My long-term goal is to understand the molecular basis for how the actin cytoskeleton of the auditory hair cell functions in the detection of sound. Initially, I will focus on the biochemical basis for how six mutations in nonmuscle gamma-actin cause autosomal dominant early hearing loss. The molecular basis for the deafness caused by these mutations is unknown, in large part because of the inability to obtain amounts of the mutant protein necessary to assess the biochemical consequences of these mutations on actin function. We have addressed this problem by cloning each of the mutations into yeast actin which is 91% identical to human nonmuscle gamma-actin. Recently, we demonstrated that these mutations cause allele-specific effects in yeast, while only one of the mutations affected actin polymerization in vitro, suggesting that the mutations instead interfere with the ability of the mutant filaments to be properly regulated by different actin- binding proteins. I will use a series of well-established biochemical, biophysical, and fluorescence microscopic techniques to study the effects that each of the deafness-causing mutations has on the ability of actin to interact with the following three candidate actin-binding proteins: cofilin, myosin, and formin. These three regulatory proteins were chosen for the following reasons. The importance of myosin and formin to proper hair cell function is underscored by the fact that mutations in either protein cause deafness. Cofilin is one of the key regulatory proteins which regulate filament dynamics and is present in virtually all cells. Moreover, several of the deafness mutations are located within the vicinity of the binding interface these three proteins use to interact with actin.
Deafness is a significant problem in the United States. My proposed work will not only provide insight into the biochemical basis for the hearing loss caused by these six actin mutations but will also provide valuable new information as to how actin functions and is regulated at the biochemical level in the normal ear. Thus, this work should enhance our understanding of the molecular basis of the normal hearing process. ? ? ?
|Bryan, Keith E; Rubenstein, Peter A (2009) Allele-specific effects of human deafness gamma-actin mutations (DFNA20/26) on the actin/cofilin interaction. J Biol Chem 284:18260-9|