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

Hearing Research xxx (2015) 1e12

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Research paper

Proteomic identification of hair cell repair proteins in the model sea anemone Nematostella vectensis Pei-Ciao Tang, Glen M. Watson* Department of Biology, University of Louisiana Lafayette, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 March 2015 Received in revised form 15 June 2015 Accepted 9 July 2015 Available online xxx

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. © 2015 Elsevier B.V. All rights reserved.

Keywords: 20S proteasome Hair bundle Hsp70s Repair proteins

1. Introduction Noise exposure is one of the most common causes of damage to hair cells of the inner ear. Exposure to intense noise can be followed by a temporary threshold shift following moderate damage or by a permanent threshold shift following more severe damage to hair cells. Loud noise produces large oscillations between the tectorial and basilar membranes, where the outer hair cells are located. If the deflections of the stereocilia are too extreme, the tip links between stereocilia are severed (Pickles et al., 1987a; Kurian et al., 2003). Acoustic trauma can ultimately lead to hair cell death because of mechanical disruption of hair bundles, oxidative stress, and excitotoxicity (Hakuba et al., 2000; Cheng et al., 2005; Henderson et al., 2006; LePrell et al., 2007; Tan et al., 2013). In birds, noise-damaged hair cells can be repaired by processes culminating in the replacement of tip links (Husbands et al., 1999; Kurian et al., 2003). However, in mammals, damaged hair cells exhibit only a limited capacity to be repaired (Sobkowicz et al.,

Abbreviations: RP, repair proteins; stellaSW, natural seawater diluted to a salinity of 16 parts per thousand; Arl5b, ADP-ribosylation factor-like 5b * Corresponding author. P.O. Box 42451, Lafayette, LA, 70504, USA. E-mail address: [email protected] (G.M. Watson).

1992; Jia et al., 2009). In laboratory cultures of cochlea explants, noise trauma is mimicked by briefly exposing hair cells to calciumdepleted buffers. Following such trauma, mammalian hair cells can recover, evidently after replacing their tip links (Jia et al., 2009; Indzhykulian et al., 2013). Initially, the replacement tip links consist solely of protocadherin 15 followed by a heterotetramer of cadherin 23 and protocadherin 15 within 48 h (Indzhykulian et al., 2013). Perhaps the most dramatic example of hair cell repair involves hair bundle mechanoreceptors located on tentacles of sea anemones (Watson et al., 1998). Anemone hair bundles consist of actinbased stereocilia interconnected by tip links, among other linkages (Watson and Hessinger, 1989; Mire-Thibodeaux and Watson, 1994; Watson et al., 1997). Anemone hair bundles detect vibrations produced by rhythmic swimming movements of nearby potential prey (Watson and Hessinger, 1989; Watson and Mire, 2004) and participate in regulating discharge of nematocysts during prey capture (Mire-Thibodeaux and Watson, 1994). During prey detection and capture, hair bundles are commonly damaged, apparently due to large deflections of the stereocilia entrained to large movements of prey appendages spanning several hundreds of microns. In general terms, the structural consequences of damage to anemone hair bundles due to prey capture may be similar to those

http://dx.doi.org/10.1016/j.heares.2015.07.005 0378-5955/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Tang, P.-C., Watson, G.M., Proteomic identification of hair cell repair proteins in the model sea anemone Nematostella vectensis, Hearing Research (2015), http://dx.doi.org/10.1016/j.heares.2015.07.005

