Periodic, moderate water flow reversibly increases hair bundle density and size in Nematostella vectensis.

Animals employ hair bundles on hair cells to detect flow, vibrations and gravity. Hair bundles on sea anemone tentacles detect nearby vibrations in the water column produced by prey movements and then regulate discharge of cnidae to capture prey. This study investigated: (1) the progressive effects of periodic water flow on hair bundle morphology and density of hair bundles and cnidae in sea anemones, (2) the reversibility of the flow response and (3) the ability of the response to be expedited with increased flow duration. Linear density of hair bundles along tentacles and each hair bundle's dimensions was measured in anemones exposed to flow and in the absence of flow. With increasing numbers of days of flow, hair bundles in anemones exposed to flow for 1 h every weekday for 20 days increased in density and grew longer and wider at bases and middles, whereas controls did not. Time courses fit to a linear function exhibited significantly larger positive slopes from animals exposed to flow compared with controls. Hair bundles in anemones exposed to flow for 3 h each day increased in linear density, length, base width and middle width after 10 days of flow and returned to control levels after 10 days following cessation of flow. In addition, there was a trend for an increase in density of cnidae with flow. Therefore, anemone hair bundles are dynamically and reversibly modified by periodic, moderate flow to become more abundant and robust. These findings may have relevance to hair cells in acoustico-lateralis systems of higher animals.

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.

The involvement of arl-5b in the repair of hair cells in sea anemones.

The subcellular processes involved in repair of hair cells are not well understood. Sea anemones repair hair bundle mechanoreceptors on their tentacles after severe trauma caused by 1-h exposure to calcium-depleted seawater. Repair is dependent on the synthesis and secretion of large protein complexes named "repair proteins." A cDNA library on traumatized anemone tissue was probed using polyclonal antibodies raised to a specific chromatographic fraction of the repair protein mixture. An ADP-ribosylation factor-like protein, Arl-5b, was identified. The amino acid sequence of the Arl-5b protein in sea anemones is similar to that among several model vertebrates and humans. A polyclonal antibody raised to a peptide of the anemone Arl-5b labels some but not all hair bundles in healthy control animals. The abundance of labeled hair bundles significantly increases above healthy controls after trauma and continuing through the first hour of recovery. Dilute anti-Arl-5b blocks the spontaneous repair of hair bundle mechanoreceptors, suggesting that Arl-5b acts on the extracellular face of the plasma membrane. Immunoelectron microscopy indicates that Arl-5b is located along the length of stereocilia including sites in the vicinity of tip links. We propose that Arl-5b is involved in installing replacement linkages into damaged hair bundle mechanoreceptors.

Hair bundle motility induced by chemoreceptors in anemones.

Most hair bundles are essentially fixed with respect to frequency specificity. However, hair bundles in sea anemones are dynamically tuned by actin-dependent changes in length. Tuning to low frequencies is accomplished by activation of chemoreceptors to N-acetylated sugars resulting in hair bundle elongation. We report here that following sugar-induced tuning of hair bundles, membrane currents reverse polarity in hair cells during unidirectional mechanical stimulation. Reversal in polarity of currents with sugar stimulation is inhibited if hair bundle elongation is blocked by pretreatment with cytochalasin D. A re-examination of morphological changes to hair bundles reveals a sugar-induced reorientation of stereocilia in addition to elongation with chemosensitization. In controls, hair bundles are noticeably twisted. With sugar stimulation stereocilia become oriented more parallel to the long axis of the hair bundle. This sugar-induced change in orientation is similarly inhibited by cytochalasin D pretreatment. Based on these results, we present a model wherein anemone hair bundle twisting serves as a built-in safety mechanism to preserve linkages likely to be subjected to potentially damaging tension during tuning. The twisted hair bundles can untwist while elongating to relieve excessive tension on extracellular linkages between stereocilia critical to mechanosensitivity.