TY - JOUR
T1 - Identification of the hair cell soma-1 antigen, hcs-1, as otoferlin
AU - Goodyear, Richard J.
AU - Legan, P. Kevin
AU - Christiansen, Jeffrey R.
AU - Xia, Bei
AU - Korchagina, Julia
AU - Gale, Jonathan E.
AU - Warchol, Mark E.
AU - Corwin, Jeffrey T.
AU - Richardson, Guy P.
N1 - Funding Information:
Dr. Janet L. Cyr was originally involved in this work when she was at West Virginia University School of Medicine. The authors would like to thank Dr. Kelli Phillips for experimental advice and dissections; Dr. Kathryn Lilley, Cambridge Centre for Proteomics, UK for her assistance; James Finley for technical support; Shirley House for instruction in monoclonal antibody production; Dr. Peter Gillespie for the anti-PMCAb antibody; and Dr. Jim Bartles for the EGFP-espin construct. This work was supported by NIH/NIDCD grants R01DC006402 (previously to JLC), RO1DC00200 (JTC), and R01DC006283 and P30DC04665 (MEW) and NIH/NCRR grant P20RR015574 (WVU Sensory Neuroscience Research Center), The Wellcome Trust (JEG, RJG, GPR), a Royal Society University Research Fellowship (JEG), and a PhD Studentship from The Royal National Institute for Deaf People (JK). A portion of this work was performed at the Marine Biological Laboratory, Woods Hole, MA.
PY - 2010/12
Y1 - 2010/12
N2 - Hair cells, the mechanosensitive receptor cells of the inner ear, are critical for our senses of hearing and balance. The small number of these receptor cells in the inner ear has impeded the identification and characterization of proteins important for hair cell function. The binding specificity of monoclonal antibodies provides a means for identifying hair cell-specific proteins and isolating them for further study. We have generated a monoclonal antibody, termed hair cell soma-1 (HCS-1), which specifically immunolabels hair cells in at least five vertebrate classes, including sharks and rays, bony fish, amphibians, birds, and mammals. We used HCS-1 to immunoprecipitate the cognate antigen and identified it as otoferlin, a member of the ferlin protein family. Mutations in otoferlin underlie DFNB9, a recessive, nonsyndromic form of prelingual deafness characterized as an auditory neuropathy. Using immunocytochemistry, we find that otoferlin is associated with the entire basolateral membrane of the hair cells and with vesicular structures distributed throughout most of the hair cell cytoplasm. Biochemical assays indicate that otoferlin is tightly associated with membranes, as it is not solubilized by alterations in calcium or salt concentrations. HCS-1 immunolabeling does not co-localize with ribeye, a constituent of synaptic ribbons, suggesting that otoferlin may, in addition to its proposed function in synaptic vesicle release, play additional roles in hair cells.
AB - Hair cells, the mechanosensitive receptor cells of the inner ear, are critical for our senses of hearing and balance. The small number of these receptor cells in the inner ear has impeded the identification and characterization of proteins important for hair cell function. The binding specificity of monoclonal antibodies provides a means for identifying hair cell-specific proteins and isolating them for further study. We have generated a monoclonal antibody, termed hair cell soma-1 (HCS-1), which specifically immunolabels hair cells in at least five vertebrate classes, including sharks and rays, bony fish, amphibians, birds, and mammals. We used HCS-1 to immunoprecipitate the cognate antigen and identified it as otoferlin, a member of the ferlin protein family. Mutations in otoferlin underlie DFNB9, a recessive, nonsyndromic form of prelingual deafness characterized as an auditory neuropathy. Using immunocytochemistry, we find that otoferlin is associated with the entire basolateral membrane of the hair cells and with vesicular structures distributed throughout most of the hair cell cytoplasm. Biochemical assays indicate that otoferlin is tightly associated with membranes, as it is not solubilized by alterations in calcium or salt concentrations. HCS-1 immunolabeling does not co-localize with ribeye, a constituent of synaptic ribbons, suggesting that otoferlin may, in addition to its proposed function in synaptic vesicle release, play additional roles in hair cells.
KW - auditory system
KW - balance
KW - hearing
KW - ribeye
KW - vestibular system
UR - http://www.scopus.com/inward/record.url?scp=78650175139&partnerID=8YFLogxK
U2 - 10.1007/s10162-010-0231-6
DO - 10.1007/s10162-010-0231-6
M3 - Article
C2 - 20809368
AN - SCOPUS:78650175139
SN - 1525-3961
VL - 11
SP - 573
EP - 586
JO - JARO - Journal of the Association for Research in Otolaryngology
JF - JARO - Journal of the Association for Research in Otolaryngology
IS - 4
ER -