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P.-C. Tang, G.M. Watson / Hearing Research xxx (2015) 1e12

observed in vertebrate hair cells exposed to noise trauma. Unlike mammals, however, sea anemones demonstrate an extraordinary capacity to repair hair bundles, even after severe trauma (Watson et al., 1998). The repair of hair bundles in anemones includes reorganizing the actin cytoskeleton in stereocilia by first depolymerizing and then repolymerizing actin (Watson and Mire, 2001). In addition, secreted proteins are involved in repair, including Arl-5b, a Gprotein that participates in ADP-ribosylation. Arl-5b occurs in a complex mixture of secreted proteins that were named “repair proteins” (RPs) (Watson et al., 1998). A secreted Hsp60 chaperone may be present in RPs (Nag and Watson, 2007). Antibodies to Hsp60 (but not non-immune serum) diluted in seawater containing traumatized anemones delayed the spontaneous recovery of vibration sensitivity (Nag and Watson, 2007). Exogenously supplied RPs sped the time course of recovery by more than 30-fold in anemone hair cells that were severely traumatized by 1 h exposure to calcium-depleted seawater (Watson et al., 1998). Exogenously supplied RPs also allowed hair cells of the acousticolateralis system to recover from trauma in blind cavefish (Repass and Watson, 2001; Berg and Watson, 2002). The biological activity of the RPs on the fish appears to result from a direct effect because biotinylated RPs bind hair cells of superficial neuromasts. If traumatized specimens are treated by a combination of RPs and beta-NAD or ATP, the recovery rate is further enhanced both in fish and in anemones (Watson et al., 1999; Berg and Watson, 2002). The finding that anemone RPs assist neuromast hair cells to recover from trauma raises the possibility that anemone RPs may have therapeutic potential. To further explore such potential, RPs were exogenously added to media containing cochlea explants from mice that had been traumatized by immersion in calciumdepleted buffer. Anemone RPs assisted outer hair cells to recover from trauma, as evidenced by a restoration of uptake of FM1-43 into the hair cells, as well as by a recovery of normal morphology of hair bundles. The results of these experiments are available in preliminary form (Tang and Watson, 2015). 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, we identified several RPs using a proteomic approach. Candidate RPs were targeted after comparing the blue-native gel profiles of mucus obtained from healthy controls with mucus obtained from experimentally traumatized specimens of the model sea anemone Nematostella vectensis. Polypeptides in four specific bands excised from the gels were identified by mass spectrometry-based sequencing. 2. Materials and methods 2.1. Anemone culture and maintenance Specimens of N. vectensis were maintained in natural seawater diluted in distilled water (“stellaSW”) to a salinity of ~16 ppt. The animals were fed freshly hatched brine shrimp twice weekly and cleaned at least two hours after feeding. During cleaning, specimens were transferred individually to a new culture dish containing clean stellaSW. 2.2. Mucus collection Mucus rings were collected from the body columns, near the oral disc, of healthy anemones two or three days after feeding. The mucus that was collected from healthy anemones will be referred to as “control mucus.” On the other hand, the mucus that was collected from traumatized anemones will be referred to as “trauma mucus”. In this study, specimens were immersed in

