Plakins are involved in the regulation of centrosome position in polarized epithelial cells.

BACKGROUND INFORMATION: The control of epithelial cell polarity is key to their function. Its dysregulation is a major cause of tissue transformation. In polarized epithelial cells,the centrosome is off-centred toward the apical pole. This asymmetry determines the main orientation of the microtubule network and intra-cellular traffic. However, the mechanism regulating centrosome positioning at the apical pole of polarized epithelial cells is still poorly undertood. RESULTS: In this study we used transcriptomic data from breast cancer cells to identify molecular changes associated with the different stages of tumour transformation. We correlated these changes with variations in centrosome position or with cell progression along the epithelial-to-mesenchymal transition (EMT), a process that involves centrosome repositioning. We found that low levels of epiplakin, desmoplakin and periplakin correlated with centrosome mispositioning in cells that had progressed through EMT or tissue transformation. We further tested the causal role of these plakins in the regulation of centrosome position by knocking down their expression in a non-tumorigenic breast epithelial cell line (MCF10A). The downregulation of periplakin reduced the length of intercellular junction, which was not affected by the downregulation of epiplakin or desmoplakin. However, down-regulating any of them disrupted centrosome polarisation towards the junction without affecting microtubule stability. CONCLUSIONS: Altogether, these results demonstrated that epiplakin, desmoplakin and periplakin are involved in the maintenance of the peripheral position of the centrosome close to inter-cellular junctions. They also revealed that these plakins are downregulated during EMT and breast cancer progression, which are both associated with centrosome mispositioning. SIGNIFICANCE: These results revealed that the down-regulation of plakins and the consequential centrosome mispositioning are key signatures of disorganised cytoskeleton networks, inter-cellular junction weakening, shape deregulation and the loss of polarity in breast cancer cells. These metrics could further be used as a new readouts for early phases of tumoral development.

Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14-mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses.

The attributes of plakins in cancer and disease: perspectives on ovarian cancer progression, chemoresistance and recurrence.

The plakin family of cytoskeletal proteins play an important role in cancer progression yet are under-studied in cancer, especially ovarian cancer. These large cytoskeletal proteins have primary roles in the maintenance of cytoskeletal integrity but are also associated with scaffolds of intermediate filaments and hemidesmosomal adhesion complexes mediating signalling pathways that regulate cellular growth, migration, invasion and differentiation as well as stress response. Abnormalities of plakins, and the closely related spectraplakins, result in diseases of the skin, striated muscle and nervous tissue. Their prevalence in epithelial cells suggests that plakins may play a role in epithelial ovarian cancer progression and recurrence. In this review article, we explore the roles of plakins, particularly plectin, periplakin and envoplakin in disease-states and cancers with emphasis on ovarian cancer. We discuss the potential role the plakin family of proteins play in regulating cancer cell growth, survival, migration, invasion and drug resistance. We highlight potential relationships between plakins, epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) and discuss how interaction of these processes may affect ovarian cancer progression, chemoresistance and ultimately recurrence. We propose that molecular changes in the expression of plakins leads to the transition of benign ovarian tumours to carcinomas, as well as floating cellular aggregates (commonly known as spheroids) in the ascites microenvironment, which may contribute to the sustenance and progression of the disease. In this review, attempts have been made to understand the crucial changes in plakin expression in relation to progression and recurrence of ovarian cancer. Video Abstract.

The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation.

While the mechanisms by which chemical signals control cell fate have been well studied, the impact of mechanical inputs on cell fate decisions is not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through linker of nucleoskeleton and cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

Giardia intestinalis coiled-coil cytolinker protein 259 interacts with actin and tubulin.

Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.

Keratins and the plakin family cytolinker proteins control the length of epithelial microridge protrusions.

