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The human macula is a highly specialized retinal region with pit‐like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone‐ and rod‐rich retinae from human and mice and identified different expression profiles of cone‐ and rod‐associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell‐derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region‐specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.

The adaptation of vertebrates to different environments was associated with changes in the molecular composition and regulation of epithelia. Whales and dolphins, together forming the clade cetaceans, have lost multiple epithelial keratins during or after their evolutionary transition from life on land to life in water. It is unknown whether the changes in keratins were accompanied by gain or loss of cytoskeletal adapter proteins of the plakin family. Here we investigated whether plakin proteins are conserved in cetaceans and other vertebrates. Comparative analysis of genome sequences showed conservation of dystonin, microtubule actin crosslinking factor 1 (MACF1), plectin, desmoplakin, periplakin and envoplakin in cetaceans. By contrast, EPPK1 (epiplakin) was disrupted by inactivating mutations in all cetaceans investigated. Orthologs of EPPK1 are present in bony and cartilaginous fishes and tetrapods, indicating an evolutionary origin of EPPK1 in a common ancestor of jawed vertebrates (Gnathostomes). In many vertebrates, EPPK1 is flanked by an as-yet uncharacterized gene that encodes protein domains homologous to the carboxy-terminal segment of MACF1. We conclude that epiplakin, unlike other plakins, was lost in cetaceans.

Lamina-associated polypeptide (LAP) 2alpha is a chromatin-associated protein that binds A-type lamins. Mutations in both LAP2alpha and A-type lamins are linked to human diseases called laminopathies, but the molecular mechanisms are poorly understood. The A-type lamin-LAP2alpha complex interacts with and regulates retinoblastoma protein (pRb), but the significance of this interaction in vivo is unknown. Here we address the function of the A-type lamin-LAP2alpha complex with the use of LAP2alpha-deficient mice. We show that LAP2alpha loss causes relocalization of nucleoplasmic A-type lamins to the nuclear envelope and impairs pRb function. This causes inefficient cell-cycle arrest in dense fibroblast cultures and hyperproliferation of epidermal and erythroid progenitor cells in vivo, leading to tissue hyperplasia. Our results support a disease-relevant model in which LAP2alpha defines A-type lamin localization in the nucleoplasm, which in turn affects pRb-mediated regulation of progenitor cell proliferation and differentiation in highly regenerative tissues.

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.

Epiplakin is a large (>725 kDa) cytoskeletal protein exclusively expressed in epithelial tissues. It has a unique structure, consisting entirely of plakin repeat domains (PRDs), one of the hallmarks of spectraplakin protein family members. Previous studies, including the phenotypic analyses of knockout mice, failed to reveal the biological function of epiplakin. Using in vitro binding assays, we show here that all but one of the 16 PRDs of mouse epiplakin bind to keratins of basal keratinocytes. Nevertheless, in primary keratinocyte cell cultures, epiplakin only partially colocalized with keratin intermediate filament networks. However, upon application of cellular stress in the form of keratin hyperphosphorylation, osmotic shock or UV irradiation, the entire cytoplasmic epiplakin pool became associated with keratin. In response to such types of stress, epiplakin initially translocated to the still-intact keratin filament network and remained associated with keratin after its disruption and transformation into granular aggregates. Time-course experiments revealed that serine/threonine (okadaic acid) and tyrosine (orthovanadate) phosphatase inhibitor-induced filament disruption in differentiated keratinocytes proceeded faster in epiplakin-deficient cells compared with wild-type cells. Our data suggest that epiplakin plays a role in keratin filament reorganization in response to stress, probably by protecting keratin filaments against disruption in a chaperone-like fashion.

