CDH23 immunocytochemistry and effects of CDH23 antibodies on vibration sensitivity in <i>Nematostella vectensis</i>.

<p>(A) Affinity purified CDH23 antibodies label stereocilia of a hair bundle (HB) with punctate fluorescence. The tip (t) and base (b) of the hair bundle are indicated. The merged image of a hair bundle is shown in transmitted light by oblique contrast (assigned green) and epifluorescence microscopy for CDH23 immunocytochemistry (assigned red). Scale bar  = 2 µm. (B) Vibration sensitivity was evaluated by means of a bioassay based on counting nematocysts discharged into test probes touched to tentacles in the presence of nearby vibrations at a key frequency. Vibration sensitivity was tested at intervals after adding 0.1 nM CDH23 antibodies (final concentration) to the seawater containing intact anemones. The mean number of nematocysts counted per field of view at 400× (±SEM, n = 10–15) is plotted (open circles). Data also are plotted for healthy, vibrating controls (closed squares) and non-vibrating controls (closed diamond) for which the animals were not exposed to exogenously supplied antibodies. (C) Vibration sensitivity was tested at intervals after adding 0.1 nM TRPA1 antibodies (final concentration) as an antibody-loading control. The mean number of nematocysts counted per field of view at 400× (±SEM, n = 5–6) is plotted (open circles). Data also are plotted for healthy, vibrating controls (solid squares). ^aSignificant difference in the mean number of nematocysts discharged between vibrating controls and 0.1 nM CDH23 antibody-treated specimens and ^bsignificant difference between the mean number of nematocysts discharged between the vibrating controls and non-vibrating control (<i>p</i><0.05).</p

Effects of CDH23 peptides on the mechanoelectric responses induced by deflecting hair bundles.

<p>Hair bundles were deflected by pressure jets delivered by a nearby puffer pipette. Recording pipettes were attached to the plasma membrane adjacent to the base of the hair bundles. The TTL signal depicting the opening and closing the solenoid valve controlling pressure delivered to a puffer pipet is shown above representative traces depicting membrane current transients induced by hair bundle deflection. (A) Recordings are shown for a perfusion control before (left) and after perfusion of potassium-enriched seawater (right). (B) Recordings are shown before (left) and 3 min after the perfusion of potassium-enriched seawater containing 10 nM CDH23 peptide (right). (C) Recordings are shown before (left) and 5 min after the perfusion of potassium-enriched seawater containing 10 nM CDH23 peptide (right). (D) The mean peak membrane current induced by deflecting hair bundles was calculated and plotted for before and after perfusion (±SEM, n = 10). Perfusion controls (empty bar) had perfusion of potassium-enriched seawater only. The CDH23 peptide-treated specimens (hatched bar) were exposed to potassium-enriched seawater containing 10 nM CDH23 peptide (final concentration). The peak membrane current induced by deflecting hair bundles was measured at two sampling times after the perfusion, 3 min and 5 min, in the CDH23 peptide-treated specimens. Asterisks indicate significant differences in mean peak membrane current between before and after perfusion (<i>p</i><0.05).</p

CDH23 peptide cytochemistry and effects of CDH23 peptides on vibration sensitivity in <i>Nematostella vectensis</i>.

<p>(A) Fluorescently tagged CDH23 peptides label stereocilia near the base of a hair bundle with punctate fluorescence. The upper micrograph depicts a merged image of a hair bundle shown in transmitted light using oblique contrast (assigned green) and epifluorescence microscopy for CDH23 peptide-FITC cytochemistry (assigned red). The tip (t) and base (b) of the hair bundle (HB) are indicated. The lower micrograph depicts immunocytochemistry for TRPN1. It is intended to show the morphology of stereocilia in the hair bundle. White arrowheads indicate the height of the tips of several small diameter stereocilia. Scale bar  = 2 µm. (B) Vibration sensitivity was tested at intervals after adding 0.1 nM CDH23 peptide (open triangles, final concentration) or 10 nM CDH23 peptide (open circles, final concentration) to the seawater containing intact anemones. The mean number of nematocysts counted per field of view (±SEM, n = 6) is plotted for the experimental animals as well as for untreated, vibrating controls (closed squares) and non-vibrating controls (closed diamond). ^aSignificant difference between mean nematocyst discharge for the 10 nM CDH23 peptide treated animals and untreated, vibrating controls. ^bSignificant difference between mean nematocyst discharge for the 0.1 nM CDH23 peptide treated animals and untreated, vibrating controls. ^cSignificant difference between non-vibrating controls and the other three treatments at time 0. (C) Vibration sensitivity was tested at intervals after adding 10 nM harmonin peptide (open circles, final concentration) as a peptide-loading control. The mean number of nematocysts discharged is plotted (±SEM, n = 6–8) for untreated vibrating controls (closed squares) and for animals exposed to the harmonin peptide (open circles). Asterisks indicate significant differences in mean nematocyst discharge (<i>p</i><0.05).</p

Effects of CDH23 antibodies on morphology and abundance of hair bundles.

<p>Morphology was assayed by measuring diameter of hair bundles at their bases (b) and tips (t) and then determining the tip/base ratio for each hair bundle. Hair bundle abundance was measured by the number of hair bundles at the tip of tentacles in a field of view (∼260 µm of tentacle length). (A) The mean tip/base ratio (±SEM, n = 8) is plotted for hair bundles imaged in untreated, healthy controls (closed squares) and in animals exposed to 0.1 nM CDH23 antibodies (open circles). Insets show images of an extremely splayed hair bundle (top) and a normal hair bundle (bottom). Scale bar  = 2 µm. (B) The mean abundance of hair bundles (±SEM, n = 3) on the tentacle epithelium is plotted for untreated, healthy controls (closed squares) and for animals exposed to 0.1 nM CDH23 antibody (open circles). Asterisks indicate significant differences based on time-adjusted pairwise comparisons between controls and experimentals (<i>p</i><0.05).</p

F-actin cytochemistry and effects of the CDH23 peptide on rhodamine-phalloidin fluorescence intensity of hair bundles.

<p>(A) Rhodamine-phalloidin labeled stereocilia (S) and rootlets (R) of hair bundles in <i>N. vectensis</i>. Scale bars  = 2 µm. (B) Untreated controls (closed squares) and specimens incubated in 10 nM CDH23 peptide (open circles) were fixed and then incubated in a rhodamine conjugate of phalloidin. Mean fluorescence intensity was obtained from analysis of digital images of hair bundles. Background fluorescence was subtracted to yield net fluorescence intensity. Mean net fluorescence intensity is plotted (±SEM, n = 3) in grey values. Asterisks indicate significant differences between mean net fluorescence intensity of untreated controls and specimens exposed to 10 nM CDH23 peptide (<i>p</i><0.05).</p

Effects of the CDH23 peptide on morphology and abundance of hair bundles.

<p>Hair bundles were evaluated in untreated, healthy controls (closed squares), in animals exposed to 0.1 nM CDH23 peptide (open triangles) or to 10 nM CDH23peptide (open circles). (A) Morphology was assayed by the tip/base ratio. The mean tip/base ratio (±SEM, n = 5–8) is plotted. Insets show representative images of an extremely splayed hair bundle (top) and normal hair bundle (bottom). The diameters of tip (t) and base (b) are indicated. Scale bar  = 2 µm. (B) The mean abundance of hair bundles; ±SEM, n = 3) along the tentacle epithelium is plotted. ^aSignificant difference between data for the 10 nM CDH23 peptide treated specimens and untreated controls and ^bSignificant difference between data for the 0.1 nM CDH23 peptide treated specimens and untreated controls.</p