Characterization of the Drosophila Ortholog of the Human Usher Syndrome Type 1G Protein Sans Fabio Demontis¤, Christian Dahmann* Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Abstract Background: The Usher syndrome (USH) is the most frequent deaf-blindness hereditary disease in humans. Deafness is attributed to the disorganization of stereocilia in the inner ear. USH1, the most severe subtype, is associated with mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15, and sans. Myosin VIIa, harmonin, cadherin 23, and protocadherin 15 physically interact in vitro and localize to stereocilia tips in vivo, indicating that they form functional complexes. Sans, in contrast, localizes to vesicle-like structures beneath the apical membrane of stereocilia-displaying hair cells. How mutations in sans result in deafness and blindness is not well understood. Orthologs of myosin VIIa and protocadherin 15 have been identified in Drosophila melanogaster and their genetic analysis has identified essential roles in auditory perception and microvilli morphogenesis, respectively. Principal Findings: Here, we have identified and characterized the Drosophila ortholog of human sans. Drosophila Sans is expressed in tubular organs of the embryo, in lens-secreting cone cells of the adult eye, and in microvilli-displaying follicle cells during oogenesis. Sans mutants are viable, fertile, and mutant follicle cells appear to form microvilli, indicating that Sans is dispensable for fly development and microvilli morphogenesis in the follicle epithelium. In follicle cells, Sans protein localizes, similar to its vertebrate ortholog, to intracellular punctate structures, which we have identified as early endosomes associated with the syntaxin Avalanche. Conclusions: Our work is consistent with an evolutionary conserved function of Sans in vesicle trafficking. Furthermore it provides a significant basis for further understanding of the role of this Usher syndrome ortholog in development and disease. Citation: Demontis F, Dahmann C (2009) Characterization of the Drosophila Ortholog of the Human Usher Syndrome Type 1G Protein Sans. PLoS ONE 4(3): e4753. doi:10.1371/journal.pone.0004753 Editor: Francois Schweisguth, Institut Pasteur, France Received October 31, 2008; Accepted February 9, 2009; Published March 9, 2009 Copyright: 2009 Demontis 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 work was supported by Grant DA586/4-1 of the Deutsche Forschungsgemeinschaft. 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: dahmann@mpi-cbg.de ¤ Current address: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America Introduction The analysis of orthologs of human disease genes in model organisms has revealed important insights into developmental mechanisms. One outstanding example is the contribution that the analysis of Usher syndrome-related genes has made to our understanding of hair cell development and auditory perception [1]. Usher syndrome is the most common hereditary disease associated with deafness and blindness in humans [2,3,4]. Three clinical subtypes, USH1, USH2, and USH3, have been defined according to the severity of the hearing impairment, the absence or presence of vestibular dysfunctions, and the age of onset of retinitis pigmentosa, which leads to blindness. USH1 is the most severe subtype and USH1 patients suffer from severe hearing impairment, balance defects, and prepubertal-onset of retinitis pigmentosa. Five USH1 genes have been identified: USH1B encodes an unconventional motor protein, myosin VIIa [5]; USH1C encodes a PDZ-domain-containing scaffolding protein, harmonin [5]; USH1D and USH1F encode two members of the cadherin family of Ca2+-dependent cell adhesion molecules, cadherin 23 and protocadherin 15, respectively [6,7,8,9]; USH1G encodes a putative scaffolding protein, sans, containing three ankyrin repeats and a sterile alpha motif (SAM) domain [10]. Hair cells of the mammalian inner ear display bundles of actin- filled, microvilli-derived projections of the apical membrane, known as stereocilia, that act as mechanosensitive devices important for the detection of sound [1]. Stereocilia develop from microvilli through the lateral addition of actin filaments to the microvillar actin bundle and the subsequent elongation of the filaments within the actin bundle [11]. The differential elongation of stereocilia leads to their characteristic staircase-like arrange- ment. Within each bundle, the stereocilia are connected by various links, including tip links connecting the tip of one stereocilium to the side of an adjacent stereocilium. Mutations in the five known USH1 genes lead to alterations in stereocilia length and orientation. Biochemical analysis further shows that the five USH1 proteins can interact with each other in vitro. Harmonin binds, through its PDZ domain, to any of the other four USH1 proteins [10,12,13,14,15] and myosin VIIa can interact with protocadherin 15 and sans [12,16]. Localization studies have PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4753