Epiplakin is a large member (700 kDa) of the plakin protein family and exclusively expressed in epithelial cell types. Compared to other plakin proteins epiplakin exhibits an unusual structure as it consists entirely of a variable number of consecutive plakin repeat domains (13 in humans, 16 in mice). The only binding partners of epiplakin identified so far are keratins of simple as well as of stratified epithelia. Epiplakin-deficient mice show no obvious spontaneous phenotype. However, ex vivo studies using epiplakin-deficient primary cells indicated protective functions of epiplakin in response to stress. Recent studies using stress models for organs of the gastrointestinal tract revealed that epiplakin-deficient mice develop more pronounced pancreas and liver injuries than their wild-type littermates. In addition, impaired stress-induced keratin network reorganization was observed in the affected organs, and primary epiplakin-deficient hepatocytes showed reduced tolerance for forced keratin overexpression which could be rescued by a chemical chaperone. These findings indicate protective functions of epiplakin in chaperoning disease-induced keratin reorganization. In this review, we describe some of the methods we used to analyze epiplakin's function with the focus on biochemical and ex vivo techniques.

Plectin, a widespread intermediate filament-based cytolinker protein capable of interacting with a variety of cytoskeletal structures and plasma membrane-bound junctional complexes, serves essential functions in maintenance of cell and tissue cytoarchitecture. We have generated a mouse line bearing floxed plectin alleles and conditionally deleted plectin in stratified epithelia. This strategy enabled us to study the consequences of plectin deficiency in this particular type of tissues in the context of the whole organism without plectin loss affecting other tissues. Conditional knockout mice died early after birth, showing signs of starvation and growth retardation. Blistering was observed on their extremities and on the oral epithelium after initial nursing, impairing food uptake. Knockout epidermis was very fragile and showed focal epidermal barrier defects caused by the presence of small skin lesions. Stratification, proliferation and differentiation of knockout skin seemed unaffected by epidermis-restricted plectin deficiency. In an additionally generated mouse model, tamoxifen-induced Cre-ERT-mediated recombination led to mice with a mosaic plectin deletion pattern in adult epidermis, combined with microblister formation and epidermal barrier defects. Our study explains the early lethality of plectin-deficient mice and provides a model to ablate plectin in adult animals which could be used for developing gene or pharmacological therapies.

Epiplakin, a giant epithelial protein of >700 kDa, belongs to the plakin family of cytolinker proteins. It represents an atypical family member, however, as it consists entirely of plakin repeat domains but lacks any of the other domains commonly shared by plakins. Hence, its putative function as a cytolinker protein remains to be shown. To investigate epiplakin's biological role, we generated epiplakin-deficient mice by gene targeting in embryonic stem cells. Epiplakin-deficient mice were viable and fertile, without developing any discernible phenotype. Ultrastructurally, their epidermis revealed no differences compared to wild-type littermates, and cornified envelopes isolated from skin showed no alterations in shape or stability. Furthermore, neither embryonal formation nor later function of the epithelial barrier was affected. In primary cultures of epiplakin-deficient keratinocytes, the organization of actin filaments, microtubules, and keratin networks was found to be normal. Similarly, no alterations in keratin network organization were observed in simple epithelia of small intestine and liver or in primary hepatocytes. We conclude that, despite epiplakin's abundant and highly specific expression in stratified and simple epithelia, its absence in mice does not lead to severe skin dysfunctions, nor has it detectable consequences for keratin filament organization and cytoarchitecture of cells.

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.

The widely expressed cytolinker protein plectin shows extensive isoform diversity both at the N-terminus and in the central part of the molecule. Judged on mRNA data, plectin variants lacking the central rod domain are expressed at a approximately 20-fold lower level than full-length proteins and their detection on the protein level can be difficult. Here we present data on the expression of plectin rodless isoforms in mouse brain and in rat glioma C6 cells on RNA and protein levels. Our data indicate that among the rodless variants expressed in neuronal tissues, those starting with exon 1c (plectin 1c) seem to be the most prominent ones. In addition, we show that similar to other monoclonal antibodies reported in the literature, the widely used mAb 7A8 recognizes an epitope within plectin's rod domain and therefore is unsuited to detect rodless variants of plectin.

