title: Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear.
abstract: The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24-48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of (15)N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips.
authors.affiliation: Laboratory of Molecular Genetics, Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Meghan C
authors.initials: MC
authors.lastname: Drummond
authors.affiliation: Laboratory of Molecular Genetics, Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Melanie
authors.initials: M
authors.lastname: Barzik
authors.affiliation: Laboratory of Molecular Genetics, Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Bird
authors.affiliation: Department of Neurobiology, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.
authors.forename: Duan-Sun
authors.initials: DS
authors.lastname: Zhang
authors.affiliation: 1] National Resource for Imaging Mass Spectrometry, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts 02139, USA [2] Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts 02139, USA.
authors.forename: Claude P
authors.initials: CP
authors.lastname: Lechene
authors.affiliation: Department of Neurobiology, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.
authors.forename: David P
authors.initials: DP
authors.lastname: Corey
authors.affiliation: Section on Sensory Cell Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Lisa L
authors.initials: LL
authors.lastname: Cunningham
authors.affiliation: Laboratory of Molecular Genetics, Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Friedman
mesh_terms: D000199:Actins / Q000502:physiology*; D000818:Animals; D001692:Biological Transport; D007758:Ear, Inner / Q000166:cytology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007930:Leucine / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D013194:Staining and Labeling; D059547:Stereocilia / Q000502:physiology*; D014162:Transfection
chemical_list: D000199:Actins; D049452:Green Fluorescent Proteins; D007930:Leucine