calcium-depleted stellaSW (50 mM KCl, 12 mM MgCl2, 178 mM NaCl, 13 mM MgSO4, 1.15 mM NaHCO3, and 8 mM EGTA; pH ¼ ~7.6) in order to traumatize hair bundles. The mucus collection method was modified from Watson and Venable-Thibodeaux (2000). Briefly, anemones were severely traumatized by 1-h incubation in calcium-depleted stellaSW. Once the anemones were returned to normal (i.e., calcium-containing) stellaSW, the recovery process was considered to have begun. Trauma mucus rings were collected after 3.5 h of recovery and stored at 70 C until the day of use. 2.3. SYTOX assay SYTOX Green Nucleic Acid Stain (Life Technologies, CA, USA) was employed for two purposes: (i) to detect possible contamination by dead anemone cells in the collected mucus and; (ii) to detect possible cell lysis in the tentacle epithelium following severe trauma caused by 1-h immersion in calcium-depleted stellaSW. One milliliter of freshly collected trauma mucus (n ¼ 3) and control mucus (n ¼ 3) was incubated with SYTOX Green stain at a final concentration of 100 nM and then examined with epifluorescence microscopy (model RP011-T, LOMO America, IL, USA) with a 100 oil immersion objective (Plan Fluorite, na ¼ 1.30) Images were captured with an STL-11000M SBIG cooled CCD camera (SBIG, CA, USA) operated using Maxim-DL software (Diffraction Limited, ON, Canada). To further verify that lysed anemone cells did not contribute proteins to the RPs in the mucus, traumatized and healthy anemones (n ¼ 4 for each treatment) were incubated in potassium-enriched stellaSW (necessary to anesthetize the specimens; 50 mM KCl, 12 mM MgCl2, 6 mM CaCl2, 166.5 mM NaCl, 13 mM MgSO4, and 1.15 mM NaHCO3) in the presence of 100 nM SYTOX Green stain for 30 min followed by fixation for 1 h in 4% paraformaldehyde prepared in a phosphate buffer modified from Sorensen's buffer (199 mM Na2HPO4, 48 mM NaH2PO4, and 75 mM NaCl). After three washes (5 min each), tentacles were excised with a scalpel and prepared as wet mounts in ProLong Gold Antifade Reagent (Life Technologies). Specimens were imaged by epifluorescence microscopy with a 20 objective (Plan Achromat, na ¼ 0.45). To serve as a positive control for the SYTOX assay, healthy anemones (n ¼ 3) were incubated in potassium-enriched stellaSW supplemented with a final concentration of 0.02% Triton X-100 to permeabilize plasma membranes. The positive controls were processed and imaged as described above. 2.4. Repair bioactivity tests using a vibration sensitivity assay Vibration sensitivity bioassays were conducted with intact anemones following the procedure described in Tang and Watson (2014). The vibration sensitivity data were analyzed with a oneway ANOVA followed by Fisher's post-hoc tests to test for significant differences between treatments at the same sampling time (p < 0.05; STATISTICA software, StatSoft, Inc., OK, USA). The repair bioactivity of anemone mucus was tested using the vibration sensitivity bioassay. First, anemones were severely traumatized by incubating them in calcium-depleted stellaSW for one hour. This was the usual means by which animals were traumatized with one exception detailed below. The repair bioactivity of the trauma anemone mucus was tested by incubating traumatized anemones in 10 ml stellaSW in the presence of 10 ml of trauma mucus. Traumatized anemones (i.e., “traumatized controls”) were allowed to recover in stellaSW alone (i.e., without the addition of exogenous trauma mucus) to determine the spontaneous rate of recovery. To test whether the repair bioactivity in the mucus from N. vectensis was associated with RPs, rather than other organic constituents, the trauma mucus was boiled for 10 min to denature proteins. This boiled mucus (10 ml) was added to 10 ml stellaSW in