We have recently identified PARP10 as a novel functional poly(ADP-ribose) polymerase . The gene encoding PARP10 is conserved in vertebrates but no orthologs were found in lower organisms. In addition to the poly(ADP-ribose) polymerase domain, PARP10 possesses several additional sequence motifs , including an RNA recognition motif and two ubiquitin interaction motifs. We characterized the murine genomic locus of the Parp10 gene. We noticed that 3′ Parp10 sequences overlapped with the plectin 1 gene in a head-to-tail arrangement. Detailed analyses revealed that the two most 3′ Parp10 exons (exons 10 and 11) are also used for plectin 1 . While these two exons code for part of the poly(ADP-ribose) polymerase domain in Parp10, they are noncoding for plectin 1 due to the lack of appropriate start codons . Furthermore our findings suggest that at least one of the plectin 1 promoters is located within intron 9 of the Parp10 gene.

Although a few experimental approaches to isolated limb perfusion (ILP) are described in the literature, none of these animal models mimics the clinical perfusion techniques adequately to improve the technique of ILP on the basis of valid preclinical data. Therefore, we developed an ILP setup in rats allowing online monitoring of essential perfusion parameters such as temperature (in perfusate, various tissues, and rectum), pH (perfusate), perfusion pressure, and O(2) concentration (in perfusate, tissue), by a tailor-made data acquisition system. This setup permits close supervision of vital parameters during ILP. Various interdependencies, concerning the flow rate and the pressure of perfusate as well as tissue oxygenation were registered. For the measurement of pO(2) values in the perfusate and in different regions of the perfused hind limb, a novel type of microoptode based on quenching of a fluorescent dye was devised. Stable normothermic (37 degrees C) perfusion conditions were maintained at a constant perfusion pressure in the range of 40-60 mm Hg by administration of the spasmo lytic moxaverine (0.5 mg/mL of perfusate as initial dose) at a perfusate flow rate of 0.5 mL/min for 60 min. At the end of an ILP, there were no signs of tissue damage, neither concerning laboratory data (K(+), myoglobin, creatine kinase, lactic dehydrogenase) nor histopathological criteria. The reported ILP model is not only well suited to investigate the effects of hyperthermia but also to assess the efficacy of new antineoplastic approaches, when nude rats, bearing human tumours in the hind limbs, are used.

Plectin is a large cytoskeletal linker protein expressed as several different isoforms from a highly complex gene. This transcript diversity is mainly caused by short 5'-sequences contained in alternative first exons. To elucidate the influence of these sequence differences and to determine potential differential functionality of the resulting protein forms, we conducted a systematic investigation of plectin isoforms on transcript and protein levels. Isoform expression was highly dependent on the different 5' ends, largely due to effects of the 5'-untranslated regions. Initiation of translation downstream of the expected start site led to loss of actin- and integrin beta4-binding in some isoforms. The small alternative N-terminal sequences (5-180 residues) profoundly affected the subcelluar localization of this >500 kDa protein. Specifically, plectin 1f was concentrated at focal adhesion contacts and plectin 1b was exclusively targeted to mitochondria, providing a connection of these organelles to intermediate filaments. Thus, with plectin as a model, we demonstrate a role for 5'-untranslated regions and alternative 5'-splicing as an important regulatory mechanism of protein expression and protein function.

The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe skin blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes. In skin sections and cultured keratinocytes, plectin 1a was shown to colocalize with hemidesmosomal structures. In contrast, a second isoform expressed in epithelia, plectin 1c, differing from 1a merely by a short N-terminal sequence, colocalized with microtubules. Expression of plectin 1a, but not of its N-terminal fragment alone, or of a third alternative full length isoform (plectin 1), restored the reduced number of hemidesmosome-like stable anchoring contacts in cultured plectin-null keratinocytes. Our results show for the first time that different isoforms of a cytolinker protein expressed in one cell type perform distinct functions. Moreover, the identification of plectin 1a as the isoform defects in which cause skin blistering in plectin-related genetic diseases, such as epidermolysis bullosa simplex MD and epidermolysis bullosa simplex Ogna, could have implications for the future development of clinical therapies for patients.

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.

