The Experts below are selected from a list of 2193 Experts worldwide ranked by ideXlab platform
Gerhard Wiche - One of the best experts on this subject based on the ideXlab platform.
-
Muscular Integrity—A Matter of Interlinking Distinct Structures via Plectin
Advances in Experimental Medicine and Biology, 2020Co-Authors: Patryk Konieczny, Gerhard WicheAbstract: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.
-
the cytolinker Plectin regulates nuclear mechanotransduction in keratinocytes
Journal of Cell Science, 2015Co-Authors: Filipe V Almeida, Gerhard Wiche, James R Mcmillan, Gernot Walko, John A Mcgrath, Asa H Barber, John T ConnellyAbstract:The transmission of mechanical forces to the nucleus is important for intracellular positioning, mitosis and cell motility, yet the contribution of specific components of the cytoskeleton to nuclear mechanotransduction remains unclear. In this study, we examine how crosstalk between the cytolinker Plectin and F-actin controls keratin network organisation and the 3D nuclear morphology of keratinocytes. Using micro-patterned surfaces to precisely manipulate cell shape, we find that cell adhesion and spreading regulate the size and shape of the nucleus. Disruption of the keratin cytoskeleton through loss of Plectin facilitated greater nuclear deformation, which depended on acto-myosin contractility. Nuclear morphology did not depend on direct linkage of the keratin cytoskeleton with the nuclear membrane, rather loss of Plectin reduced keratin filament density around the nucleus. We further demonstrate that keratinocytes have abnormal nuclear morphologies in the epidermis of Plectin-deficient, epidermolysis bullosa simplex patients. Taken together, our data demonstrate that Plectin is an essential regulator of nuclear morphology in vitro and in vivo and protects the nucleus from mechanical deformation.
-
Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks
Journal of Cell Science, 2015Co-Authors: Selma Osmanagicmyers, Siegfried Reipert, Irmgard Fischer, Navid Bonakdar, Gernot Walko, Wolfgang H Goldmann, Michael Wolfram, Daniela Brunner, Aurora Zuzuarregui, Gerhard WicheAbstract:Mutations in the cytoskeletal linker protein Plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established Plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, Plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in Plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional Plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a Plectin-organized vimentin scaffold keeps actomyosin contractility 'in-check' and maintains AJ homeostasis.
-
Plectin isoform p1b and p1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle
Human Molecular Genetics, 2015Co-Authors: Lilli Winter, Irmgard Fischer, Aniko Zeold, Andrey V Kuznetsov, Michael Grimm, Gerhard WicheAbstract: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.
-
determining the mechanical properties of Plectin in mouse myoblasts and keratinocytes
Experimental Cell Research, 2015Co-Authors: Navid Bonakdar, Gerhard Wiche, Achim Schilling, Marina Sporrer, Pablo Lennert, Astrid Mainka, Lilli Winter, Gernot Walko, Ben Fabry, Wolfgang H GoldmannAbstract:Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human Plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine Plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, Plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and Plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated para- magnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in Plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between Plectin- deficient and wild-type keratinocytes, indicating that Plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of Plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in Plectin-deficient and wild-type myoblasts, but was significantly increased in Plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of Plectin has divergent implications on biomechanical properties depending on the respective cell type.
Arnoud Sonnenberg - One of the best experts on this subject based on the ideXlab platform.
-
The rod domain is not essential for the function of Plectin in maintaining tissue integrity
Molecular Biology of the Cell, 2015Co-Authors: Mirjam Ketema, Pablo Secades, Maaike Kreft, Leila Nahidiazar, Hans Janssen, Kees Jalink, José M. De Pereda, Arnoud SonnenbergAbstract:Plectin is a cytoskeletal linker protein that consists of a central rod domain connecting two globular domains. Rodless Plectin is able to functionally compensate for the loss of full-length plecti...
