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G Wiche

Publications and source records attributed to G Wiche.

At least 37 records · Page 2Linked to original sources
PubMed1999

Unusual 5' transcript complexity of plectin isoforms: novel tissue-specific exons modulate actin binding activity.

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.

Actins↗
PubMed1999

Plectin: a cytolinker by design.

Plectin is a cytoskeletal protein of >500 kDa that forms dumbbell-shaped homodimers comprising a central parallel alpha-helical coiled coil rod domain flanked by globular domains, thus providing a molecular backbone ideally suited to mediate the protein's interactions with an array of other cytoskeletal elements. Plectin self-associates and interacts with actin and intermediate filament cytoskeleton networks at opposite ends, and it binds at both ends to the hemidesmosomal transmembrane protein integrin beta-4, and likely to other junctional proteins. The central coiled coil rod domain can form bridges over long stretches and serves as a flexible linker between the structurally diverse N-terminal domain and the highly conserved C-terminal domain. Plectin is also a target of p34cdc2 kinase that regulates its dissociation from intermediate filaments during mitosis.

Actin Cytoskeleton↗
PubMed1998

Novel features of the light chain of microtubule-associated protein MAP1B: microtubule stabilization, self interaction, actin filament binding, and regulation by the heavy chain.

Previous studies on the role of microtubule-associated protein 1B (MAP1B) in adapting microtubules for nerve cell-specific functions have examined the activity of the entire MAP1B protein complex consisting of heavy and light chains and revealed moderate effects on microtubule stability. Here we have analyzed the effects of the MAP1B light chain in the absence or presence of the heavy chain by immunofluorescence microscopy of transiently transfected cells. Distinct from all other MAPs, the MAP1B light chain-induced formation of stable but apparently flexible microtubules resistant to the effects of nocodazole and taxol. Light chain activity was inhibited by the heavy chain. In addition, the light chain was found to harbor an actin filament binding domain in its COOH terminus. By coimmunoprecipitation experiments using epitope-tagged fragments of MAP1B we showed that light chains can dimerize or oligomerize. Furthermore, we localized the domains for heavy chain-light chain interaction to regions containing sequences homologous to MAP1A. Our findings assign several crucial activities to the MAP1B light chain and suggest a new model for the mechanism of action of MAP1B in which the heavy chain might act as the regulatory subunit of the MAP1B complex to control light chain activity.

3T3 Cells↗
PubMed1998

Not just scaffolding: plectin regulates actin dynamics in cultured cells.

Plectin, a major linker and scaffolding protein of the cytoskeleton, has been shown to be essential for the mechanical integrity of skin, skeletal muscle, and heart. Studying fibroblast and astroglial cell cultures derived from plectin (-/-) mice, we found that their actin cytoskeleton, including focal adhesion contacts, was developed more extensively than in wild-type cells. Also it failed to show characteristic short-term rearrangments in response to extracellular stimuli activating the Rho/Rac/Cdc42 signaling cascades. As a consequence, cell motility, adherence, and shear stress resistance were altered, and morphogenic processes were delayed. Furthermore, we show that plectin interacts with G-actin in vitro in a phosphatidylinositol-4,5-biphosphate-dependent manner and associates with actin stress fibers in living cells. The actin stress fiber phenotype of plectin-deficient fibroblasts could be reversed to a large degree by transient transfection of full-length plectin or plectin fragments containing the amino-terminal actin-binding domain (ABD). These results reveal a novel role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its proposed role in scaffolding and mechanical stabilization of cells.

Actins↗
PubMed1998

The mouse and rat MAP1B genes: genomic organization and alternative transcription.

We report the genomic organization of the mouse and rat genes coding for the 2460-amino-acid microtubule-associated protein (MAP) 1B. In addition to seven exons that encode full-length MAP1B, we have identified two alternative exons, exon 3A and the novel exon 3U. We demonstrate that alternative MAP1B transcripts containing either exon 3A or exon 3U are expressed in a variety of mouse and rat tissues at about 1 to 10% of the level of regular transcripts. The alternative transcripts, if translated, would give rise to MAP1B isoforms truncated at the N-terminus. The exon/intron organization underlying the alternative transcripts and the N-terminal amino acid sequence of the putative truncated MAP1B isoforms resemble those of MAP1A, providing further evidence for an evolutionary relationship. The detection of alternative transcripts has implications for the interpretation of conflicting results recently obtained in MAP1B knockout mice.

