Proteomic identification of hair cell repair proteins in the model sea anemone Nematostella vectensis.

Sea anemones have an extraordinary capability to repair damaged hair bundles, even after severe trauma. A group of secreted proteins, named repair proteins (RPs), found in mucus covering sea anemones significantly assists the repair of damaged hair bundle mechanoreceptors both in the sea anemone Haliplanella luciae and the blind cavefish Astyanax hubbsi. The polypeptide constituents of RPs must be identified in order to gain insight into the molecular mechanisms by which repair of hair bundles is accomplished. In this study, several polypeptides of RPs were isolated from mucus using blue native PAGE and then sequenced using LC-MS/MS. Thirty-seven known polypeptides were identified, including Hsp70s, as well as many polypeptide subunits of the 20S proteasome. Other identified polypeptides included those involved in cellular stress responses, protein folding, and protein degradation. Specific inhibitors of Hsp70s and the 20S proteasome were employed in experiments to test their involvement in hair bundle repair. The results of those experiments suggested that repair requires biologically active Hsp70s and 20S proteasomes. A model is proposed that considers the function of extracellular Hsp70s and 20S proteasomes in the repair of damaged hair cells.

Cadherin-23 may be dynamic in hair bundles of the model sea anemone Nematostella vectensis.

Cadherin 23 (CDH23), a component of tip links in hair cells of vertebrate animals, is essential to mechanotransduction by hair cells in the inner ear. A homolog of CDH23 occurs in hair bundles of sea anemones. Anemone hair bundles are located on the tentacles where they detect the swimming movements of nearby prey. The anemone CDH23 is predicted to be a large polypeptide featuring a short exoplasmic C-terminal domain that is unique to sea anemones. Experimentally masking this domain with antibodies or mimicking this domain with free peptide rapidly disrupts mechanotransduction and morphology of anemone hair bundles. The loss of normal morphology is accompanied, or followed by a decrease in F-actin in stereocilia of the hair bundles. These effects were observed at very low concentrations of the reagents, 0.1-10 nM, and within minutes of exposure. The results presented herein suggest that: (1) the interaction between CDH23 and molecular partners on stereocilia of hair bundles is dynamic and; (2) the interaction is crucial for normal mechanotransduction and morphology of hair bundles.

Rheotaxis in larval zebrafish is mediated by lateral line mechanosensory hair cells.

The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration.

Evidence for involvement of TRPA1 in the detection of vibrations by hair bundle mechanoreceptors in sea anemones.

A homolog of TRPA1 was identified in the genome of the anemone, Nematostella vectensis (nv-TRPA1a), and predicted to possess six ankyrin repeat domains at the N-terminus and an ion channel domain near the C-terminus. Transmembrane segments of the ion channel domain are well conserved among several known TRPA1 polypeptides. Inhibitors of TRPA1 including ruthenium red decrease vibration-dependent discharge of nematocysts in N. vectensis and Haliplanella luciae. Activators of TRPA1 including URB-597 and polygodial increase nematocyst discharge in the absence of vibrations. Co-immunoprecipitation yields a band on SDS-PAGE gels at the predicted mass of the nv-TRPA1a polypeptide among other bands. Co-immunoprecipitation performed in the presence of antigenic peptide decreases the yield of this and several other polypeptides. In untreated controls, anti-nv-TRPA1a primarily labels the base of the hair bundle with some labeling also distributed along the length of stereocilia. Tissue immunolabeled in the presence of the antigenic peptide exhibits reduced labeling. Activating chemoreceptors for N-acetylated sugars induce immunolabel to distribute distally in stereocilia. In anemones, activating chemoreceptors for N-acetylated sugars induce hair bundles to elongate among several other structural and functional changes. Taken together, these results are consistent with the possibility that nv-TRPA1a participates in signal transduction of anemone hair bundles.

Cadherin 23-like polypeptide in hair bundle mechanoreceptors of sea anemones.

We investigated hair bundle mechanoreceptors in sea anemones for a homolog of cadherin 23. A candidate sequence was identified from the database for Nematostella vectensis that has a shared lineage with vertebrate cadherin 23s. This cadherin 23-like protein comprises 6,074 residues. It is an integral protein that features three transmembrane alpha-helices and a large extracellular loop with 44 contiguous, cadherin (CAD) domains. In the second half of the polypeptide, the CAD domains occur in a quadruple repeat pattern. Members of the same repeat group (i.e., CAD 18, 22, 26, and so on) share nearly identical amino acid sequences. An affinity-purified antibody was generated to a peptide from the C-terminus of the cadherin 23-like polypeptide. The peptide is expected to lie on the exoplasmic side of the plasma membrane. In LM, the immunolabel produced punctate fluorescence in hair bundles. In TEM, immunogold particles were observed medially and distally on stereocilia of hair bundles. Dilute solutions of the antibody disrupted vibration sensitivity in anemones. We conclude that the cadherin 23-like polypeptide likely contributes to the mechanotransduction apparatus of hair bundle mechanoreceptors of anemones.

Rapid recovery of sensory function in blind cave fish treated with anemone repair proteins.

Blind cave fish employ superficial neuromasts to detect currents [Baker, C.F. and J.C. Montgomery, J. Comp. Physiol. A 184 (1999) 519-527]. Briefly exposing fish to calcium-free water significantly reduces the ability of the fish to perform rheotaxis (i.e., to orient properly in currents). Spontaneous recovery to control levels of rheotaxis requires 9 days. However, if the fish are treated with fraction beta immediately after exposure to calcium-free water, recovery to control levels of rheotaxis occurs within 1.3 h, the first time point tested. Fraction beta is a chromatographic fraction of 'repair proteins' isolated from sea anemones. The benefits of fraction beta on restoring rheotaxis exhibit dose dependency with the minimum effective dose estimated at 1 ng/ml. Exogenously supplied ATP augments the efficacy of fraction beta. Such augmentation is abolished by PPADS, an inhibitor of purinoceptors. Immunocytochemistry confirms the presence of purinoceptors in superficial neuromasts. The present results suggest that 'repair proteins' obtained from anemones significantly augment intrinsic repair mechanisms in fish. Furthermore, the data obtained in the fish system strongly parallel our previously published findings on sea anemones, raising the possibility that mechanisms of hair bundle repair may be evolutionarily conserved.