-
nesprin 3 connects Plectin and vimentin to the nuclear envelope of sertoli cells but is not required for sertoli cell function in spermatogenesis
Molecular Biology of the Cell, 2013Co-Authors: Mirjam Ketema, Pablo Secades, Maaike Kreft, Hans Janssen, Arnoud SonnenbergAbstract:Nesprin-3 is a nuclear envelope protein that connects the nucleus to intermediate filaments by interacting with Plectin. To investigate the role of nesprin-3 in the perinuclear localization of Plectin, we generated nesprin-3–knockout mice and examined the effects of nesprin-3 deficiency in different cell types and tissues. Nesprin-3 and Plectin are coexpressed in a variety of tissues, including peripheral nerve and muscle. The expression level of nesprin-3 in skeletal muscle is very low and decreases during myoblast differentiation in vitro. Of interest, Plectin was concentrated at the nuclear envelope in only a few cell types. This was most prominent in Sertoli cells of the testis, in which nesprin-3 is required for the localization of both Plectin and vimentin at the nuclear perimeter. Testicular morphology and the position of the nucleus in Sertoli cells were normal, however, in the nesprin-3–knockout mice and the mice were fertile. Furthermore, nesprin-3 was not required for the polarization and migration of mouse embryonic fibroblasts. Thus, although nesprin-3 is critical for the localization of Plectin to the nuclear perimeter of Sertoli cells, the resulting link between the nuclear envelope and the intermediate filament system seems to be dispensable for normal testicular morphology and spermatogenesis.
-
structural basis of the interaction between integrin α6β4 and Plectin at the hemidesmosomes
The EMBO Journal, 2009Co-Authors: José M. De Pereda, Pilar M Lillo, Arnoud SonnenbergAbstract:The interaction between the integrin α6β4 and Plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 A resolution of the primary α6β4–Plectin complex, formed by the first pair of fibronectin type III domains and the N-terminal region of the connecting segment of β4 and the actin-binding domain of Plectin. Two missense mutations in β4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 A resolution of the β4 moiety in the absence of Plectin, which reveal a major rearrangement of the connecting segment of β4 on binding to Plectin. This conformational switch is correlated with the way α6β4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.
-
requirements for the localization of nesprin 3 at the nuclear envelope and its interaction with Plectin
Journal of Cell Science, 2007Co-Authors: Mirjam Ketema, Hans Janssen, Ingrid Kuikman, Kevin Wilhelmsen, Didier Hodzic, Arnoud SonnenbergAbstract:The outer nuclear membrane proteins nesprin-1 and nesprin-2 are retained at the nuclear envelope through an interaction of their klarsicht/ANC-1/syne homology (KASH) domain with Sun proteins present at the inner nuclear membrane. We investigated the requirements for the localization of nesprin-3α at the outer nuclear membrane and show that the mechanism by which its localization is mediated is similar to that reported for the localization of nesprin-1 and nesprin-2: the last four amino acids of the nesprin-3α KASH domain are essential for its interaction with Sun1 and Sun2. Moreover, deletion of these amino acids or knockdown of the Sun proteins results in a redistribution of nesprin-3α away from the nuclear envelope and into the endoplasmic reticulum (ER), where it becomes colocalized with the cytoskeletal crosslinker protein Plectin. Both nesprin-3α and Plectin can form dimers, and dimerization of Plectin is required for its interaction with nesprin-3α at the nuclear envelope, which is mediated by its N-terminal actin-binding domain. Additionally, overexpression of the Plectin actin-binding domain stabilizes the actin cytoskeleton and prevents the recruitment of endogenous Plectin to the nuclear envelope. Our studies support a model in which the actin cytoskeleton influences the binding of Plectin dimers to dimers of nesprin-3α, which in turn are retained at the nuclear envelope through an interaction with Sun proteins.