Alternative Splicing↗
PubMed1998

Linking integrin alpha6beta4-based cell adhesion to the intermediate filament cytoskeleton: direct interaction between the beta4 subunit and plectin at multiple molecular sites.

Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the beta4 subunit of the basement membrane laminin receptor integrin alpha6beta4 that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin beta4 cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin beta4 subunits and cytokeratin filaments.

Animals↗
PubMed1998

Evidence against structural and functional identity of microtubule-associated protein 1B and proteoglycan claustrin.

Recently, the concept of microtubule-associated protein 1B as an intracellular 2460 amino acid protein was challenged by the suggestion that only the N-terminal 1022 codons are utilized and encode the core protein of the extracellular proteoglycan claustrin (Burg and Cole (1994) J. Neurobiol. 25, 1-22). We expressed this N-terminal MAP1B fragment in tissue culture cells and found that it bound to microtubules and was not localized in the extracellular matrix. In addition, epitope mapping demonstrated that MAP1B consisted of more than 1022 amino acids and that the reported cDNA of claustrin is incomplete.

3T3 Cells↗
PubMed1998

Role of plectin in cytoskeleton organization and dynamics.

Plectin and its isoforms are versatile cytoskeletal linker proteins of very large size (>500 kDa) that are abundantly expressed in a wide variety of mammalian tissues and cell types. Earlier studies indicated that plectin molecules were associated with and/or directly bound to subcomponents of all three major cytoskeletal filament networks, the subplasma membrane protein skeleton, and a variety of plasma membrane-cytoskeleton junctional complexes, including those found in epithelia, various types of muscle, and fibroblasts. In conjunction with biochemical data, this led to the concept that plectin plays an important role in cytoskeleton network organization, with consequences for viscoelastic properties of the cytoplasm and the mechanical integrity and resistance of cells and tissues. Several recent findings lent strong support to this concept. One was that a hereditary disease, epidermolysis bullosa simplex (EBS)-MD, characterized by severe skin blistering combined with muscular dystrophy, is caused by defects in the plectin gene. Another was the generation of plectin-deficient mice by targeted inactivation of the gene. Dying shortly after birth, these animals exhibited severe defects in skin, skeletal muscle and heart. Moreover, in vitro studies with cells derived from such animals unmasked an essential new role of plectin as regulator of cellular processes involving actin stress fibers dynamics. Comprehensive analyses of the gene locus in man, mouse, and rat point towards a complex gene expression machinery, comprising an unprecedented diversity of differentially spliced transcripts with distinct 5' starting exons, probably regulated by different promoters. This could provide a basis for cell type-dependent and/or developmentally-controlled expression of plectin isoforms, exerting different functions through binding to distinct partners. Based on its versatile functions and structural diversification plectin emerges as a prototype cytolinker protein among a family of proteins sharing partial structural homology and functions.

Animals↗
PubMed1997

Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture.

Previous studies suggest that plectin, a versatile cytoskeletal linker protein, has an important role in maintaining the structural integrity of diverse cells and tissues. To establish plectin's function in a living organism, we have disrupted its gene in mice. Plectin (-/-) mice died 2-3 days after birth exhibiting skin blistering caused by degeneration of keratinocytes. Ultrastructurally, hemidesmosomes and desmosomes appeared unaffected. In plectin-deficient mice, however, hemidesmosomes were found to be significantly reduced in number and apparently their mechanical stability was altered. The skin phenotype of these mice was similar to that of patients suffering from epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the plectin gene. In addition, plectin (-/-) mice revealed abnormalities reminiscent of minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart. Our results clearly demonstrate a general role of plectin in the reinforcement of mechanically stressed cells. Plectin (-/-) mice will provide a useful tool for the study of EBS-MD, and possibly other types of plectin-related myopathies involving skeletal and cardiac muscle, in an organism amenable to genetic manipulation.

Animals↗
PubMed1997

Plectin transcript diversity: identification and tissue distribution of variants with distinct first coding exons and rodless isoforms.

