Date of Degree
PhD (Doctor of Philosophy)
Daniel F. Eberl
Na+/K+ ATPase (also referred to as the Na pump) maintains ionic homoeostasis in most biological systems. The catalytic alpha subunit requires a beta subunit for its transport to the plasma membrane and for regulating its activity. In the vertebrate inner ear, the Na pump is thought to maintain the endocochlear potential by enriching the endolymph with K+ ions and also maintains fluid volume homeostasis. I hypothesis that the Na pump plays a important functional role in establishing and maintaining ionic gradient in the scolopale cell of the Johnston's organ in Drosophila melanogaster and thereby is critical to auditory mechanotransduction in the fly ear.
We show that alpha and beta subunits are expressed in Johnston's Organ (JO), the Drosophila auditory organ. We knocked down expression of alpha subunits (ATP-alpha and alpha-like;) and beta subunits (nrv1, nrv2 and nrv3) individually in JO with UAS/Gal4-mediated RNA-interference. ATP-alpha shows elevated expression in the ablumenal membrane of scolopale cells, which enwrap JO neuronal dendrites in endolymph-like compartments. Knocking down ATP-alpha, but not alpha-like, in the entire JO or only in scolopale cells using specific drivers, resulted in complete deafness. We also show that the scolopale cell specific knockdown of the ATP-alpha can be partially rescued with nompA-Gal80. Among beta subunits, nrv2 is expressed in scolopale cells and nrv3 in JO neurons. Knocking down nrv2 in scolopale cells blocked Nrv2 expression, reduced ATP-alpha expression in the scolopale cells and caused almost complete deafness. Furthermore, knockdown of either nrv2 or ATP-alpha specifically in scolopale cells causes abnormal electron-dense material accumulation in the scolopale space. Similarly, nrv3 functions in JO but not in scolopale cells, suggesting neuron-specificity that parallels nrv2 scolopale cell-specific support of the catalytic ATPα. We also characterized two nrv3 deletion mutations and have shown that they cause homozygous lethality at early larval stages. In addition, our genetic interaction studies with nrv3 allele in a homozygous Na pump hypomorphic background (2206) show a small but significant interaction between the Nrv3 and the ATP-alpha subunits in the JO. Our studies provide an amenable model to investigate generation of endolymph-like extracellular compartments and sets the stage for future studies to elucidate the pathways and mediators of ion transport and fluid regulation in more detail in the auditory system.
xiv, 120 pages
Includes bibliographical references (pages 113-120).
Copyright 2012 Madhuparna Roy