title: DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes.
abstract: Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Gerard W
authors.initials: GW
authors.lastname: Dougherty
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Niki T
authors.initials: NT
authors.lastname: Loges
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Klinkenbusch
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Heike
authors.initials: H
authors.lastname: Olbrich
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Petra
authors.initials: P
authors.lastname: Pennekamp
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Tabea
authors.initials: T
authors.lastname: Menchen
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Johanna
authors.initials: J
authors.lastname: Raidt
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Julia
authors.initials: J
authors.lastname: Wallmeier
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Claudius
authors.initials: C
authors.lastname: Werner
authors.affiliation: 2 Gerhard-Domagk-Institut for Pathology, University Hospital Muenster, and.
authors.forename: Cordula
authors.initials: C
authors.lastname: Westermann
authors.affiliation: 3 Department of Human Genetics, University of Muenster, Muenster, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Ruckert
authors.affiliation: 4 Department of Translational Medical Sciences, Federico II University, Naples, Italy.
authors.forename: Virginia
authors.initials: V
authors.lastname: Mirra
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Rim
authors.initials: R
authors.lastname: Hjeij
authors.affiliation: 5 Wellcome Trust Sanger Institute, Cambridge, United Kingdom.
authors.forename: Yasin
authors.initials: Y
authors.lastname: Memari
authors.affiliation: 5 Wellcome Trust Sanger Institute, Cambridge, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Durbin
authors.affiliation: 5 Wellcome Trust Sanger Institute, Cambridge, United Kingdom.
authors.forename: Anja
authors.initials: A
authors.lastname: Kolb-Kokocinski
authors.affiliation: 6 Center for Human Disease Modeling, Duke University, Durham, North Carolina.|7 Regeneron Genetics Center, Tarrytown, New York; and.
authors.forename: Kavita
authors.initials: K
authors.lastname: Praveen
authors.affiliation: 8 Allergy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.|9 Baystate Medical Center, Springfield, Massachusetts.
authors.forename: Mohammad A
authors.initials: MA
authors.lastname: Kashef
authors.affiliation: 8 Allergy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Sara
authors.initials: S
authors.lastname: Kashef
authors.affiliation: 10 Australian Capital Territory Health, Canberra, Australian Capital Territory, Australia.
authors.forename: Fardin
authors.initials: F
authors.lastname: Eghtedari
authors.affiliation: 11 Department of General Pediatrics, Adolescent Medicine and Neonatology, University of Freiburg, Freiburg, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Häffner
authors.affiliation: 12 Department of Pediatrics, Lapland Central Hospital, Rovaniemi, Finland.
authors.forename: Pekka
authors.initials: P
authors.lastname: Valmari
authors.affiliation: 13 Department of Bronchology, Pediatric Institute Svábhegy, Budapest, Hungary.
authors.forename: György
authors.initials: G
authors.lastname: Baktai
authors.affiliation: 14 Soroka Medical Center, Beer-Sheva, Israel.
authors.forename: Micha
authors.initials: M
authors.lastname: Aviram
authors.affiliation: 15 Rambam Medical Center, Haifa, Israel.
authors.forename: Lea
authors.initials: L
authors.lastname: Bentur
authors.affiliation: 16 Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Israel
authors.initials: I
authors.lastname: Amirav
authors.affiliation: 6 Center for Human Disease Modeling, Duke University, Durham, North Carolina.
authors.forename: Erica E
authors.initials: EE
authors.lastname: Davis
authors.affiliation: 6 Center for Human Disease Modeling, Duke University, Durham, North Carolina.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Katsanis
authors.affiliation: 17 Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Martina
authors.initials: M
authors.lastname: Brueckner
authors.affiliation: 18 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Artem
authors.initials: A
authors.lastname: Shaposhnykov
authors.affiliation: 18 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Gaia
authors.initials: G
authors.lastname: Pigino
authors.affiliation: 3 Department of Human Genetics, University of Muenster, Muenster, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Dworniczak
authors.affiliation: 1 Department of General Pediatrics and.
authors.forename: Heymut
authors.initials: H
authors.lastname: Omran
mesh_terms: D056444:Axonemal Dyneins / Q000378:metabolism*; D001483:Base Sequence; D002923:Cilia / Q000378:metabolism* / Q000648:ultrastructure; D004398:Dyneins / Q000378:metabolism* / Q000648:ultrastructure; D006720:Homozygote; D006801:Humans; D007619:Kartagener Syndrome / Q000235:genetics; D008168:Lung / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D021381:Protein Transport
chemical_list: D056444:Axonemal Dyneins; C465433:DNAH11 protein, human; C430745:DNAH9 protein, human; D004398:Dyneins
keywords: immunofluorescence microscopy; left–right dynein; normal ciliary ultrastructure; primary ciliary dyskinesia; transmission electron microscopy