Related Experiment Video
Updated: Jun 24, 2026
Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish
Published on: October 17, 2012
Proteomic and functional analysis of NCS-1 binding proteins reveals novel signaling pathways required for inner ear
Jessica A Petko1, Nadine Kabbani, Colleen Frey
1Department of Pharmacology, Penn State College of Medicine, 500 University Drive, Hershey PA 17033, USA. jac554@psu.edu
Background:
The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. Little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. Zebrafish represent a particularly tractable model system for the study of inner ear development. This is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. We have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor-1 (NCS-1) is required for semicircular canal formation. The function of NCS-1 in regulating semicircular canal formation has not yet been elucidated.
Results:
We initiated a multistep functional proteomic strategy to identify neuronal calcium sensor-1 (NCS-1) binding partners (NBPs) that contribute to inner ear development in zebrafish. By performing a Y2H screen in combination with literature and database searches, we identified 10 human NBPs. BLAST searches of the zebrafish EST and genomic databases allowed us to clone zebrafish orthologs of each of the human NBPs. By investigating the expression profiles of zebrafish NBP mRNAs, we identified seven that were expressed in the developing inner ear and overlapped with the ncs-1a expression profile. GST pulldown experiments confirmed that selected NBPs interacted with NCS-1, while morpholino-mediated knockdown experiments demonstrated an essential role for arf1, pi4kbeta, dan, and pink1 in semicircular canal formation.
Conclusion:
Based on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4kbeta/Arf1 form a signaling pathway that regulates secretion of molecular components, including Dan and Bmp4, that are required for development of the vestibular apparatus. A second set of NBPs, consisting of Pink1, Hint2, and Slc25a25, are destined for localization in mitochondria. Our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for NCS-1 in mitochondrial function via its association with several mitochondrial proteins.
