Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

https://doi.org/10.1016/j.ejcb.2010.11.013Get rights and content

Abstract

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A–2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.

Introduction

The plakin family consists of proteins critically involved in the cytoskeletal organization, acting as versatile cytolinkers and scaffolding proteins. They cross-link intermediate filaments (IFs), microfilaments and microtubules with each other and further connect these cytoskeletal networks to the plasma and outer nuclear membranes at distinct sites. The various plakins share a similar structural multi-domain organization. They all harbour spectrin repeats in their amino (NH2)-terminal region, which form the plakin domain. Some members bear additional spectrin repeats towards their carboxyl (COOH) terminus and are thus referred to as spectraplakins (Boyer et al., 2010, Sonnenberg and Liem, 2007). In mammals, the plakin family comprises six canonical members, including plectin and desmoplakin. These two proteins constitute important structural elements for resilience to mechanical stress and integrity of the cytoskeleton architecture in tissues such as stratified epithelia and striated muscles.

Plectin is a ubiquitous large phosphoprotein with several splice variants (Wiche, 1998). Its NH2-terminus contains an actin-binding domain, which can also associate with other proteins than actin (Sonnenberg and Liem, 2007). Among the eight isoforms with alternative NH2-termini described so far (Rezniczek et al., 2003), the isoforms 1, 1b, 1d and 1f are those predominantly expressed in skeletal and cardiac muscles, where they have specific subcellular distributions and functions (Hijikata et al., 2008, Konieczny et al., 2008, Rezniczek et al., 2007). The COOH-terminal part of plectin, which contains six plakin repeat domains (PRDs), has been shown to bind to various IF proteins, including vimentin and desmin (Reipert et al., 1999, Wiche, 1998) (see Fig. 1). In skeletal and cardiac myocytes, plectin is found in filamentous bridges between individual myofibrils at inter-Z-band spaces, between peripheral myofibrils and the sarcolemma or spanning from myofibrils to adjacent mitochondria. In cardiac myocytes, plectin is also found in the intercalated disc region (Hijikata et al., 1999, Reipert et al., 1999, Schröder et al., 1999). In addition, plectin is also able to link IFs to costameres through dystrophin–glycoprotein complexes (Hijikata et al., 2008, Rezniczek et al., 2007). After birth, plectin null-mutant mice show widespread epithelial detachment and muscular dystrophy with necrotic muscle fibres, streaming of Z-discs, focal ruptures of the sarcolemmal membrane, and subsarcolemmal accumulation of mitochondria (Andrä et al., 1997). In humans, mutations in the plectin gene (PLEC) cause a distinct form of epidermolysis bullosa simplex associated with muscular dystrophy (EBS-MD) (Pfendner et al., 2005, Schröder et al., 2002).

Desmin is detected in the early phase of skeletal and cardiac muscle differentiation together with vimentin and nestin (Capetanaki et al., 1997). These proteins belong to the IF family and share a similar structure constituted by a central coiled-coil domain flanked by globular non α-helical NH2-terminal head and COOH-terminal tail domains (see Fig. 1). The assembly of IF proteins into long 10–12 nm caliber polymers is initiated by dimerization of the protein central rod domain. The process progresses by the formation of tetramers, which further associate by following three principal assembly modes to build IFs. While their head domain controls IF assembly and stabilization, their tail regulates lateral packing and stabilization of higher order filament structures (Herrmann and Aebi, 2004). Engineered disruption of the desmin gene in mice and the discovery of pathogenic mutations in humans have demonstrated the importance of desmin for the integrity of the cytoarchitecture of skeletal and cardiac muscle cells. Desmin null-mutant mice show disorganization of the myofibril architecture in mechanically stressed striated muscles, including the heart (reviewed in Paulin and Li, 2004). Furthermore, desmin gene mutations underlie certain forms of muscular dystrophies, with or without cardiac involvement. While several mutations appear to have an impact on the ability of desmin to form an IF network and/or lead to the disruption of the pre-existing endogenous filament networks, in other cases the pathogenic mechanisms are unclear (Bär et al., 2005, Bär et al., 2006a, Bär et al., 2006b, Goudeau et al., 2006). Certain desmin mutations may critically affect the IF–plakin interaction, such as suggested for desmoplakin (Lapouge et al., 2006), and thus the tethering of the IF system to plasma membrane sites.