Actin filaments and microtubules create diverse cellular protrusions, but intermediate filaments, the strongest and most stable cytoskeletal elements, are not known to directly participate in the formation of protrusions. Here we show that keratin intermediate filaments directly regulate the morphogenesis of microridges, elongated protrusions arranged in elaborate maze-like patterns on the surface of mucosal epithelial cells. We found that microridges on zebrafish skin cells contained both actin and keratin filaments. Keratin filaments stabilized microridges, and overexpressing keratins lengthened them. Envoplakin and periplakin, plakin family cytolinkers that bind F-actin and keratins, localized to microridges, and were required for their morphogenesis. Strikingly, plakin protein levels directly dictate microridge length. An actin-binding domain of periplakin was required to initiate microridge morphogenesis, whereas periplakin-keratin binding was required to elongate microridges. These findings separate microridge morphogenesis into distinct steps, expand our understanding of intermediate filament functions, and identify microridges as protrusions that integrate actin and intermediate filaments.

Cells adopt a wide array of irregular and bumpy shapes, which are scaffolded by an internal structure called the cytoskeleton. This network of filaments can deform the cell membrane the way tent poles frame a canvas. Cells contain three types of cytoskeleton elements (actin filaments, intermediate filaments, and microtubules), each with unique chemical and mechanical properties. One of the main roles of the cytoskeleton is to create protrusions, a range of structures that 'stick out' of a cell to allow movement and interactions with the environment. Both actin filaments and microtubules help form protrusions, but the role of intermediate filaments remains unclear. Microridges are a type of protrusion found on cells covered by mucus, for instance on the surface of the eye, inside the mouth, or on fish skin. These small bumps are organised on the membrane of a cell in fingerprint-like arrangements. Scientists know that actin networks are necessary for microridges to form; yet, many structures supported by actin filaments are not stable over time, suggesting that another component of the cytoskeleton might be lending support. Intermediate filaments are the strongest, most stable type of cytoskeleton element, and they can connect to actin filaments via linker proteins. However, research has yet to show that this kind of cooperation happens in any membrane protrusion. Here, Inaba et al. used high-resolution microscopy to monitor microridge development in the skin of live fish. In particular, they focused on a type of intermediate filaments known as keratin filaments. This revealed that, inside microridges, the keratin and actin networks form alongside each other, with linker proteins called Envoplakin and Periplakin connecting the two structures together. Genetic experiments revealed that Envoplakin and Periplakin must attach to actin for microridges to start forming. However, the two proteins bind to keratin for protrusions to grow. This work therefore highlights how intermediate filaments and linker proteins contribute to the formation of these structures. Many tissues must be covered in mucus to remain moist and healthy. As microridges likely contribute to mucus retention, the findings by Inaba et al. may help to better understand how disorders linked to problems in mucus emerge.

Evaluation of predictive role of carcinoembryonic antigen and salivary mRNA biomarkers in gastric cancer detection.

We explored the potential of combining carcinoembryonic antigen (CEA) and salivary mRNAs for gastric cancer (GC) detection.This study included 2 phases of study: a biomarker discovery phase and an independent validation phase. In the discovery phase, we measured CEA levels in blood samples and expression level of messenger RNAs (SPINK7, PPL, SEMA4B, SMAD4) in saliva samples of 140 GC patients and 140 healthy controls. We evaluated the clinical performance of each biomarker and developed a predictive model using machine-learning algorithm to differentiate GC patients and healthy controls.Our biomarker panel successfully discriminated GC patients from healthy controls with both high sensitivity (0.94) and high specificity (0.91). We next applied our biomarker panel in the independent validation phase, in which we recruited a new patient cohort of 60 GC patients and 60 healthy controls. Using our biomarker panel, the GC patients were discriminated from healthy controls in the validation phase, with sensitivity of 0.92 and specificity of 0.87.A combination of blood CEA and salivary messenger RNA could be a promising approach to detect GC.

Binding of the periplakin linker requires vimentin acidic residues D176 and E187.

Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle.

Adaptor protein-3 complex is required for Vangl2 trafficking and planar cell polarity of the inner ear.

