No abstract

Extracellular protein-protein interactions are essential for both intercellular communication and cohesion within multicellular organisms. Approximately a fifth of human genes encode membrane-tethered or secreted proteins, but they are largely absent from recent large-scale protein interaction datasets, making current interaction networks biased and incomplete. This discrepancy is due to the unsuitability of popular high-throughput methods to detect extracellular interactions because of the biochemical intractability of membrane proteins and their interactions. For example, cell surface proteins contain insoluble hydrophobic transmembrane regions, and their extracellular interactions are often highly transient, having half-lives of less than a second. To detect transient extracellular interactions on a large scale, we developed AVEXIS (avidity-based extracellular interaction screen), a high-throughput assay that overcomes these technical issues and can detect very transient interactions (halflives Յ 0.1 sec) with a low false-positive rate. We used it to systematically screen for receptor-ligand pairs within the zebrafish immunoglobulin superfamily and identified novel ligands for both well-known and orphan receptors. Genes encoding receptor-ligand pairs were often clustered phylogenetically and expressed in the same or adjacent tissues, immediately implying their involvement in similar biological processes. Using AVEXIS, we have determined the first systematic low-affinity extracellular protein interaction network, supported by independent biological data. This technique will now allow large-scale extracellular protein interaction mapping in a broad range of experimental contexts.

The stone-like otoliths from the ears of teleost fishes are involved in balance and hearing and consist of calcium carbonate crystallites embedded in a protein framework. We report that a previously unknown gene, starmaker, is required in zebrafish for otolith morphogenesis. Reduction of starmaker activity by injection of modified antisense oligonucleotides causes a change in the crystal lattice structure and thus a change in otolith morphology. The expression pattern of starmaker, along with the presence of the protein on the growing otolith, suggest that the expression levels of starmaker control the shape of the otoliths.

Hair cells have highly organized bundles of apical projections, or stereocilia, that are deflected by sound and movement. Displacement of stereocilia stretches linkages at the tips of stereocilia that are thought to gate mechanosensory channels. To identify the molecular machinery that mediates mechanotransduction in hair cells, zebrafish mutants were identified with defects in balance and hearing. In sputnik mutants, stereociliary bundles are splayed to various degrees, with individuals displaying reduced or absent mechanotransduction. Here we show that the defects in sputnik mutants are caused by mutations in cadherin 23 (cdh23). Mutations in Cdh23 also cause deafness and vestibular defects in mice and humans, and the protein is present in hair bundles. We show that zebrafish Cdh23 protein is concentrated near the tips of hair bundles, and that tip links are absent in homozygous sputnik(tc317e) larvae. Moreover, tip links are absent in larvae carrying weak alleles of cdh23 that affect mechanotransduction but not hair bundle integrity. We conclude that Cdh23 is an essential tip link component required for hair-cell mechanotransduction.