Plectin is a widely expressed protein that is very large in size and that has all the attributes of a multifunctional crosslinking and organizing element of the cytoskeleton. It displays a multidomain structure, versatile binding activities, and subcellular localizations that enable it to strengthen cells against mechanical stress forces. Moreover, hereditary gene defects in plectin cause epidermolysis bullosa simplex (EBS)-MD, a severe skin blistering disease with muscular dystrophy. Here we report the analysis of the exonintron organization of the rat plectin gene and the identification of several different isoforms on the transcriptional level. We show that of 35 coding exons identified, 4 serve as alternative first exons splicing into the same successive exon 2, which is the first of 7 exons encoding a highly conserved actin-binding domain. RNase protection mapping of transcripts containing 3 of the identified 4 alternate first exons revealed their coexpression in rat glioma C6 cells and in a series of different rat tissues that we examined. Significant variations in expression levels of first exons indicated the possibility of tissue-specific promoter usage. In addition, plectin splice variants lacking exon 31 (> 3 kb), which encodes the entire rod domain of the molecule, were identified in a variety of rat tissues. This study provides first insights into a complex plectin gene regulatory machinery with similarities to that of dystrophin.

Alternative Splicing↗
PubMed1997

Plectin abnormality in epidermolysis bullosa simplex Ogna: non-responsiveness of basal keratinocytes to some anti-rat plectin antibodies.

Epidermolysis bullosa (EB) is a heterogeneous group of genetic bullous skin diseases. The EB simplex group (EBS) is characterized by intraepidermal blistering. EBS-Ogna was first described as a separate entity based on clinical studies. Later genetic linkage of EBS-Ogna to the GPT locus for glutamate pyruvate transaminase (alanine transaminase) was detected and GPT was assigned to chromosome 8, then to the terminal long arm band 8q24. Plectin is an abundant and widespread cytoskeletal protein which has been proposed as a general crosslinking element of intermediate filaments. Human plectin has recently been cloned and in situ hybridized to chromosome 8q24. To examine whether plectin could be associated with EBS-Ogna we performed an immunohistochemical study with a panel of mAbs to rat plectin. Interestingly, 2 of these mAbs showed strong intracellular staining of the suprabasal and basal layer of the epidermis in all control samples, whereas no reactivity of the basal layer was found in the Ogna group. These results strongly suggest that plectin is involved in the pathogenesis of EBS-Ogna.

Animals↗
PubMed1997

Polarisation-dependent association of plectin with desmoplakin and the lateral submembrane skeleton in MDCK cells.

The intermediate filament-binding protein plectin and cytokeratin were localised at the cellular periphery of fully polarised Madin-Darby canine kidney (MDCK) cells, whereas vimentin was primarily found in a perinuclear network. Confocal and immunoelectron microscopy revealed that plectin was restricted to areas underlying the lateral plasma membrane. It colocalised with fodrin, a component of the submembrane skeleton, and was closely associated with desmosomal plaque structures. Biochemically, plectin was shown to interact directly with immunoprecipitated desmoplakin in vitro. Upon loss of cell polarity in low calcium medium, plectin redistributed to a cytoplasmic vimentin- and cytokeratin-related network, clearly distinct from diffusely distributed fodrin and internalised desmoplakin structures. The structural reorganisation of plectin was also reflected by an increased solubility of the protein in Triton X-100/high salt, and a decrease in its half-life from approximately 20 to approximately 5 hours. Furthermore, unlike cytokeratins and vimentin, desmoplakin and fodrin did not associate with plectin attached to magnetic beads in cell lysates of unpolarised cells, while all proteins formed a stable complex in polarised cells. Altogether, these data indicate that plectin is involved in the anchorage of intermediate filaments to desmosomes and to the submembrane skeleton in polarised MDCK cells.

Animals↗
PubMed1997

Recessive epidermolysis bullosa simplex associated with plectin mutations: infantile respiratory complications in two unrelated cases.