which traumatized anemones were incubated to serve as a negative control (“boiled-RP controls”). Healthy, untreated anemones were also tested to serve as “healthy controls”. The vibration sensitivity assay was used to test the function of hair bundles at thirty-minute intervals during the recovery period. The vibration sensitivity assay was also employed to test the repair activity of polypeptides eluted from gel-isolated RP bands (see Section 2.5). The polypeptides in RP bands and bands of comparable size from the control mucus proteins lane (i.e., “control bands”) were eluted in stellaSW. It was assumed a priori that the concentrations of the RPs eluted from the RP bands in native gels were less than those of the trauma mucus; therefore, biological activity was evaluated in anemones exposed to a less severe, ‘moderate’ trauma. Specifically, for these experiments only, animals were incubated in calcium-depleted stellaSW for 15 min instead of 1 h. To determine whether the gel itself had an effect on the anemones, protein-free gel slices were incubated in stellaSW overnight, and the supernatant was collected. The healthy and traumatized controls were incubated in stellaSW enriched with the supernatant collected from the protein-free gel. The vibration sensitivity assay was performed (n ¼ 5e6 anemones for each treatment at each sampling time for each of 3 replicate experiments) for the duration of the 3-h incubation. 2.5. Isolation of RPs Rather than submitting fully intact trauma mucus for proteomic analysis, the strategy for identifying polypeptide constituents of RP was based on substantially reducing the number of candidate polypeptides to those most likely to be enriched in RPs or unique to RPs. Although we realized that such a strategy would likely generate an incomplete list of the polypeptides in RPs, we reasoned that such an approach might quickly allow for the identification of key polypeptides in the RP mixture. Samples of control mucus (“control mucus proteins”) and trauma mucus (“trauma mucus proteins”) were homogenized in protein extraction buffer (50 mM Tris [pH 7.2], 250 mM sucrose, and 10% protease inhibitor cocktail [SigmaeAldrich, MO, USA]) on ice with a Dounce Homogenizer (Kimble/Kontes LLC, NJ, USA) for 25 min. Mucus homogenates were centrifuged at 20,000 g for 60 min. The supernatant containing soluble proteins was transferred to a new tube, quantified with the Bradford protein assay (Bradford, 1976), and stored at 70 C. Blue native PAGE was conducted following the protocol detailed in the Native PAGE (Life Technologies) instruction manual. Briefly, approximately 2 mg of each control and trauma mucus protein samples were loaded into the native gel, and electrophoresis was performed at 150 V for ~1.5 h. The bands in the native gel were visualized using a modified silver staining protocol (Mahoney et al., 2011). Bands in the lane for trauma mucus that were absent or very weak in the control mucus lane were considered likely to contain RPs (i.e., “RP bands”). Upon examination of 10 native gels prepared from three separate batches of proteins, four bands were identified with masses estimated to be 696, 407, 281, and 144 kDa, respectively (Fig. 1). The approximate masses of the bands were estimated using a polynomial curve fit as recommended in the manual for the NativeMark Unstained Protein Standard (Life Technologies). These RP bands were used for the remainder of the experiments described in this manuscript. 2.6. Biotinylation of polypeptides in RP bands In order to localize RPs during hair bundle repair, polypeptides in trauma mucus were biotinylated using the EZ-LinkMicro NHSPEG4-Biotinylation kit (Thermo Scientific, MA, USA) according to protocol detailed in the manufacturer's instructions. Biotinylated

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Fig. 1. Illustration of NativeMarker Unstained Protein Standard (Life Technologies, CA, USA) and repair protein (RP) bands (gray). Four bands were enriched/unique to the trauma mucus and designated as RP bands according to the results of 10 native PAGE gels based on three different sets of mucus proteins. Two representative native PAGE gels are shown after siliver-staining. Enriched/unique bands in the anemone trauma mucus are indicated by black arrow heads.

polypeptides in trauma mucus proteins were then separated using blue native PAGE as described in Section 2.5. The positions of the bands in the gels were not dramatically different between nonbiotinylated and biotinylated-trauma mucus protein samples (data not shown). Four bands with approximately the same mass as the RP bands (biotinylated-RP bands) were excised and eluted in potassium-enriched stellaSW. In each gel, eight wells were loaded with 25 ml biotinylated trauma mucus protein, and a total of 32 bands were excised, pooled, and eluted in 2 ml potassium-enriched stellaSW. 2.7. Immunocytochemistry Anemones (n ¼ 3) were traumatized by exposure to calciumdepleted stellaSW for 1 h. Then, traumatized anemones were transferred to 25 ml of potassium-enriched stellaSW with 1 ml of the potassium-enriched stellaSW containing eluted biotinylated RP bands (potassium þ RP band-enriched stellaSW). After 1 h of recovery in the potassium þ RP band-enriched stellaSW, oral discs were dissected from the anemones and fixed for 1 h in 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer. After fixation, specimens were washed three times in phosphate buffer (5 min each wash) and then pre-blocked for 30 min in phosphate buffer augmented with 3% BSA. Affinity-purified, rabbit anti-biotin primary antibody (Bethyl Laboratories, Inc., TX, USA) was used to detect biotinylated-RPs. Specimens were incubated in a 1/200 dilution of the primary antibody at 4 C overnight. The next day, the specimens were washed three times in phosphate buffer followed by a 1-h incubation in a 1/500 dilution of a secondary antibody (goat anti-rabbit IgG conjugated to Alexa Fluor 555) in the dark at room temperature. Afterwards, the specimens were washed three times in phosphate buffer. Tentacles were excised with a scalpel and prepared as wet mounts in ProLong Gold Antifade Reagent. Specimens were imaged using epifluorescence microscopy with a 100 oil immersion objective. Images were captured as described in Section 2.3. Transmitted light microscopy was performed using


