Student Theses and Dissertations

Date of Award

2017

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hudspeth Laboratory

Abstract

Hair cells are the mechanosensory detectors that underlie our senses of audition and balance. Their mechanosensitivity arises from direct gating of force-sensitive ion channels by the tension in tip links located at the tops of the stereocilia. The study of hair cells and their mechanically gated channels has heretofore required physical displacement of a hair bundle to open its mechanotransduction channels. The work presented here describes a novel method of hair-bundle stimulation: irradiation with light. The original aim of this study was to elucidate the role of Ca2+ in fast adaptation, a process thought to underlie a hair bundle’s ability to amplify stimuli. I aimed to do this by generating intracellular surges in Ca2+ concentration without altering the state of the mechanotransduction channel or its associated elements. This procedure would permit me to study the mechanical correlates of fast adaptation independently from those associated with transduction. I used a light-sensitive, Ca2+-loaded cage compound to release Ca2+ in hair cells by ultraviolet irradiation. To my surprise, irradiation both in the presence and absence of the caged compound caused rapid motion of hair bundles towards their tall edge, a motion typically associated with excitatory stimulation. Tight-seal, whole-cell recording disclosed that the motion was associated with rapid opening of mechanotransduction channels. Both the light-evoked movement and the channel gating disappeared when tip links were ruptured, indicating that the mechanotransduction apparatus is involved in a hair cell’s response to light. Blockage of the mechanotransduction channels in their open state with gentamicin abolished the light-evoked electrical response but incompletely reduced the light-evoked displacement, suggesting that channel gating is only partially responsible for light-evoked hair-bundle motion. I sought to identify the cellular components underlying light-evoked hair-bundle motion. Using an illumination system containing a digital micromirror device, I localized the absorptive element responsible for hair-bundle motion to the region directly below the bundle. The absorbers appeared to be mitochondrial chromophores that release the absorbed energy as heat. In experiments combining ultraviolet and infrared irradiation, I found that heat pulses of 0.5 K are sufficient to elicit hair-bundle motion. These heat pulses may alter the mechanical properties of tip links, leading to hair-bundle motion and mechanotransduction-channel gating. Thus the hair bundle confers thermal sensitivity upon a hair cell. The ability to stimulate hair cells using light will benefit physiological investigations of hair-cell function, and may have a role in non0invasively boosting hair-cell function in patients with hearing loss.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Julien Babak Azimzadeh June

Available for download on Friday, May 10, 2019

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