title: Fascin 2b is a component of stereocilia that lengthens actin-based protrusions.
abstract: Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a K(d)≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Shih-Wei
authors.initials: SW
authors.lastname: Chou
authors.forename: Philsang
authors.initials: P
authors.lastname: Hwang
authors.forename: Gustavo
authors.initials: G
authors.lastname: Gomez
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fernando
authors.forename: Megan C
authors.initials: MC
authors.lastname: West
authors.forename: Lana M
authors.initials: LM
authors.lastname: Pollock
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lin-Jones
authors.forename: Beth
authors.initials: B
authors.lastname: Burnside
authors.forename: Brian M
authors.initials: BM
authors.lastname: McDermott
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified; D019556:COS Cells; D002522:Chlorocebus aethiops; D002923:Cilia / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006198:Hair Cells, Auditory / Q000166:cytology / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D011554:Pseudopodia / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012694:Serine; D015027:Zebrafish / Q000235:genetics
chemical_list: D000199:Actins; D005136:Eye Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; C518611:fscn2b protein, zebrafish; D012694:Serine