Previous immunoelectron microscopy studies have provided strong evidence that plectin and desmin are co-localized at distinct subcellular sites in skeletal and cardiac myocytes, such as intercalated discs and inter-Z-band spaces between the peripheral myofibrils and the sarcolemma (Hijikata et al., 2003, McMillan et al., 2007, Reipert et al., 1999). Furthermore, in vitro binding assays have demonstrated a direct interaction between the COOH-terminal region of plectin and desmin (Reipert et al., 1999). However, little information is available regarding functionally important sequences implicated in binding of these molecules to each other. Therefore, we have sought to better characterize the interaction of plectin with both desmin and vimentin. Furthermore, we also assessed the potential impact of some pathogenic mutations in plectin and desmin on these interactions.

Section snippets

Cloning

Plasmid inserts were generated by restriction digestion or PCR using the proofreading Pfu DNA polymerase (Promega) and cDNA-specific sense and antisense primers containing restriction site tags. Primer design (and amino acid numbering in the text) was based on mouse or human plectin (GenBank accession number: NM_201389 and NM_201380, respectively), human desmin (NM_001927), vimentin (NM_003380), and BPAG1e (NM_001723) sequences. Mutagenesis of alanine 213 to a valine (A213V), L385P and I385M in

The COOH-terminal region of plectin encompassing the fifth PRD and the linker region co-localizes with desmin and vimentin

Previous studies identified sequences within plectin important for its interaction with several types of IF proteins, such as cytokeratins and vimentin (Nikolic et al., 1996), but the interaction of plectin with desmin has not yet been characterized in detail. To assess the co-localization potential of the COOH-region of plectin with desmin, we first performed transfection studies in the IF-free cell line SW13vim with constructs encompassing various domains of the COOH-extremity of mouse or

Discussion

Desmin forms a filamentous network connecting myofibrils to each other at the level of Z-discs, to the sarcolemma, to mitochondria and, in heart, to intercalated discs (Capetanaki et al., 1997, Goldfarb and Dalakas, 2009, Paulin and Li, 2004). Although the importance of desmin/plectin connection in the maintenance of cytoarchitecture and thus cell resilience to mechanical stress in skeletal and cardiac myocytes are well established (Goldfarb and Dalakas, 2009, Konieczny et al., 2008, Paulin and

Acknowledgements

The authors are indebted to R. Evans, University of Colorado (USA) for kindly providing the SW13vim cell line, T. Magin, University of Bonn (Germany) and A. Sonnenberg, The Netherlands Cancer Institute (Amsterdam) for human plectin cDNAs, H. Herrmann, German Cancer Research Center (Heidelberg) for MEFvim−/− cells, recombinant vimentin and desmin proteins, various cDNAs, and critical reading of the manuscript, Z. Li, Pierre and Marie Curie University (France) and D. Paulin, Paris VII University

References (54)

  • T.S. Stappenbeck et al.

    Phosphorylation of the desmoplakin COOH terminus negatively regulates its interaction with keratin intermediate filament networks

    J. Biol. Chem.

    (1994)
  • K. Andrä et al.

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

    Genes Dev.

    (1997)
  • H. Bär et al.

    Conspicuous involvement of desmin tail mutations in diverse cardiac and skeletal myopathies

    Hum. Mutat.

    (2007)
  • H. Bär et al.

    Severe muscle disease-causing desmin mutations interfere with in vitro filament assembly at distinct stages

    Proc. Natl. Acad. Sci. U.S.A.