Planar cell polarity (PCP) regulates coordinated cellular polarity among neighboring cells to establish a polarity axis parallel to the plane of the tissue. Disruption in PCP results in a range of developmental anomalies and diseases. A key feature of PCP is the polarized and asymmetric localization of several membrane PCP proteins, which is essential to establish the polarity axis to orient cells coordinately. However, the machinery that regulates the asymmetric partition of PCP proteins remains largely unknown. In the present study, we show Van gogh-like 2 (Vangl2) in early and recycling endosomes as made evident by colocalization with diverse endosomal Rab proteins. Vangl2 biochemically interacts with adaptor protein-3 complex (AP-3). Using short hairpin RNA knockdown, we found that Vangl2 subcellular localization was modified in AP-3-depleted cells. Moreover, Vangl2 membrane localization within the cochlea is greatly reduced in AP-3-deficient mocha mice, which exhibit profound hearing loss. In inner ears from AP-3-deficient mocha mice, we observed PCP-dependent phenotypes, such as misorientation and deformation of hair cell stereociliary bundles and disorganization of hair cells characteristic of defects in convergent extension that is driven by PCP. These findings demonstrate a novel role of AP-3-mediated sorting mechanisms in regulating PCP proteins.

SUMOylation of periplakin is critical for efficient reorganization of keratin filament network.

The architecture of the cytoskeleton and its remodeling are tightly regulated by dynamic reorganization of keratin-rich intermediate filaments. Plakin family proteins associate with the network of intermediate filaments (IFs) and affect its reorganization during migration, differentiation, and response to stress. The smallest plakin, periplakin (PPL), interacts specifically with intermediate filament proteins K8, K18, and vimentin via its C-terminal linker domain. Here, we show that periplakin is SUMOylated at a conserved lysine in its linker domain (K1646) preferentially by small ubiquitin-like modifier 1 (SUMO1). Our data indicate that PPL SUMOylation is essential for the proper reorganization of the keratin IF network. Stresses perturbing intermediate-filament and cytoskeletal architecture induce hyper--SUMOylation of periplakin. Okadaic acid induced hyperphosphorylation-dependent collapse of the keratin IF network results in a similar hyper-SUMOylation of PPL. Strikingly, exogenous overexpression of a non-SUMOylatable periplakin mutant (K1646R) induced aberrant bundling and loose network interconnections of the keratin filaments. Time-lapse imaging of cells expressing the K1646R mutant showed the enhanced sensitivity of keratin filament collapse upon okadaic acid treatment. Our data identify an important regulatory role for periplakin SUMOylation in dynamic reorganization and stability of keratin IFs.

Identification of periplakin as a major regulator of lung injury and repair in mice.

Periplakin is a component of the desmosomes that acts as a cytolinker between intermediate filament scaffolding and the desmosomal plaque. Periplakin is strongly expressed by epithelial cells in the lung and is a target antigen for autoimmunity in idiopathic pulmonary fibrosis. The aim of this study was to determine the role of periplakin during lung injury and remodeling in a mouse model of lung fibrosis induced by bleomycin. We found that periplakin expression was downregulated in the whole lung and in alveolar epithelial cells following bleomycin-induced injury. Deletion of the Ppl gene in mice improved survival and reduced lung fibrosis development after bleomycin-induced injury. Notably, Ppl deletion promoted an antiinflammatory alveolar environment linked to profound changes in type 2 alveolar epithelial cells, including overexpression of antiinflammatory cytokines, decreased expression of profibrotic mediators, and altered cell signaling with a reduced response to TGF-ß1. These results identify periplakin as a previously unidentified regulator of the response to injury in the lung.

Mammalian Plakins, Giant Cytolinkers: Versatile Biological Functions and Roles in Cancer.

Cancer is a highly lethal disease that is characterized by aberrant cell proliferation, migration, and adhesion, which are closely related to the dynamic changes of cytoskeletons and cytoskeletal-adhesion. These will further result in cell invasion and metastasis. Plakins are a family of giant cytolinkers that connect cytoskeletal elements with each other and to junctional complexes. With various isoforms composed of different domain structures, mammalian plakins are broadly expressed in numerous tissues. They play critical roles in many cellular processes, including cell proliferation, migration, adhesion, and signaling transduction. As these cellular processes are key steps in cancer development, mammalian plakins have in recent years attracted more and more attention for their potential roles in cancer. Current evidence shows the importance of mammalian plakins in various human cancers and demonstrates mammalian plakins as potential biomarkers for cancer. Here, we introduce the basic characteristics of mammalian plakins, review the recent advances in understanding their biological functions, and highlight their roles in human cancers, based on studies performed by us and others. This will provide researchers with a comprehensive understanding of mammalian plakins, new insights into the development of cancer, and novel targets for cancer diagnosis and therapy.

