Advanced microscope technologies for single-molecule functional analyses of stereocilia components necessary for normal human hearing - Project Summary/Abstract Sensorineural hearing loss affects 20% of the population in the United States at all ages. A normal acoustic sense requires a variety of cells in the inner ear, in the brain and on neuronal pathways. In the inner ear, stereocilia on the apical surface of hair cells function as biological mechanical switches, and their degeneration is one of the causes of sensorineural hearing loss. Presently, there are no clinically useful treatments to regenerate or repair damaged stereocilia. In this K99 proposal, I describe my ongoing and future studies using single-molecule microscopy techniques (1) in live hair cells and (2) in stereocilia F-actin cores. I am also developing (3) a novel mouse line to visualize the developmental transition of nascent microvilli to mature stereocilia and to identify novel candidate “deafness genes”. Single-molecule microscopy in live hair cells has already yielded a novel model to explain the molecular dynamics in stereocilia and succeeded in detecting abnormal kinetics of mutant molecules. With these techniques, I introduce the concept of “single-molecule functional analysis” to hearing research and analyze the functions of “deafness gene” variants discovered in families segregating hearing loss. The training objective in this K99 award is to develop novel microscope methodologies. Specifically, I will develop single-molecule microscopy in live stereocilia by learning diSPIM light-sheet microscopy and new ways of high-resolution imaging using deep learning from Dr. Hari Shroff (NIBIB/NIH), my co-mentor, who is a world-renowned engineer of novel microscopes. I will learn cryo-electron microscopy (CryoET) from Dr. Dennis C. Winkler (NIDCD/NIH) to localize single molecules in F-actin cores and STED super-resolution microscopy from Dr. Christian A. Combs (NHLBI/NIH) to visualize the change of molecular dynamics using my novel mouse models that I am establishing in the laboratory of my NIDCD/NIH mentor, Dr. Thomas B. Friedman. The research aims during the mentored (K99) phase are to elucidate the molecular dynamics of stereocilia components using these advanced microscope techniques. Single-molecule microscopy in live stereocilia will visualize dynamics over the short time span of seconds to minutes. Two other techniques, CryoET and STED in combination with our novel mouse line, will elucidate dynamics over a longer time span during repair processes and development. Molecular dynamics elucidated by these techniques will be a control to detect abnormal functions of “deafness gene” variants as I provide in the preliminary data section. The aim during the independent (R00) phase is to identify and study candidate “deafness genes” and potential therapeutic targets for human deafness. I will use my novel mouse model to study the cessation of treadmilling during the transition of microvilli to stereocilia. By combining transcriptomics, I will identify molecules localized in microvilli which have ceased normal F-actin treadmilling as they transit to nascent stereocilia. With these microscopy techniques, I will (1) establish a pipeline to analyze functions of stereocilia molecular components and (2) reveal therapeutic targets for sensorineural hearing loss.