Cytolinker proteins stabilize cells mechanically, regulate cytoskeleton dynamics, and provide scaffolds for signaling molecules. For plectin, the prototype of these proteins, an unusual diversity of isoforms has been reported, which show distinct expression patterns, subcellular localizations, and functions. Plectin has been shown to have important functions in skin and muscle, but little is known about its role in neural cells. To address this issue, we generated two knock-out mouse lines, one which was selectively lacking plectin 1c (P1c), the major isoform expressed in neural cells, and another in which plectin was conditionally deleted in neuronal precursor cells. Using isoform-specific antibodies, we found P1c to be expressed late in development and to associate with postsynaptic dendrites of central nervous system neurons, motorneurons of spinal cord, sciatic nerve axons, and Schwann cells. Motor nerve conduction velocity was found significantly reduced in sciatic nerve from P1c-deficient as well as from conditional knock-out mice. This defect was traceable to an increased number of motor nerve fibers with small cross-sectional areas; the thicknesses of axons and of myelin sheaths were unaffected. This is the first report demonstrating an important role of plectin in a major nerve function.

Plectin, a cytolinker protein greater than 500 kDa in size, has an important role as a mechanical stabiliser of cells. It interlinks the various cytoskeletal filament systems and anchors intermediate filaments to peripheral junctional complexes. In addition, there is increasing evidence that plectin acts as a scaffolding platform that controls the spatial and temporal localisation and interaction of signaling proteins. In this study we show that, in differentiated mouse myotubes, plectin binds to the regulatory gamma1 subunit of AMP-activated protein kinase (AMPK), the key regulatory enzyme of energy homeostasis. No interaction was observed in undifferentiated myoblasts, and plectin-deficient myotubes showed altered positioning of gamma1-AMPK. In addition we found that plectin affects the subunit composition of AMPK, because isoform alpha1 of the catalytic subunit decreased in proportion to isoform alpha2 during in vitro differentiation of plectin(-/-) myotubes. In plectin-deficient myocytes we could also detect a higher level of activated (Thr172-phosphorylated) AMPK, compared with wild-type cells. Our data suggest a differentiation-dependent association of plectin with AMPK, where plectin selectively stabilises alpha1-gamma1 AMPK complexes by binding to the gamma1 regulatory subunit. The distinct plectin expression patterns in different fibre types combined with its involvement in the regulation of isoform compositions of AMPK complexes could provide a mechanism whereby cytoarchitecture influences energy homeostasis.

Based on cDNA cloning and sequencing, human epiplakin has been classified as a member of the plakin protein family of cytolinkers. We report here the characterization of the mouse epiplakin gene locus and the isolation of full-length mouse epiplakin cDNA using BAC vectors. We found that the protein is encoded by a single remarkably large exon (>20 kb) that consists of a series of 0.8–1.5-kb-long DNA repeats, eight of which are virtually identical. Consequently, mouse epiplakin contains 16 plakin repeat domains, three more than reported for the human protein and eight more than predicted for the mouse protein based on the contig characterized by the Mouse Genome Sequencing Consortium. Using antibodies raised to a highly conserved repeating epiplakin sequence domain, we show that the protein in cells is expressed in its full length (725 kDa), and we provide evidence that the size of human epiplakin previously may have been underestimated. In addition we show on transcript and protein levels that epiplakin is restricted to epithelial tissues and that its gene maps to mouse chromosome 15 (human chromosome 8). This study lays the groundwork for future genetic approaches aimed at defining the biological role of this unique protein.

Signalling through the transmembrane receptor Notch is triggered by ligand binding, which induces the proteolytic cleavage of the Notch protein. This cleavage generates an intracellular fragment of the Notch protein (Notch-IC), which translocates into the nucleus and modifies transcription of target genes through its association with the RBP-J protein. Thus, the isolated Notch-IC protein represents the constitutively activated receptor. We have performed a deletion analysis of Notch IC in order to identify the transferable transactivation domain of Notch-IC and the minimal domain of Notch-IC required for RBP-J dependent transactivational activation. Functionally, Notch-IC has been linked to cell fate decision in development and oncogenesis in vivo. In vitro, Notch-IC can cooperate in neoplastic transformation of baby rat kidney cells with the adenoviral E1A protein. We have defined the minimal domain of Notch-IC required for E1A cotransformation. This domain, consisting of the ankyrin repeats of Notch-IC only, can neither activate RBP-J dependent transcription nor does it carry a transactivation domain. Therefore, the ankyrin repeat domain of Notch-IC might trigger novel pathways relevant for transformation but unrelated to RBP-J signalling.