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets.

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents.

Position matters: multiple functions of LINC-dependent chromosome positioning during meiosis.

Chromosome positioning is crucial for multiple chromosomal events, including DNA replication, repair, and recombination. The linker of nucleoskeleton and cytoskeleton (LINC) complexes, which consist of conserved nuclear membrane proteins, were shown to control chromosome positioning and facilitate various biological processes by interacting with the cytoskeleton. However, the precise functions and regulation of LINC-dependent chromosome positioning are not fully understood. During meiosis, the LINC complexes induce clustering of telomeres, forming the bouquet chromosome arrangement, which promotes homologous chromosome pairing. In fission yeast, the bouquet forms through LINC-dependent clustering of telomeres at the spindle pole body (SPB, the centrosome equivalent in fungi) and detachment of centromeres from the SPB-localized LINC. It was recently found that, in fission yeast, the bouquet contributes to formation of the spindle and meiotic centromeres, in addition to homologous chromosome pairing, and that centromere detachment is linked to telomere clustering, which is crucial for proper spindle formation. Here, we summarize these findings and show that the bouquet chromosome arrangement also contributes to nuclear fusion during karyogamy. The available evidence suggests that these functions are universal among eukaryotes. The findings demonstrate that LINC-dependent chromosome positioning performs multiple functions and controls non-chromosomal as well as chromosomal events, and that the chromosome positioning is stringently regulated for its functions. Thus, chromosome positioning plays a much broader role and is more strictly regulated than previously thought.

[Current approaches to the morphologic diagnosis of different types of congenital epidermolysis bullosa]. / Sovremennye podkhody k morfologicheskoi diagnostike razlichnykh tipov vrozhdennogo bulleznogo epidermoliza.

Congenital epidermolysis bullosa (CEB) is an extensive group of hereditary skin diseases, the differential diagnosis of which is a challenge due to the rarity of this pathology and the diversity of its clinical manifestations. The determination of the type of CEB makes it possible to estimate its prognosis and to facilitate a prenatal diagnosis. AIM: to optimize the morphological diagnosis of different types of CEB. MATERIAL AND METHODS: 28 skin biopsies from 14 patients with different types of CEB were investigated. The investigators performed routine histological examination of skin fragments taken from a bullous area and immunofluorescence antigen mapping using the indirect immunofluorescence test (IIFT) with antibodies against structural proteins of the dermal-epidermal junction (laminin α3, ß3, and γ2 chains, keratins 5 and 14, types VII and XVII collagen, α6 and ß4 integrin subunits, desmoplakin, plectin, kindlin-1, and plakophillin) of the apparently unaffected skin. The intact skin of healthy individuals, which had been obtained during cosmetic operations, was used as controls in IIFT. RESULTS: Immunofluorescence antigen mapping could determine the type of CEB in all cases and in 86% of cases identify the protein, the impaired production of which was responsible for the development of the disease. CONCLUSION: Immunofluorescence antigen mapping is an integral part of the comprehensive morphological diagnosis of CEB, acting as an intermediate between the morphological verification of CEB diagnosis and the targeted search for mutations by a molecular genetic method.

One gene but different proteins and diseases: the complexity of dystonin and bullous pemphigoid antigen 1.

Since the immunochemical identification of the bullous pemphigoid antigen 230 (BP230) as one of the major target autoantigens of bullous pemphigoid (BP) in 1981, our understanding of this protein has significantly increased. Cloning of its gene, development and characterization of animal models with engineered gene mutations or spontaneous mouse mutations have revealed an unexpected complexity of the gene encoding BP230. The latter, now called dystonin (DST), is composed of at least 100 exons and gives rise to three major isoforms, an epithelial, a neuronal and a muscular isoform, named BPAG1e (corresponding to the original BP230), BPAG1a and BPAG1b, respectively. The various BPAG1 isoforms play a key role in fundamental processes, such as cell adhesion, cytoskeleton organization, and cell migration. Genetic defects of BPAG1 isoforms are the culprits of epidermolysis bullosa and complex, devastating neurological diseases. In this review, we summarize recent advances of our knowledge about several BPAG1 isoforms, their role in various biological processes and in human diseases.