-
modeling and experimental validation of the binary complex of the Plectin actin binding domain and the first pair of fibronectin type iii fniii domains of the β4 integrin
Journal of Biological Chemistry, 2005Co-Authors: Sandy H M Litjens, José M. De Pereda, Kevin Wilhelmsen, Anastassis Perrakis, Arnoud SonnenbergAbstract:Abstract The binding of Plectin to the β4 subunit of the α6β4 integrin is a critical step in the formation of hemidesmosomes. An important interaction between these two proteins occurs between the actin-binding domain (ABD) of Plectin and the first pair of fibronectin type III (FNIII) domains and a small part of the connecting segment of β4. Previously, a few amino acids, critical for this interaction, were identified in both Plectin and β4 and mapped on the crystal structures of the ABD of Plectin and the first pair of FNIII domains of β4. In the present study, we used this biochemical information and protein-protein docking calculations to construct a model of the binary complex between these two protein domains. The top scoring computational model predicts that the calponin-homology 1 (CH1) domain of the ABD associates with the first and the second FNIII domains of β4. Our mutational analysis of the residues at the proposed interface of both the FNIII and the CH1 domains is in agreement with the suggested interaction model. Computational simulations to predict protein motions suggest that the exact model of FNIII and Plectin CH1 interaction might well differ in detail from the suggested model due to the conformational plasticity of the FNIII domains, which might lead to a closely related but different mode of interaction with the Plectin-ABD. Furthermore, we show that Ser-1325 in the connecting segment of β4 appears to be essential for the recruitment of Plectin into hemidesmosomes in vivo. This is consistent with the proposed model and previously published mutational data. In conclusion, our data support a model in which the CH1 domain of the Plectin-ABD associates with the groove between the two FNIII domains of β4.
José M. De Pereda - One of the best experts on this subject based on the ideXlab platform.
-
The rod domain is not essential for the function of Plectin in maintaining tissue integrity
Molecular Biology of the Cell, 2015Co-Authors: Mirjam Ketema, Pablo Secades, Maaike Kreft, Leila Nahidiazar, Hans Janssen, Kees Jalink, José M. De Pereda, Arnoud SonnenbergAbstract:Plectin is a cytoskeletal linker protein that consists of a central rod domain connecting two globular domains. Rodless Plectin is able to functionally compensate for the loss of full-length plecti...
-
structural basis of the interaction between integrin α6β4 and Plectin at the hemidesmosomes
The EMBO Journal, 2009Co-Authors: José M. De Pereda, Pilar M Lillo, Arnoud SonnenbergAbstract:The interaction between the integrin α6β4 and Plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 A resolution of the primary α6β4–Plectin complex, formed by the first pair of fibronectin type III domains and the N-terminal region of the connecting segment of β4 and the actin-binding domain of Plectin. Two missense mutations in β4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 A resolution of the β4 moiety in the absence of Plectin, which reveal a major rearrangement of the connecting segment of β4 on binding to Plectin. This conformational switch is correlated with the way α6β4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.
-
modeling and experimental validation of the binary complex of the Plectin actin binding domain and the first pair of fibronectin type iii fniii domains of the β4 integrin
Journal of Biological Chemistry, 2005Co-Authors: Sandy H M Litjens, José M. De Pereda, Kevin Wilhelmsen, Anastassis Perrakis, Arnoud SonnenbergAbstract:Abstract The binding of Plectin to the β4 subunit of the α6β4 integrin is a critical step in the formation of hemidesmosomes. An important interaction between these two proteins occurs between the actin-binding domain (ABD) of Plectin and the first pair of fibronectin type III (FNIII) domains and a small part of the connecting segment of β4. Previously, a few amino acids, critical for this interaction, were identified in both Plectin and β4 and mapped on the crystal structures of the ABD of Plectin and the first pair of FNIII domains of β4. In the present study, we used this biochemical information and protein-protein docking calculations to construct a model of the binary complex between these two protein domains. The top scoring computational model predicts that the calponin-homology 1 (CH1) domain of the ABD associates with the first and the second FNIII domains of β4. Our mutational analysis of the residues at the proposed interface of both the FNIII and the CH1 domains is in agreement with the suggested interaction model. Computational simulations to predict protein motions suggest that the exact model of FNIII and Plectin CH1 interaction might well differ in detail from the suggested model due to the conformational plasticity of the FNIII domains, which might lead to a closely related but different mode of interaction with the Plectin-ABD. Furthermore, we show that Ser-1325 in the connecting segment of β4 appears to be essential for the recruitment of Plectin into hemidesmosomes in vivo. This is consistent with the proposed model and previously published mutational data. In conclusion, our data support a model in which the CH1 domain of the Plectin-ABD associates with the groove between the two FNIII domains of β4.
