Plectin dysfunction in neurons leads to tau accumulation on microtubules affecting neuritogenesis, organelle trafficking, pain sensitivity and memory.

AIMS: Plectin, a universally expressed multi-functional cytolinker protein, is crucial for intermediate filament networking, including crosstalk with actomyosin and microtubules. In addition to its involvement in a number of diseases affecting skin, skeletal muscle, heart, and other stress-exposed tissues, indications for a neuropathological role of plectin have emerged. Having identified P1c as the major isoform expressed in neural tissues in previous studies, our aim for the present work was to investigate whether, and by which mechanism(s), the targeted deletion of this isoform affects neuritogenesis and proper nerve cell functioning. METHODS: For ex vivo phenotyping, we used dorsal root ganglion and hippocampal neurons derived from isoform P1c-deficient and plectin-null mice, complemented by in vitro experiments using purified proteins and cell fractions. To assess the physiological significance of the phenotypic alterations observed in P1c-deficient neurons, P1c-deficient and wild-type littermate mice were subjected to standard behavioural tests. RESULTS: We demonstrate that P1c affects axonal microtubule dynamics by isoform-specific interaction with tubulin. P1c deficiency in neurons leads to altered dynamics of microtubules and excessive association with tau protein, affecting neuritogenesis, neurite branching, growth cone morphology, and translocation and directionality of movement of vesicles and mitochondria. On the organismal level, we found P1c deficiency manifesting as impaired pain sensitivity, diminished learning capabilities and reduced long-term memory of mice. CONCLUSIONS: Revealing a regulatory role of plectin scaffolds in microtubule-dependent nerve cell functions, our results have potential implications for cytoskeleton-related neuropathies.

Laryngeal lesion associated with epidermolysis bullosa secondary to congenital plectin deficiency.

INTRODUCTION: Epidermolysis bullosa (EB) is a congenital disease characterized by fragility of epithelial structures. The skin is the organ primarily affected, resulting in the formation of skin blisters. Some forms of EB may also present mucosal lesions. CASE REPORT: We report the case of a girl with epidermolysis bullosa simplex (EBS) associated with muscular dystrophy secondary to congenital plectin deficiency. She presented severe respiratory tract lesions extending from the oral cavity to the larynx. In particular, we describe our medical and surgical management of the laryngeal lesions, responsible for several episodes of respiratory distress and feeding difficulties. DISCUSSION: Epidermolysis bullosa simplex associated with muscular dystrophy is a rare hereditary form of EB, as fewer than 50 cases have been reported in the literature. This form is characterized by mucosal lesions involving the upper aerodigestive tract, with consequences for feeding, phonation and breathing. Special care must be taken when performing diagnostic and therapeutic procedures to avoid worsening the lesions of this very fragile mucosa. Tracheotomy is a harmful procedure in these patients and should only be considered as a last resort.

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment.

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

Plectin controls biliary tree architecture and stability in cholestasis.

BACKGROUND & AIMS: Plectin, a highly versatile cytolinker protein, controls intermediate filament cytoarchitecture and cellular stress response. In the present study, we investigate the role of plectin in the liver under basal conditions and in experimental cholestasis. METHODS: We generated liver-specific plectin knockout (PleΔalb) mice and analyzed them using two cholestatic liver injury models: bile duct ligation (BDL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Primary hepatocytes and a cholangiocyte cell line were used to address the impact of plectin on keratin filament organization and stability in vitro. RESULTS: Plectin deficiency in hepatocytes and biliary epithelial cells led to aberrant keratin filament network organization, biliary tree malformations, and collapse of bile ducts and ductules. Further, plectin ablation significantly aggravated biliary damage upon cholestatic challenge. Coincidently, we observed a significant expansion of A6-positive progenitor cells in PleΔalb livers. After BDL, plectin-deficient bile ducts were prominently dilated with more frequent ruptures corresponding to an increased number of bile infarcts. In addition, more abundant keratin aggregates indicated less stable keratin filaments in PleΔalb hepatocytes. A transmission electron microscopy analysis revealed a compromised tight junction formation in plectin-deficient biliary epithelial cells. In addition, protein profiling showed increased expression of the adherens junction protein E-Cadherin, and inefficient upregulation of the desmosomal protein desmoplakin in response to BDL. In vitro analyses revealed a higher susceptibility of plectin-deficient keratin networks to stress-induced collapse, paralleled by elevated activation of p38 MAP kinase. CONCLUSION: Our study shows that by maintaining proper keratin network cytoarchitecture and biliary epithelial stability, plectin plays a critical role in protecting the liver from stress elicited by cholestasis. LAY SUMMARY: Plectin is a cytolinker protein capable of interconnecting all three cytoskeletal filament systems and linking them to plasma membrane-bound junctional complexes. In liver, the plectin-controlled cytoskeleton mechanically stabilizes epithelial cells and provides them with the capacity to adapt to increased bile pressure under cholestasis.

Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle.

Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. 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. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion-fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways.

Plectin-related skin diseases.

Plectin has been characterized as a linker protein that is expressed in many cell types and is distinctive in various isoforms in the N-terminus and around the rod domain due to complicated alternative splicing of PLEC, the gene encoding plectin. Plectin deficiency causes autosomal recessive epidermolysis bullosa simplex (EBS) with involvement of the skin and other organs, such as muscle and gastrointestinal tract, depending on the expression pattern of the defective protein. In addition, a point mutation in the rod domain of plectin leads to autosomal dominant EBS, called as EBS-Ogna. Plectin can be targeted by circulating autoantibodies in subepidermal autoimmune blistering diseases. This review summarizes plectin-related skin diseases, from congenital to autoimmune disorders.

Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle.

The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD.

Epidermolysis bullosa with late-onset muscular dystrophy and plectin deficiency.

Epidermolysis bullosa associated with muscular dystrophy is a rare, autosomal recessive form of epidermolysis bullosa simplex caused by mutations in the plectin gene, PLEC1. We describe a phenotypically mild case due to compound heterozygous mutations in PLEC1 (2677_2685del and the novel mutation Q1644X). Clinical features included mild skin blistering since birth, slowly progressive and late-onset upper limb-predominant weakness, facial weakness, ptosis, incomplete ophthalmoplegia, and paroxysmal atrial fibrillation.

Plectin deficiency on cytoskeletal disorganization and transformation of human liver cells in vitro.

Plectin is a versatile cytoplasmic cross-linking protein that connects intermediate filaments to microfilaments, microtubules, and membrane adhesion sites. The cross-linking functions of plectin help organize the cytoskeleton into a stable meshwork important for maintaining uniformity in cell size and shape. As cells of hepatocellular carcinoma are morphologically different from normal human hepatocytes, we hypothesized that altered plectin expression and cytoskeletal organization underlies this pleomorphic transformation. To test this hypothesis, we analyzed expression levels and organization of all cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after plectin knockdown in human Chang liver cells. We found that expression of cytokeratin 18, but not actin or tubulin, was downregulated by suppression of plectin protein. Furthermore, cytokeratin networks were partially collapsed and actin-rich stress fibers were increased. The organization of microtubule networks, by contrast, was unaltered. These findings support our hypothesis that, via effects on cytoskeletal organization, plectin deficiency might play an important role in the transformation of human liver cells.

Ptosis and ophthalmoplegia associated with epidermolysis bullosa simplex-muscular dystrophy.

The association of epidermolysis bullosa simplex and muscular dystrophy (EBS-MD) has rarely been discussed in ophthalmology literature. This case report offers a brief summary of epidermolysis bullosa and describes what is known about EBS-MD. The case involves a patient with EBS-MD who presented with ptosis and ophthalmoplegia, suggesting that these may be complications of EBS-MD.

Plectin deficiency leads to both muscular dystrophy and pyloric atresia in epidermolysis bullosa simplex.

Plectin is a cytoskeletal linker protein which has a long central rod and N- and C-terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS-MD), and EBS with pyloric atresia (EBS-PA). Previous studies have demonstrated that loss of full-length plectin with residual expression of the rodless isoform leads to EBS-MD, whereas complete loss or marked attenuation of expression of full-length and rodless plectin underlies the more severe EBS-PA phenotype. However, muscular dystrophy has never been identified in EBS-PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon-causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient.

Respiratory tract involvement in a child with epidermolysis bullosa simplex with plectin deficiency: a case report.

