Mutations of the Mouse ELMO Domain Containing 1 Gene (Elmod1 ) Link Small GTPase Signaling to Actin Cytoskeleton Dynamics in Hair Cell Stereocilia Kenneth R. Johnson*, Chantal M. Longo-Guess, Leona H. Gagnon The Jackson Laboratory, Bar Harbor, Maine, United States of America Abstract Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda2J). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1–5 of Elmod1, and rda2J is an intragenic duplication of exons 3–8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase- activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia. Citation: Johnson KR, Longo-Guess CM, Gagnon LH (2012) Mutations of the Mouse ELMO Domain Containing 1 Gene (Elmod1) Link Small GTPase Signaling to Actin Cytoskeleton Dynamics in Hair Cell Stereocilia. PLoS ONE 7(4): e36074. doi:10.1371/journal.pone.0036074 Editor: Yoshiro Ishimaru, University of Tokyo, Japan Received February 8, 2012; Accepted March 25, 2012; Published April 27, 2012 Copyright: 2012 Johnson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by grant number R01DC004301 from the National Institute On Deafness And Other Communication Disorders (NIDCD). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIDCD or the National Institutes of Health (NIH). Jackson Laboratory Shared Services are supported by grant number CA34196 from the National Cancer Institute (NCI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ken.johnson@jax.org Introduction Many medically important hearing and balance disorders are caused by defects in the sensory hair cells of the inner ear. Modified microvilli called stereocilia project from the apical surfaces of hair cells and are tethered together by fibrous links to form hair bundles. Hair bundle deflections, caused by fluid movements induced by sound pressure or head movements, open mechanically gated transduction channels located at the stereocilia tips [1]. The resulting influx of cations depolarizes the hair cell and triggers neurotransmitter release, which initiates the transfer of auditory or vestibular information to the brain. Hair bundles thus are essential to the mechanoelectrical transduction process, and proper development and maintenance of these key structures is critical for normal hearing and balance. Each hair cell has a single hair bundle composed of tens to hundreds of stereocilia that are arranged in precise rows of increasing height, forming a uniform staircase pattern. Each stereocilium consists of a core of actin filaments surrounded by the plasma membrane. Molecular genetic analyses of human and mouse deafness mutations and the analysis of hair-bundle proteins have identified several constituents of the hair bundle and have provided insight into its development and function [2,3,4,5]. In addition to the cytoskeletal proteins associated with its actin core, stereocilia contain motor proteins, scaffolding proteins, adhesion proteins, integral membrane proteins, calcium regulatory proteins, and proteins involved in ATP synthesis. Protein complexes forming stereociliary links are particularly important in the development and cohesion of the hair bundle [6,7]. Although much progress has been made in identifying the structural components of the hair bundle [8], little is known about the signaling pathways that regulate hair bundle development. Small guanosine triphosphatases (GTPases) of the Rho and Arf families are key regulators of actin cytoskeleton dynamics in many cell types and species [9,10]. Several lines of evidence suggest that small GTPases also may be involved in regulating the actin dynamics of hair cells [11,12,13]. For example, diaphanous proteins can act as effectors for Rho GTPases to regulate actin polymerization [14,15], and a mutation of the human DIAPH1 gene underlies the nonsyndromic deafness disorder DFNA1 [16]. Recently, an analysis of late stage mouse embryos with an inner ear-specific knockout of the Rac1 gene demonstrated an essential role for this Rho-family GTPase in cochlear development and hair cell morphogenesis [13]. PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e36074