title: Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants.
abstract: Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.
authors.affiliation: E. & H. Klessmann Institute for Cardiovascular Research & Development, Ruhr-University Bochum, 32545 Bad Oeynhausen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Brodehl
authors.forename: Per Niklas
authors.initials: PN
authors.lastname: Hedde
authors.forename: Mareike
authors.initials: M
authors.lastname: Dieding
authors.forename: Azra
authors.initials: A
authors.lastname: Fatima
authors.forename: Volker
authors.initials: V
authors.lastname: Walhorn
authors.forename: Susan
authors.initials: S
authors.lastname: Gayda
authors.forename: Tomo
authors.initials: T
authors.lastname: Šarić
authors.forename: Bärbel
authors.initials: B
authors.lastname: Klauke
authors.forename: Jan
authors.initials: J
authors.lastname: Gummert
authors.forename: Dario
authors.initials: D
authors.lastname: Anselmetti
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.forename: Gerd Ulrich
authors.initials: GU
authors.lastname: Nienhaus
authors.forename: Hendrik
authors.initials: H
authors.lastname: Milting
mesh_terms: D000818:Animals; D009202:Cardiomyopathies / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D003893:Desmin / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D015151:Immunoblotting; D007382:Intermediate Filaments / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008853:Microscopy / Q000379:methods; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D014162:Transfection
chemical_list: D003893:Desmin; D008164:Luminescent Proteins; D050505:Mutant Proteins