title: Distribution of formins in cardiac muscle: FHOD1 is a component of intercalated discs and costameres.
abstract: The formin homology domain-containing protein1 (FHOD1) suppresses actin polymerization by inhibiting nucleation, but bundles actin filaments and caps filament barbed ends. Two polyclonal antibodies against FHOD1 were generated against (i) its N-terminal sequence (residues 1-339) and (ii) a peptide corresponding the sequence from position 358-371, which is unique for FHOD1 and does not occur in its close relative FHOD3. After affinity purification both antibodies specifically stain purified full length FHOD1 and a band of similar molecular mass in homogenates of cardiac muscle. The antibody against the N-terminus of FHOD1 was used for immunostaining cells of established lines, primary neonatal (NRC) and adult (ARC) rat cardiomyocytes and demonstrated the presence of FHOD1 in HeLa and fibroblastic cells along stress fibers and within presumed lamellipodia and actin arcs. In NRCs and ARCs we observed a prominent staining of presumed intercalated discs (ICD). Immunostaining of sections of hearts with both anti-FHOD1 antibodies confirmed the presence of FHOD1 in ICDs and double immunostaining demonstrated its colocalisation with cadherin, plakoglobin and a probably slightly shifted localization to connexin43. Similarly, immunostaining of isolated mouse or pig ICDs corroborated the presence of FHOD1 and its colocalisation with the mentioned cell junctional components. Anti-FHOD1 immunoblots of isolated ICDs demonstrated the presence of an immunoreactive band comigrating with purified FHOD1. Conversely, an anti-peptide antibody specific for FHOD3 with no cross-reactivity against FHOD1 immunostained on sections of cardiac muscle and ARCs the myofibrils in a cross-striated pattern but not the ICDs. In addition, the anti-peptide-FHOD1 antibody stained the lateral sarcolemma of ARCs in a banded pattern. Double immunostaining with anti-cadherin and -integrin-ß1 indicated the additional localization of FHOD1 in costameres. Immunostaining of cardiac muscle sections or ARCs with antibodies against mDia3-FH2-domain showed colocalisation with cadherin along the lateral border of cardiomyocytes suggesting also its presence in costameres.
authors.affiliation: Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany.
authors.forename: Abdulatif
authors.initials: A
authors.lastname: Al Haj
authors.affiliation: Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany; Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Poland.
authors.forename: Antonina J
authors.initials: AJ
authors.lastname: Mazur
authors.affiliation: Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany; Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Radaszkiewicz
authors.affiliation: Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany; Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Radaszkiewicz
authors.affiliation: Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany; Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Makowiecka
authors.affiliation: Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany; Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Stopschinski
authors.affiliation: Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Schönichen
authors.affiliation: Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany; Center of Advanced European Studies and Research (CAESAR), Bonn, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Geyer
authors.affiliation: Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany. Electronic address: hans.g.mannherz@rub.de.
authors.forename: Hans Georg
authors.initials: HG
authors.lastname: Mannherz
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D018031:Connexin 43 / Q000378:metabolism; D054974:Costameres / Q000378:metabolism*; D005326:Fetal Proteins / Q000378:metabolism*; D000080865:Formins; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D022502:Stress Fibers / Q000378:metabolism; D013552:Swine; D051185:gamma Catenin / Q000378:metabolism
chemical_list: D000906:Antibodies; D015820:Cadherins; D018031:Connexin 43; C120021:FHOD1 protein, human; C485994:FHOD3 protein, human; D005326:Fetal Proteins; D000080865:Formins; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D051185:gamma Catenin
keywords: Actin; Cardiomyocyte; FHOD1; Intercalated discs