Plectin is a 500 kDa protein involved in cytoskeleton-plasma membrane attachment with a wide tissue distribution including cutaneous and airway epithelia, muscle and neuronal tissue. Recently, mutations in the gene encoding plectin (PLEC1) have been implicated in the pathogenesis of an autosomal recessive variant of epidermolysis bullosa simplex in which cutaneous blistering starting in the neonatal period is associated with muscular dystrophy in later life. In this study, we report two unrelated patients, both of consanguineous parentage, who presented with cutaneous blistering and a hoarse cry from birth. Both experienced inspiratory stridor and respiratory distress, necessitating emergency tracheostomy in one case. Immunoreactivity to monoclonal antibodies against plectin was absent or markedly reduced in skin biopsies from both patients. Electron microscopy revealed a low intraepidermal plane of cleavage and hypoplastic hemidesmosomes with a reduced association with keratin intermediate filaments. Direct sequencing of PLEC1 in each case demonstrated two novel homozygous frameshift deletion mutations, 5069del19 and 5905del2, which both create downstream premature termination codons. Although currently neither patient has symptoms of muscle disease, the identification of mutations in PLEC1 may be predictive for the future development of muscular dystrophy. Recessive epidermolysis bullosa simplex resulting from abnormalities in plectin should be considered in the differential diagnosis blistering, hoarseness and stridor in infancy.

Child, Preschool↗
PubMed1996

Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy.

Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.

Adult↗
PubMed1996

Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24).

Plectin, a 500-kDa intermediate filament binding protein, has been proposed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton. To set the basis for future studies on gene regulation, tissue-specific expression, and pathological conditions involving this protein, we have cloned the human plectin gene, determined its coding sequence, and established its genomic organization. The coding sequence contains 32 exons that extend over 32 kb of the human genome. Most of the introns reside within a region encoding the globular N-terminal domain of the molecule, whereas the entire central rod domain and the entire C-terminal globular domain were found to be encoded by single exons of remarkable length, >3 kb and >6 kb, respectively. Overall, the organization of the human plectin gene was strikingly similar to that of human bullous pemphigoid antigen 1 (BPAG1), confirming that both proteins belong to the same gene family. Comparison of the deduced protein sequences for human and rat plectin revealed that they were 93% identical. By using fluorescence in situ hybridization, we have mapped the plectin gene to the long arm of chromosome 8 within the telomeric region. This gene locus (8q24) has previously been implicated in the human blistering skin disease epidermolysis bullosa simplex Ogna. Detailed knowledge of the structure of the plectin gene and its chromosome localization will aid in the elucidation of whether this or any other pathological conditions are linked to alterations in the plectin gene.

Amino Acid Sequence↗
PubMed1996

Identification of plectin as a substrate of p34cdc2 kinase and mapping of a single phosphorylation site.

Plectin is an in vitro substrate for various kinases present in cell lysates from mitotic and interphase Chinese hamster ovary cells. Sensitivity of plectin kinase activity to the inhibitor olomoucine, and two-dimensional tryptic peptide mapping of plectin phosphorylated by various kinase preparations suggested that the major plectin kinase activity in mitotic extracts is related to the cell cycle regulator kinase p34cdc2. Bacterial expression of various truncated plectin mutant proteins comprising different domains of the molecule and their phosphorylation by purified p34cdc2kinase revealed that the target site of this kinase resided within plectin's C-terminal globular domain. Among the subdomains of the C-terminal region (six repeats and a short tail sequence), only repeat 6 and the tail were phosphorylated by p34cdc2 kinase. As shown by two-dimensional phosphopeptide mapping, repeat 6, but not the tail, contained a mitosis-specific phosphorylation site targeted by p34cdc2 kinase in intact plectin molecules. By performing site-directed mutagenesis of a potential p34cdc2 recognition sequence motif within the repeat 6 domain, threonine 4542 was identified as the major target for the kinase. Protein kinase A, phosphorylating plectin also within repeat 6, targeted sites that were clearly different from those of p34cdc2 kinase.

Animals↗
PubMed1996

Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.

We report that mutation in the gene for plectin, a cytoskeleton-membrane anchorage protein, is a cause of autosomal recessive muscular dystrophy associated with skin blistering (epidermolysis bullosa simplex). The evidence comes from absence of plectin by antibody staining in affected individuals from four families, supportive genetic analysis (localization of the human plectin gene to chromosome 8q24.13-qter and evidence for disease segregation with markers in this region) and finally the identification of a homozygous frameshift mutation detected in plectin cDNA. Absence of the large multifunctional cytoskeleton protein plectin can simultaneously account for structural failure in both muscle and skin.

Amino Acid Sequence↗