M6). Using a pull-down assay, we found that GST-PLEC M1 and a GST-C-terminal region fusion protein (which comprised PLEC M6, along with an adjacent vimentin site) interacted with BRCA2. Since each PLEC module exhibits high homology to the others, the possibility of all six domains participating in this interaction was indicated. Moreover, when PLEC M1 was overexpressed in HeLa cells, it competed with endogenous plectin and inhibited the BRCA2-plectin interaction. This inhibitory effect resulted in dissociation of the centrosomes from the nucleus and increased the rate of micronuclei formation which may lead to carcinogenesis. In addition, when either BRCA2 or plectin was suppressed by the appropriate siRNA, a similar change in centrosomal positioning was observed. We suggest that the BRCA2-plectin interaction plays an important role in the regulation of centrosome localization and also that displacement of the centrosome may result in genomic instability and cancer development.

Skin and heart: une liaison dangereuse.

Both skin and heart are subject to shear mechanical stress and need to be stress-resistant in a flexible way. The intercellular connecting structures in skin and heart, the desmosomes, that have to resist these forces show remarkable resemblance in epidermis and myocardium. Mutations in desmosomal proteins lead to inherited desmosomal cardiocutaneous syndromes (DCCS): une liaison dangereuse. This article will critically review the cutaneous and cardiac features as well as the molecular background of DCCS, such as Naxos disease and Carvajal syndrome caused by deficiencies of plakoglobin and desmoplakin respectively. In addition, potential other desmosomal gene candidates for an involvement in cardiocutaneous syndromes are considered. The skin features in these syndromes may be the hallmark for the presence of progressive and ultimately lethal cardiac disease. Knowledge of these skin features and early recognition of such a syndrome may provide opportunities to halt or slow down cardiac disease progression, treat arrhythmias and even prevent sudden death.

Plectin deficiency affects precursor formation and dynamics of vimentin networks.

Plectin is a typical cytolinker protein that connects intermediate filaments to the other cytoskeletal filament systems and anchors them at membrane-associated junctional sites. One of the most important binding partners of plectin in fibroblasts is the intermediate filament subunit protein vimentin. Previous studies have demonstrated that vimentin networks are highly dynamic structures whose assembly and disassembly is accomplished stepwise via several intermediates. The precursor forms as well as polymerized (filamentous) vimentin are found in the cells in a dynamic equilibrium characterized by the turnover of the subunits within the polymer and the movement of the smaller precursors. To examine whether plectin plays a role in intermediate filament dynamics, we studied vimentin filament formation in plectin-deficient compared to wild-type fibroblasts using GFP-tagged vimentin. Monitoring vimentin and plectin in spreading and dividing cells, we demonstrate that plectin is associated with vimentin from the early stages of assembly and is required for vimentin motility as well as for the stepwise formation of stable filaments. Furthermore, plectin prevents vimentin networks from complete disassembly during mitosis, facilitating the rebuilding of the intermediate filament network in daughter cells.

Immunofluorescence analysis of villous trophoblasts: a tool for prenatal diagnosis of inherited epidermolysis bullosa with pyloric atresia.

Genetic mutations invalidating the genes for integrin alpha6beta4 and, in some cases, plectin are associated with junctional and simplex epidermolysis bullosa with pyloric atresia (PA-JEB and PA-EBS), respectively. These recessive inherited conditions are characterized by pregnancies with fetal bullae, pyloric atresia, polyhydramnios, and neonatal mucocutaneous blistering, which often results in early postnatal demise. To date, first-trimester DNA-based prenatal diagnosis is not applicable to affected kindred carrying as yet unidentified genetic mutations. Here, we show that first-trimester chorionic villi strongly express both integrin alpha6beta4 and plectin, which persist throughout the pregnancy. Based on this observation, we implemented 25 prenatal diagnoses in kindred at risk for PA-EB by immunomapping, which identified three PA-JEB-affected fetuses and 22 healthy ones. In 19 cases, including the three PA-JEB pregnancies that were prematurely terminated, the results were confirmed by chorionic villous DNA-based tests, which also led to the identification of seven previously unreported mutations in the alpha6beta4 integrin genes. Our prediction was further sustained by the birth of 22 healthy babies. These results validate chorionic villi immunofluorescence examination as a tool for prenatal diagnosis of PA-JEB and PA-EBS and indicate that this procedure could be devised for EB with muscular dystrophy, which is also associated with genetic mutations in plectin.

A maturational model for the study of airway smooth muscle adaptation to mechanical oscillation.

It has been shown that mechanical stretches imposed on airway smooth muscle (ASM) by deep inspiration reduce the subsequent contractile response of the ASM. This passive maneuver of lengthening and retraction of the muscle is beneficial in normal subjects to counteract bronchospasm. However, it is detrimental to hyperresponsive airways because it triggers further bronchoconstriction. Although the exact mechanisms for this contrary response by normal and hyperresponsive airways are unclear, it has been suggested that the phenomenon is related to changes in ASM adaptability to mechanical oscillation. Healthy immature airways of both human and animal exhibit hyperresponsiveness, but whether the adaptative properties of hyperresponsive airway differ from normal is still unknown. In this article, we review the phenomenon of ASM adaptation to mechanical oscillation and its relevance and implication to airway hyperresponsiveness. We demonstrate that the age-specific expression of ASM adaptation is prominent using an established maturational animal model developed in our laboratory. Our data on immature ASM showed potentiated contractile force shortly after a length oscillation compared with the maximum force generated before oscillation. Several potential mechanisms such as myogenic response, changes in actin polymerization, or changes in the quantity of the cytoskeletal regulatory proteins plectin and vimentin, which may underlie this age-specific force potentiation, are discussed. We suggest a working model of the structure of smooth muscle associated with force transmission, which may help to elucidate the mechanisms responsible for the age-specific expression of smooth muscle adaptation. It is important to study the maturational profile of ASM adaptation as it could contribute to juvenile hyperresponsiveness.