-
specificity of binding of the Plectin actin binding domain to β4 integrin
Molecular Biology of the Cell, 2003Co-Authors: Sandy H M Litjens, José M. De Pereda, Jan Koster, Ingrid Kuikman, Sandra Van Wilpe, Arnoud SonnenbergAbstract:Plectin is a major component of the cytoskeleton and links the intermediate filament system to hemidesmosomes by binding to the integrin β4 subunit. Previously, a binding site for β4 was mapped on the actin-binding domain (ABD) of Plectin and binding of β4 and F-actin to Plectin was shown to be mutually exclusive. Here we show that only the ABDs of Plectin and dystonin bind to β4, whereas those of other actin-binding proteins do not. Mutations of the ABD of Plectin-1C show that Q131, R138, and N149 are critical for tight binding of the ABD to β4. These residues form a small cavity, occupied by a well-ordered water molecule in the crystal structure. The β4 binding pocket partly overlaps with the actin-binding sequence 2 (ABS2), previously shown to be essential for actin binding. Therefore, steric interference may render binding of β4 and F-actin to Plectin mutually exclusive. Finally, we provide evidence indicating that the residues preceding the ABD in Plectin-1A and -1C, although unable to mediate binding to β4 themselves, modulate the binding activity of the ABD for β4. These studies demonstrate the unique property of the Plectin-ABD to bind to both F-actin and β4, and explain why several other ABD-containing proteins that are expressed in basal keratinocytes are not recruited into hemidesmosomes.
-
structural and functional analysis of the actin binding domain of Plectin suggests alternative mechanisms for binding to f actin and integrin β4
Structure, 2003Co-Authors: Begona Garciaalvarez, Arnoud Sonnenberg, Andrey A Bobkov, José M. De PeredaAbstract:Abstract Plectin is a widely expressed cytoskeletal linker. Here we report the crystal structure of the actin binding domain of Plectin and show that this region is sufficient for interaction with F-actin or the cytoplasmic region of integrin α6β4. The structure is formed by two calponin homology domains arranged in a closed conformation. We show that binding to F-actin induces a conformational change in Plectin that is inhibited by an engineered interdomain disulfide bridge. A two-step induced fit mechanism involving binding and subsequent domain rearrangement is proposed. In contrast, interaction with integrin α6β4 occurs in a closed conformation. Competitive binding of Plectin to F-actin and integrin α6β4 may rely on the observed alternative binding mechanisms and involve both allosteric and steric factors.
Gunther A Rezniczek - One of the best experts on this subject based on the ideXlab platform.
-
unexpected gain of function for the scaffolding protein Plectin due to mislocalization in pancreatic cancer
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Soo J. Shin, Gunther A Rezniczek, Gerhard Wiche, Jeffrey A. Smith, Ru Chen, Teresa A Brentnall, Kimberly A. KellyAbstract:We recently demonstrated that Plectin is a robust biomarker for pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive malignancies. In normal physiology, Plectin is an intracellular scaffolding protein, but we have demonstrated localization on the extracellular surface of PDAC cells. In this study, we confirmed cell surface localization. Interestingly, we found that Plectin cell surface localization was attributable to its presence in exosomes secreted from PDAC cells, which is dependent on the expression of integrin β4, a protein known to interact with cytosolic Plectin. Moreover, Plectin expression was necessary for efficient exosome production and was required to sustain enhanced tumor growth in immunodeficient and in immunocompetent mice. It is now clear that this PDAC biomarker plays a role in PDAC, and further understanding of Plectin’s contribution to PDAC could enable improved therapies.
