Semester

Spring

Date of Graduation

2007

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Janet L Cyr

Abstract

Specialized epithelial cells, called hair cells, detect stimuli perceived by the organs of the inner ear. Such stimuli deflect the sensing organelle of the hair cell, the hair bundle. This force is proposed to create tension in the tip link, an extracellular filamentous structure, and open mechanically-gated ion channels. This process, called mechanotransduction, allows ions to enter the hair cell and results in its depolarization. During prolonged stimuli, the sensitivity of the hair cell is maintained by adaptation. To date, little is known about the molecular mechanisms that regulate either mechanotransduction or adaptation.;Myosin-1c (Myo1c) has been proposed to be the adaptation motor. As such, it must interact with other components of the mechanotransduction complex. Using an in situ binding assay, we previously demonstrated that Myo1c interacts with molecules in the hair bundle, the site of mechanotransduction, through the calmodulin (CaM)-binding IQ domains in its neck region. In the current study, we identify the second CaM-binding IQ domain as a region of Myo1c that mediates CaM-sensitive binding to the hair bundle. As well, we show that the binding of Myo1c in the hair bundle is disrupted by treatments that break tip links.;Recent data indicate that Myo1c interacts in vitro with Cadherin-23 (Cdh23), a component of the tip link, and phosphotidyl-inositol bisphosphate, a phospholipid recently shown to be essential to mechanotransduction. It is not known, however, whether these interactions occur in hair cells. To determine if either of these components contribute to the binding observed in in situ binding assays, we examined Myo1c binding in mice whose hair cells lack Cdh23. In such samples, Myo1c binding was not observed. Additional experiments confirm the correct localization of PIP2 in Cdh23 mutant mice. Collectively, our data suggest that Myo1c and Cdh23 interact in the hair bundle and that this interaction is modulated by CaM.;Myosin-1a (Myo1a), the predominant unconventional myosin expressed in the enteryocyte, has also been implicated in hearing disorders. Using antibodies raised against Myo1a we failed to detect Myo1a in the hair cells of murine cochlea. However, alternative, potentially novel, antigens were detected and will be pursued in future work.

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