We report a rare case of a child with epidermolysis bullosa simplex (EBS) with plectin deficiency but without muscular dystrophy, with severe lesions of the oral cavity, oropharyngeal, hypopharyngeal, laryngeal, tracheal and bronchial mucosa. Case report and a review of the world literature are used. The literature review revealed only five similar patients with EBS without muscular dystrophy complicated by respiratory involvement. This paper highlights the potentially serious complications of the EB in the form of breathing, swallowing and speech difficulties and describes the specific problems encountered in the treatment of this patient. Epidermolysis bullosa (EB) is a group of severe hereditary diseases, primarily of the skin, but which can also involve the respiratory and gastrointestinal tract mucosa. Respiratory tract involvement is usually only found in certain types of EB. The oral cavity and oropharynx are involved more frequently than the hypopharynx, larynx and trachea. Involvement of laryngeal and tracheal mucosa is generally associated with an increased morbidity and mortality, numerous complications and therapeutic difficulties, and is more common in junctional EB and dystrophic EB than in EBS. We present a rare case of a child with EBS and plectin deficiency with pronounced lesions of respiratory tract mucosa from the oral cavity to the bronchi and even extending into the trachea. Deciding on tracheotomy requires thorough consideration and should not be taken lightly.

BPAG1 isoform-b: complex distribution pattern in striated and heart muscle and association with plectin and alpha-actinin.

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein alpha-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like alpha-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and alpha-actinin-2, and its NH(2)-terminus, which contains an actin-binding domain, directly interacted with that of plectin and alpha-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.

Plectin contributes to mechanical properties of living cells.

Plectin is a 500-kDa cross-linking protein that plays important roles in a number of cell functions including migration and wound healing. We set out to characterize the role of plectin in mechanical properties of living cells. Plectin(-/-) cells were less stiff than plectin(+/+) cells, but the slopes of the two power laws in response to loading frequencies (0.002-1,000 Hz) were similar. Plectin(-/-) cells lost the capacity to propagate mechanical stresses to long distances in the cytoplasm; traction forces in plectin(-/-) cells were only half of those in plectin(+/+) cells, suggesting that plectin deficiency compromised prestress generation, which, in turn, resulted in the inhibition of long distance stress propagation. Both plectin(+/+) and plectin(-/-) cells exhibited nonlinear stress-strain relationships. However, plectin(+/+) cells, but not plectin(-/-) cells, further stiffened in response to lysophosphatidic acid (LPA). Dynamic fluorescence resonance energy transfer analysis revealed that RhoA GTPase proteins were activated in plectin(+/+) cells but not in plectin(-/-) cells after treatment with LPA. Expression in plectin(-/-) cells of constitutively active RhoA (RhoA-V14) but not a dominant negative mutant of RhoA (RhoA-N19) or an empty vector restored the long distance force propagation behavior, suggesting that plectin is important in normal functions of RhoA. Our findings underscore the importance of plectin for mechanical properties, stress propagation, and prestress of living cells, thereby influencing their biological functions.

Plectin isoform 1b mediates mitochondrion-intermediate filament network linkage and controls organelle shape.

Plectin is a versatile intermediate filament (IF)-bound cytolinker protein with a variety of differentially spliced isoforms accounting for its multiple functions. One particular isoform, plectin 1b (P1b), remains associated with mitochondria after biochemical fractionation of fibroblasts and cells expressing exogenous P1b. Here, we determined that P1b is inserted into the outer mitochondrial membrane with the exon 1b-encoded N-terminal sequence serving as a mitochondrial targeting and anchoring signal. To study P1b-related mitochondrial functions, we generated mice that selectively lack this isoform but express all others. In primary fibroblasts and myoblasts derived from these mice, we observe a substantial elongation of mitochondrial networks, whereas other mitochondrial properties remain largely unaffected. Normal morphology of mitochondria could be restored by isoform-specific overexpression of P1b in P1b-deficient as well as plectin-null cells. We propose a model where P1b both forms a mitochondrial signaling platform and affects organelle shape and network formation by tethering mitochondria to IFs.

Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms.

Dysfunction of plectin, a 500-kD cytolinker protein, leads to skin blistering and muscular dystrophy. Using conditional gene targeting in mice, we show that plectin deficiency results in progressive degenerative alterations in striated muscle, including aggregation and partial loss of intermediate filament (IF) networks, detachment of the contractile apparatus from the sarcolemma, profound changes in myofiber costameric cytoarchitecture, and decreased mitochondrial number and function. Analysis of newly generated plectin isoform-specific knockout mouse models revealed that IF aggregates accumulate in distinct cytoplasmic compartments, depending on which isoform is missing. Our data show that two major plectin isoforms expressed in muscle, plectin 1d and 1f, integrate fibers by specifically targeting and linking desmin IFs to Z-disks and costameres, whereas plectin 1b establishes a linkage to mitochondria. Furthermore, disruption of Z-disk and costamere linkages leads to the pathological condition of epidermolysis bullosa with muscular dystrophy. Our findings establish plectin as the major organizer of desmin IFs in myofibers and provide new insights into plectin- and desmin-related muscular dystrophies.