    (2005)
  • J.G. Boyer et al.

    Plakins in striated muscle

    Muscle Nerve

    (2010)
  • Y. Capetanaki et al.

    Desmin in muscle formation and maintenance: knockouts and consequences

    Cell Struct. Funct.

    (1997)
  • R. Foisner et al.

    Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins

    J. Cell Biol.

    (1988)
  • R. Foisner et al.

    M-phase-specific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase

    Mol. Biol. Cell

    (1996)
  • L. Fontao et al.

    Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus

    Mol. Biol. Cell

    (2003)
  • G.I. Gallicano et al.

    Rescuing desmoplakin function in extra-embryonic ectoderm reveals the importance of this protein in embryonic heart, neuroepithelium, skin and vasculature

    Development

    (2001)
  • D. Geerts et al.

    Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding

    J. Cell Biol.

    (1999)
  • L.G. Goldfarb et al.

    Tragedy in a heartbeat: malfunctioning desmin causes skeletal and cardiac muscle disease

    J. Clin. Invest.

    (2009)
  • B. Goudeau et al.

    Variable pathogenic potentials of mutations located in the desmin alpha-helical domain

    Hum. Mutat.

    (2006)
  • H. Herrmann et al.

    Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular scaffolds

    Annu. Rev. Biochem.

    (2004)
  • H. Herrmann et al.

    Intermediate filaments: from cell architecture to nanomechanics

    Nat. Rev. Mol. Cell Biol.

    (2007)
  • T. Hijikata et al.

    Plectin is a linker of intermediate filaments to Z-discs in skeletal muscle fibers

    J. Cell Sci.

    (1999)
  • T. Hijikata et al.

    Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

    Histochem. Cell Biol.

    (2003)
  • Cited by (40)

    • Different desmin peptides are distinctly deposited in cytoplasmic aggregations and cytoplasm of desmin-related cardiomyopathy patients

      2017, Biochimica et Biophysica Acta - Proteins and Proteomics
      Citation Excerpt :

      It has also been reported that the 2B domain is important for elongation of desmin and the tail domain-controlled lateral packing, stabilization, and elongation of the higher order filament structure. Recent studies have indicated that the desmin head domain interacts with myospryn [26] and binds to integral membrane proteins via ankyrin [5], the 1B domain interacts with nebulin [27], and segments 1A–2A are required to interact with plectin [28]. In the present study, two trypsin-digested desmin peptides containing the head and 1B domains were scattered in the myocardium, resulting in varied distributions across large areas.

    • Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins

      2016, Methods in Enzymology
      Citation Excerpt :

      Several methods have been used to study the interaction of plakins with IFs and to map their reciprocal binding sites. These include transfection of mammalian cells for co-localization studies by indirect immunofluorescence microscopy (Nikolic et al., 1996; Stappenbeck et al., 1993; Stappenbeck & Green, 1992; Wiche et al., 1993), yeast two/three-hybrid assays (Geerts et al., 1999; Fontao et al., 2003; Meng et al., 1997), overlay assays on purified IF proteins with purified mammalian plakins (Foisner et al., 1988; Kouklis et al., 1994; Meng et al., 1997), with recombinant plakin domains, fused or not to glutathione S-transferase (Favre et al., 2011; Kalinin et al., 2005), or with in vitro transcribed-translated, tagged, or radioactively labeled plakin regions (Fontao et al., 2003; Karashima & Watt, 2002), and co-sedimentation assays of plakins or plakin fragments with IFs (Choi et al., 2002; Foisner et al., 1988; Kalinin et al., 2005). Since detection of fluorescence is sensitive and very convenient, we have recently expressed enhanced green fluorescent protein (EGFP)-tagged plectin domains to analyze their interaction with IFs.

    View all citing articles on Scopus
    1

    Contributed equally to the work.

    View full text