Myosin VIIA, Important for Human Auditory Function, Is Necessary for Drosophila Auditory Organ Development

BACKGROUND: Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition [1]-[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnston's Organ, JO) organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. PRINCIPAL FINDINGS: Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. CONCLUSIONS: Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research

Mutations in the Catalytic Loop HRD Motif Alter the Activity and Function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele

Role for a Novel Usher Protein Complex in Hair Cell Synaptic Maturation

The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23), protocadherin-15 (PCDH15) and the very large G-protein coupled receptor 1 (VLGR1) have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1−/− mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzerav3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well

Characterization of the Drosophila Ortholog of the Human Usher Syndrome Type 1G Protein Sans

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

Genes, deafness, and balance disorders

The mammalian auditory sense organ is subdivided into three principle compartments, the outer-, middle- and inner ear. The main task of the outer- and middle ear is to channel sound waves towards the cochlea within the inner ear. The inner ear also contains the vestibule, the end organ for the perception of gravity and acceleration. Hair cells within the sensory epithelia of the cochlea and the vestibule contain stereocilia that harbor mechanically gated ion channels. These ion channels open or close upon deflection of the stereocilia leading to changes in cell polarization and the rate of neurotransmitter release from hair cells onto sensory neurons. In this way, mechanical signals evoked by sound waves or head movement are transformed into electrochemical signals. The positional cloning of human disease genes and the analysis of mouse mutants has led to the identification of numerous genes that cause deafness and balance disorders. These findings provide insights into the molecular and cellular requirements for mechanosensory transduction and establish an entry point to understand deafness at the molecular level. We will summarize results that have shed light on the function of extracellular matrix glycoproteins, cell adhesion molecules, and components of the actin cytoskeleton in the inner ear

A quantitative RNA code for mRNA target selection by the germline fate determinant GLD-1

RNA-binding proteins (RBPs) are critical regulators of gene expression. To understand and predict the outcome of RBP-mediated regulation a comprehensive analysis of their interaction with RNA is necessary. The signal transduction and activation of RNA (STAR) family of RBPs includes developmental regulators and tumour suppressors such as Caenorhabditis elegans GLD-1, which is a key regulator of germ cell development. To obtain a comprehensive picture of GLD-1 interactions with the transcriptome, we identified GLD-1-associated mRNAs by RNA immunoprecipitation followed by microarray detection. Based on the computational analysis of these mRNAs we generated a predictive model, where GLD-1 association with mRNA is determined by the strength and number of 7-mer GLD-1-binding motifs (GBMs) within UTRs. We verified this quantitative model both in vitro, by competition GLD-1/GBM-binding experiments to determine relative affinity, and in vivo, by \u27transplantation\u27 experiments, where \u27weak\u27 and \u27strong\u27 GBMs imposed translational repression of increasing strength on a non-target mRNA. This study demonstrates that transcriptome-wide identification of RBP mRNA targets combined with quantitative computational analysis can generate highly predictive models of post-transcriptional regulatory networks