The influence of plectin deficiency on stability of cytokeratin18 in hepatocellular carcinoma.

Intermediate filaments are important in building the cellular architecture. Previously we found cytokeratin18 was modulated in human hepatocellular carcinoma. Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork, which can maintain the uniform size and shape of hepatocytes. Because the cells of hepatocellular carcinoma were morphologically different from the hepatocytes, we speculated that expression of plectin and organization of intermediate filament might play roles in the pleomorphism of hepatocellular carcinoma cells. In this paper, we studied the plectin expression of hepatocellular carcinoma and liver tissues by immunohistochemistry and immunoblot. The results revealed that plectin was deficient and cytokeratin18 was modulated in hepatocellular carcinoma. Furthermore, we knockdown the plectin mRNA in Chang cells, the result revealed the plectin was deficient and the organization of cytokeratin18 was altered. Conclusively, this study offers a hypothesis that plectin deficient might play an important role in the tumorigenesis of hepatocellular carcinoma.

Muscular integrity--a matter of interlinking distinct structures via plectin.

Myocytes are characterized by the presence of highly specialized cytoskeletal structures that are part of regularly spaced functional units distributed over long distances. In this chapter we discuss previously published evidence as well as novel data showing that the proper positioning and architecture of Z-disks and of sarcolemma-associated costameric structures are largely dependent on the cytolinker protein plectin and its associated intermediate filament (desmin) cytoskeleton. Deficiency in either plectin or desmin lead to muscular dystrophies of similar pathology. However, while in the absence of plectin, desmin networks collapse and form aggregates, when desmin is missing, plectin retains its typical localization. This suggests that plectin recruits and anchors desmin filaments to both Z-disks and costameres and thus is a key element for maintaining and reinforcing myocyte cytoarchitecture. We hypothesize that as an essential link of the Z-disk-costamere axis, plectin is likely to play also a crucial role in myofiber signaling.

Pleomorphism of cancer cells with the expression of plectin and concept of filament bundles in human hepatocellular carcinoma.

Intermediate filaments are important in building the architecture of liver cells and are proposed to interact with other cellular components. Among intermediate filament associated proteins, plectin is a versatile cytoskeletal linkage protein which has been shown to interact with a variety of cytoskeletal structures. Intermediate filament and plectin might play some roles in tumorigenesis of human hepatocellular carcinoma since cells of hepatocellular carcinoma were morphologically different from normal liver. Plectin exhibited wide distribution spectrum among various tissues, however, it was poorly investigated in human liver and hepatoma tissues. In this paper, we studied the plectin expression in 18 cases of human hepatocellular carcinoma and normal hepatocytes by immunohistochemistry. The results revealed that plectin expression was deficient in human hepatocellular carcinoma and was probably through post-translational modification. Many 0.4 to 0.8 microm-thick keratin bundles were found in intermediate filament extracts of liver and hepatoma tissues. These bundles were greater in diameter about 40 to 80 times of single intermediate filament. We speculated that intermediate filament organized into "filament bundles" to maintain the shape of normal cells. In cancer cells, plectin was deficient and the irregularly loosened filament bundles could cause pleomorphism of cancer cells.

Targeted ablation of plectin isoform 1 uncovers role of cytolinker proteins in leukocyte recruitment.

Plectin, a typical cytolinker protein, is essential for skin and skeletal muscle integrity. It stabilizes cells mechanically, regulates cytoskeleton dynamics, and serves as a scaffolding platform for signaling molecules. A variety of isoforms expressed in different tissues and cell types account for this versatility. To uncover the role of plectin 1, the major isoform expressed in tissues of mesenchymal origin, against the background of all other variants, we raised plectin isoform 1-specific antibodies and generated isoform-deficient mice. In contrast to plectin-null mice (lacking all plectin isoforms), which die shortly after birth because of severe skin blistering, plectin isoform 1-deficient mice were viable at birth, had a normal lifespan, and did not display the skin blistering phenotype. However, dermal fibroblasts isolated from plectin 1-deficient mice exhibited abnormalities in their actin cytoskeleton and impaired migration potential. Similarly, plectin 1-deficient T cells isolated from nymph nodes showed diminished chemotactic migration in vitro. Most strikingly, in vivo we found that leukocyte infiltration during wound healing was reduced in the mutant mice. These data show a specific role of a cytolinker protein in immune cell motility. Single isoform-deficient mice thus represent a powerful tool to unravel highly specific functions of plectin variants.