-
linking cytoarchitecture to metabolism sarcolemma associated Plectin affects glucose uptake by destabilizing microtubule networks in mdx myofibers
Skeletal Muscle, 2013Co-Authors: Marianne Raith, Gunther A Rezniczek, Irmgard Fischer, Rocio G Valencia, Michael Orthofer, Josef M Penninger, Simone Spuler, Gerhard WicheAbstract:Background Duchenne muscular dystrophy (DMD) is one of the most frequent forms of muscular disorders. It is caused by the absence of dystrophin, a core component of the sarcolemma-associated junctional complex that links the cytoskeleton to the extracellular matrix. We showed previously that Plectin 1f (P1f), one of the major muscle-expressed isoforms of the cytoskeletal linker protein Plectin, accumulates at the sarcolemma of DMD patients as well as of mdx mice, a widely studied animal model for DMD. Based on Plectin’s dual role as structural protein and scaffolding platform for signaling molecules, we speculated that the dystrophic phenotype observed after loss of dystrophin was caused, at least to some extent, by excess Plectin. Thus, we hypothesized that elimination of Plectin expression in mdx skeletal muscle, while probably resulting in an overall more severe phenotype, may lead to a partial phenotype rescue. In particular, we wanted to assess whether excess sarcolemmal Plectin contributes to the dysregulation of sugar metabolism in mdx myofibers.
-
Abstract A79: Plectin trafficking in oncosomes contributes to pancreatic cancer proliferation and invasion.
Cancer Research, 2012Co-Authors: Soo J. Shin, Gunther A Rezniczek, Gerhard Wiche, Jeffrey A. Smith, Kimberly A. KellyAbstract:Background and Aims: We recently showed that the aberrant extracellular localization of the cytoskeletal linker protein Plectin is a biomarker for the progression of pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive malignancies. In this study, we assess the molecular mechanism for Plectin deregulation and its functional role in PDAC progression. Methods: To examine their potential contribution to aberrant extracellular localization of Plectin, oncosomes (secreted membrane-derived microvesicles) produced by PDAC cells were collected by ultracentrifugation of PDAC-conditioned media and evaluated by immunoblotting. RT-PCR was used to define which Plectin isoforms showed altered expression in PDAC compared to normal pancreatic ducts. Based on this information, we tested the impact of isoform-specific interacting proteins on Plectin trafficking. Finally, we examined the impact of modulation of Plectin on PDAC migration and invasion via transwell migration assay. Results: Plectin was localized on the cell surface as well as secreted through oncosomes in PDAC cell lines but not in non-transformed human pancreatic ductal epithelial (HPDE) cells. shRNA-mediated knockdown of integrin β4, a Plectin 1a/1f interacting protein that were shown to be expressed in PDAC, impaired extracellular trafficking of Plectin in PDAC cells. shRNA and overexpression studies collectively demonstrated a positive role of Plectin proliferation, migration, and invasion of PDAC cells, and showed that Plectin-positive oncosomes derived from PDAC are taken up by and induce migration and invasion in co cultured fibroblasts and endothelial cells. Conclusions: Plectin isoforms are differentially expressed in PDAC and are translocated to the cell surface in this cancer via oncosomes in an integrin β4-dependent manner. Plectin inactivation impairs PDAC cell proliferation, migration, and invasion of PDAC cells. Since cell surface Plectin is a biomarker for transition of pre-invasive to invasive PDAC, these new insights into mechanisms of its deregulation will be important for the clinical development of Plectin targeting diagnostic approaches. Our data also reveal unexpected direct functional roles for Plectin in PDAC pathogenesis. Citation Format: Soo J. Shin, Jeffrey A. Smith, Gunther Rezniczek, Gerhard Wiche, Kimberly A. Kelly. Plectin trafficking in oncosomes contributes to pancreatic cancer proliferation and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A79.
