Benjamin J. Perrin, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Actin is one of the most abundant proteins in nature and plays essential roles in almost every cellular function including muscle contraction, cell migration, vesicle trafficking, and polarized growth. Two cytoplasmic actin isoforms, [unreadable]cyto- and ?cyto-actin, are ubiquitously expressed. Although encoded by separate genes, these cytoplasmic actin isoforms vary at only 4 of 375 amino acid residues; however, these small sequence differences are conserved between birds and mammals, suggesting that [unreadable]cyto-- and ?cyto-actin nave distinct cellular functions. Consistent with this idea, the cellular localization of [unreadable]cyto-- and ?cyto-actin differ, which is thought to contribute to cell polarization. Additionally, some ?cyto-actin mutations lead to progressive deafness in humans, indicating an essential cellular role for this isoform that cannot be filled by [unreadable]cyto-actin. Although impressive gains have been made in understanding how actins are regulated by other proteins, the distinct functions of [unreadable]- and ?cyto--actin remain poorly understood. The goals of this fellowship include elucidating the specific functions of [unreadable]cyto- and ?cyto--actin in the mammalian inner ear. Preliminary evidence suggests that ?cyto--actin deficient mice display progressive hearing loss. A [unreadable]cyto-actin-deficient mouse will be generated to allow for a comparison of the effect [unreadable]cyto- and ?cyto-actin deficiency on auditory physiology. Additionally, the structure and function of polarized inner ear cells in both models will be analyzed by both light and electron microscopy to understand the cellular mechanism underling changes in hearing. This fellowship will contribute to improving public health by furthering understanding of the distinct contributions of [unreadable]cyto- and ?cyto--actin to normal cell biology. Additionally, progressive deafness caused by mutation of the human ?cyto-actin 9ene is Paralleled by the phenotype of the ?cyto--actin deficient mouse, characterization of which will likely provide insight into the pathomechanism of this form of human deafness. [unreadable] [unreadable] [unreadable]

Actins; Laboratories; Maintenance; Muscle Mitochondria; Muscular Dystrophies;

DESCRIPTION (provided by applicant): Progressive hearing loss is a common human health problem that often stems from the loss or dysfunction of sensory hair cells in the inner ear. Actin proteins are central to hair cell structure and function; consequently, understanding the molecular mechanisms of cytoskeletal maintenance is a key step in considering rationally designed therapies for age-related or noise-induced hearing loss. ¿-Actin and ?-actin, two distinct yet closely related actin isoforms, each contribute to the population of filamentous actin in stereocilia. We have previously shown that mice with hair cells lacking either actin isoform form normal stereocilia, but go on to develop different forms of progressive hearing loss. This suggests that ¿-Actin and ?-actin each make unique contributions to stereocilia stability. This proposal is focused on two aspects of actin biology in stereocilia. In Aim 1, we will use existing conditional ¿-Actin and ?-actin knockout mouse lines and a newly developed transgenic actin reporter line to assess actin dynamics in stereocilia in vivo. In Aim 2, we will use cell biologica and biochemical approaches to determine if differential interactions between the actin bundling protein fascin 2 and ¿-actin and ?-actin contribute to distinct stereocilia maintenance mechanisms. Together, completion of these Aims will support a new model of actin regulation during stereocilia maintenance. In the long term, we will use the same suite of universal platforms established in this proposal to assess the roles of other deafness-causing mutant actin binding proteins in modulating stereocilia actin dynamics at the level of molecules, cells and mice. PUBLIC HEALTH RELEVANCE: Age-related hearing loss is the most common form of hearing impairment in humans and is often caused by degeneration of specialized sensory cells in the inner ear. Many of these cells depend on different kinds of actin proteins for normal structure and function. We are proposing to elucidate the mechanisms through which each type of actin contributes to cell maintenance to provide the foundation for rationale design of therapeutic interventions.

PROJECT SUMMARY Progressive hearing loss is a prevalent health problem that often stems from the loss or dysfunction of sensory hair cells in the inner ear. Hair cell function depends on actin-­based protrusions called stereocilia to detect the physical movement of sound. Functional stereocilia must be maintained for the life of an organism since hair cells are not renewed or regenerated. Thus, stereocilia homeostasis is critical for continued auditory function. Tip links transmit force from stereocilia deflection to gate associated mechanotransduction channels found at stereocilia tips. Rows of stereocilia that normally have active channels also have more actin incorporation at their tips. In addition, mutations in the Cdh23 gene, which encodes the tip link component cadherin-­23, strongly influence progressive hearing loss and stereocilia length maintenance in mouse models. Together, these data suggest that tip links or mechanotransduction regulates actin dynamics in stereocilia. Recently, we and others found that stereocilia actin cores are highly stable, except at stereocilia tips, where there is more dynamic actin turnover. However, several unanswered questions remain including whether actin binding proteins in stereocilia are similarly stable, how the stereocilia actin core is made to be stable and whether actin core instability directly influences progressive hearing loss. It is also unclear how the dynamic region of actin at stereocilia tips is regulated and if it contributes to stereocilia length maintenance. To address these questions, we will use transgenic reporters to monitor the dynamics of actin and the actin crosslinker fascin-­2 in vivo and ex vivo, both in normal mice and in mice with deafness causing mutations in actin binding proteins found in stereocilia. We also hypothesize that stereocilia stability is particularly critical for stereocilia maintenance when tip links break. This model will be tested by measuring actin incorporation as Cdh23 expression is varied. If stereocilia shorten following tip link loss, then actin polymerization would be required to extend stereocilia back to their original length. We have found that the actin severing proteins destrin and cofilin localize to stereocilia tips, and we will determine if they promote new actin incorporation within the dynamic tip zone. Together, these experiments will provide critical insights into the roles of both extraordinarily stable and more dynamic regions of the actin core in maintaining functional stereocilia.