ANALYSIS OF VACUOLAR TYPE - H+ - ATPASE FUNCTION IN NEUROMAST HAIR CELLS IN THE ZEBRAFISH EMBRYO

Abstract

Vacuolar type H+-ATPase (V-ATPase) is a ubiquitously expressed enzyme complex that pumps protons across membranes. The proton-motive force generated by V-ATPase is used by cells to acidify intracellular compartments. Additionally, certain specialized tissue types have V-ATPase on plasma membranes where it secretes H+into the extracellular space. While V-ATPase activity is essential for several cellular functions, our understanding of cell-type specific functions for V-ATPase remains limited. Here, I focused on investigating V-ATPase functions in mechanosensory hair cells. Hair cells are functional units of mammalian auditory and vestibular systems. Consequently, hair cell loss leads to permanent deafness. Mutation in specific V-ATPase subunits causes sensorineural deafness in human, however, the mechanism is not well understood. I used zebrafish as model vertebrate to investigate how loss of V-ATPase function impacts hair cells. Using a combination of genetic mutations, pharmacological manipulations and live imaging of hair cells in vivo, I found that V-ATPase activity is critical for hair cell survival. Analysis of molecular markers and cellular morphologies indicates hair cells in V-ATPase mutants undergo a caspase-independent, necrosis-like death. V-ATPase mutant hair cells show a significant decrease in mitochondrial membrane potential (mPTP). On modulating mPTP pharmacologically, V-ATPase mutants show a modest but consistent improvement of hair cell survival. These results indicate mitochondrial dysfunction contributes to hair cell death in V-ATPase mutants. Next, I generated a novel cilia pH biosensor and found that hair cell kinocilia have a more basic pH than other primary cilia in zebrafish embryos. Interestingly, my collaborators and I discovered that V-ATPase subunits localize to hair cell kinocilia in zebrafish and mice, which suggests cell-type specific functions for V-ATPase in kinocilia. pH maintenance in kinocilia may be an essential function that contributes to proper kinocilia length and/or function. In conclusion, this work has uncovered a function for V-ATPase activity that is critical for hair cell survival, in part by maintaining mitochondrial health, and a function that mediates hair cell kinocilia form and function. The work presented in this thesis advances our understanding of V-ATPase functioning in hearing loss, more broadly elucidates new in vivo cell-type specific V-ATPase functions.