-
Plectin 1f scaffolding at the sarcolemma of dystrophic mdx muscle fibers through multiple interactions with beta dystroglycan
Journal of Cell Biology, 2007Co-Authors: Gunther A Rezniczek, Patryk Konieczny, Branislav Nikolic, Siegfried Reipert, Doris Schneller, Christina Abrahamsberg, Kay E Davies, Steve J Winder, Gerhard WicheAbstract:In skeletal muscle, the cytolinker Plectin is prominently expressed at Z-disks and the sarcolemma. Alternative splicing of Plectin transcripts gives rise to more than eight protein isoforms differing only in small N-terminal sequences (5–180 residues), four of which (Plectins 1, 1b, 1d, and 1f) are found at substantial levels in muscle tissue. Using Plectin isoform–specific antibodies and isoform expression constructs, we show the differential regulation of Plectin isoforms during myotube differentiation and their localization to different compartments of muscle fibers, identifying Plectins 1 and 1f as sarcolemma-associated isoforms, whereas Plectin 1d localizes exclusively to Z-disks. Coimmunoprecipitation and in vitro binding assays using recombinant protein fragments revealed the direct binding of Plectin to dystrophin (utrophin) and β-dystroglycan, the key components of the dystrophin–glycoprotein complex. We propose a model in which Plectin acts as a universal mediator of desmin intermediate filament anchorage at the sarcolemma and Z-disks. It also explains the Plectin phenotype observed in dystrophic skeletal muscle of mdx mice and Duchenne muscular dystrophy patients.
-
Plectin 5 transcript diversity short alternative sequences determine stability of gene products initiation of translation and subcellular localization of isoforms
Human Molecular Genetics, 2003Co-Authors: Gunther A Rezniczek, Christina Abrahamsberg, Daniel Spazierer, Peter Fuchs, Gerhard WicheAbstract: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.
Gernot Walko - One of the best experts on this subject based on the ideXlab platform.
-
the cytolinker Plectin regulates nuclear mechanotransduction in keratinocytes
Journal of Cell Science, 2015Co-Authors: Filipe V Almeida, Gerhard Wiche, James R Mcmillan, Gernot Walko, John A Mcgrath, Asa H Barber, John T ConnellyAbstract:The transmission of mechanical forces to the nucleus is important for intracellular positioning, mitosis and cell motility, yet the contribution of specific components of the cytoskeleton to nuclear mechanotransduction remains unclear. In this study, we examine how crosstalk between the cytolinker Plectin and F-actin controls keratin network organisation and the 3D nuclear morphology of keratinocytes. Using micro-patterned surfaces to precisely manipulate cell shape, we find that cell adhesion and spreading regulate the size and shape of the nucleus. Disruption of the keratin cytoskeleton through loss of Plectin facilitated greater nuclear deformation, which depended on acto-myosin contractility. Nuclear morphology did not depend on direct linkage of the keratin cytoskeleton with the nuclear membrane, rather loss of Plectin reduced keratin filament density around the nucleus. We further demonstrate that keratinocytes have abnormal nuclear morphologies in the epidermis of Plectin-deficient, epidermolysis bullosa simplex patients. Taken together, our data demonstrate that Plectin is an essential regulator of nuclear morphology in vitro and in vivo and protects the nucleus from mechanical deformation.
-
Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks
Journal of Cell Science, 2015Co-Authors: Selma Osmanagicmyers, Siegfried Reipert, Irmgard Fischer, Navid Bonakdar, Gernot Walko, Wolfgang H Goldmann, Michael Wolfram, Daniela Brunner, Aurora Zuzuarregui, Gerhard WicheAbstract:Mutations in the cytoskeletal linker protein Plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established Plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, Plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in Plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional Plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a Plectin-organized vimentin scaffold keeps actomyosin contractility 'in-check' and maintains AJ homeostasis.
-
determining the mechanical properties of Plectin in mouse myoblasts and keratinocytes
Experimental Cell Research, 2015Co-Authors: Navid Bonakdar, Gerhard Wiche, Achim Schilling, Marina Sporrer, Pablo Lennert, Astrid Mainka, Lilli Winter, Gernot Walko, Ben Fabry, Wolfgang H GoldmannAbstract:Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human Plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine Plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, Plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and Plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated para- magnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in Plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between Plectin- deficient and wild-type keratinocytes, indicating that Plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of Plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in Plectin-deficient and wild-type myoblasts, but was significantly increased in Plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of Plectin has divergent implications on biomechanical properties depending on the respective cell type.
-
targeted proteolysis of Plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease ebs ogna
PLOS Genetics, 2011Co-Authors: Gernot Walko, Siegfried Reipert, Peter Fuchs, Irmgard Fischer, Nevena Vukasinovic, Karin Gross, Sabrina Sibitz, Ute Jungwirth, Walter Berger, Ulrich SalzerAbstract:Autosomal recessive mutations in the cytolinker protein Plectin account for the multisystem disorders epidermolysis bullosa simplex (EBS) associated with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and congenital myasthenia (EBS-CMS). In contrast, a dominant missense mutation leads to the disease EBS-Ogna, manifesting exclusively as skin fragility. We have exploited this trait to study the molecular basis of hemidesmosome failure in EBS-Ogna and to reveal the contribution of Plectin to hemidesmosome homeostasis. We generated EBS-Ogna knock-in mice mimicking the human phenotype and show that blistering reflects insufficient protein levels of the hemidesmosome-associated Plectin isoform 1a. We found that Plectin 1a, in contrast to Plectin 1c, the major isoform expressed in epidermal keratinocytes, is proteolytically degraded, supporting the notion that degradation of hemidesmosome-anchored Plectin is spatially controlled. Using recombinant proteins, we show that the mutation renders Plectin's 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains and other proteases activated in the epidermis but not in skeletal muscle. Accordingly, treatment of cultured EBS-Ogna keratinocytes as well as of EBS-Ogna mouse skin with calpain inhibitors resulted in increased Plectin 1a protein expression levels. Moreover, we report that Plectin's rod domain forms dimeric structures that can further associate laterally into remarkably stable (paracrystalline) polymers. We propose focal self-association of Plectin molecules as a novel mechanism contributing to hemidesmosome homeostasis and stabilization.
-
Plectin isoform dependent regulation of keratin integrin α6β4 anchorage via ca2 calmodulin
Journal of Biological Chemistry, 2009Co-Authors: Julius Kostan, Martin Gregor, Gernot Walko, Gerhard WicheAbstract:The detachment of epithelial cells from the basal matrix during wound healing and differentiation of keratinocytes requires the disassembly of the hemidesmosomal multiprotein adhesion complex. Integrin α6β4-Plectin interaction plays a major role in the formation of hemidesmosomes, and thus the mechanisms regulating this interaction should be critical also for the disassembly process. Here we show that a particular Plectin isoform (1a) interacts with the Ca2+-sensing protein calmodulin in a Ca2+-dependent manner. As a result of this interaction, binding of the hemidesmosome-associated Plectin isoform 1a to integrin β4 is substantially diminished. Calmodulin-binding inhibits also the interaction of Plectin with F-actin. Further, we found that, during Ca2+-induced keratinocyte differentiation, Plectin 1a is first relocated within the cell and later down-regulated, suggesting that Ca2+ affects the fate of Plectin 1a upon its release from hemidesmosomes. We propose a novel model for the disassembly of hemidesmosomes during keratinocyte differentiation, where both, binding of calmodulin to Plectin 1a and phosphorylation of integrin β4 by protein kinases, are required for disruption of the integrin α6β4-Plectin complex.