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oblique contrast. Overlays of images obtained using oblique contrast and epifluorescence microscopy were generated using Maxim-DL software. Non-traumatized anemones (n ¼ 3) were processed and imaged in an identical fashion to serve as negative controls. 2.8. Protein identification Polypeptide constituents of the four RP bands were sequenced with liquid chromatography-mass spectrometry (LC-MS/MS) at the Proteomics and Metabolomics Facility at Colorado State University (CSU). The acrylamide gel slices were stored in 1% acetic acid and then subjected to partial digestion by trypsin. Samples were prepared according to instructions provided by the Proteomics and Metabolomics Facility at CSU (http://www.pmf. colostate.edu/proteomics_services.html). Two to three gel fragments for each band were provided and obtained from the same biological sample. The SEQUEST and/or Mascot search engine(s) was/were used to search MS/MS spectra against the N. vectensis genome on the NCBI database. The identified proteins were examined using Scaffold 4 Viewer (http://www.proteomesoftware. com/products/free-viewer/). NCBI's BLASTp program was used to elucidate the potential function of the sequenced proteins. 2.9. Inhibition experiments Both a 20S proteasome protein complex and heat shock protein-70 (Hsp70) were identified from the RP bands, and inhibitors of these two proteins/protein complexes were employed to test their involvement in hair bundle repair. Healthy control and traumatized anemones (n ¼ 8e12 for each treatment at each sampling time) were tested for vibration sensitivity in the presence of 1 mM and 10 mM concentrations (final) of epoxomicin and VER155008, respectively. Epoxomicin (APExBIO, TX, USA) is a selective and irreversible inhibitor of the 20S proteasome (Meng et al., 1999). Epoxomicin potently inhibits chymotrypsin activity in the 20S proteasome but does not inhibit cellular proteases including papain, chymotrypsin, cathepsin B, or calpain at concentrations of up to 50 mM (Sin et al., 1999). Futhermore, the molecular basis of specific interactions between epoxomicin and the proteasome have been identified based on crystal structures (Groll et al., 2000). VER155008 (APExBIO) is an inhibitor of Hsp70 family chaperones that binds to the nucleotide binding, ATPase domain to inhibit chaperone activity (Massey et al., 2010). VER155008 is an adenosine analog that competes with ADP and ATP for binding the nucleotide binding domain of Hsp70 chaperones (Williamson et al., 2009). Crystal structures confirm VER155008 binds the nucleotide binding domain of Hsp70 (Schlecht et al., 2013). Because this domain is highly conserved among members of the Hsp70 family, VER155008 is considered to be a pan-inhibitor of Hsp70 family chaperones (Goloudina et al., 2012; Schlecht et al., 2013). After a 1-h immersion in calcium-depleted stellaSW, traumatized anemones were returned to stellaSW in the presence of the appropriate inhibitor. The recovery of vibration sensitivity during incubation in each inhibitor was examined at intervals over a 6-h period. As a control, healthy anemones were incubated in 1 mM epoxomicin or 10 mM VER155008 (n ¼ 8e11). Vibration sensitivity was tested at 1 h intervals over a 4-h period. Because both of these inhibitors must be dissolved in DMSO, an additional control was performed to test for possible effects of DMSO on the recovery of vibration sensitivity. We performed the vibration sensitivity assay on healthy and traumatized anemones (n ¼ 4e6 for each treatment) that were allowed to recover in stellaSW containing the

same final concentration (0.1%, v/v) of DMSO as was used for the inhibitors, as well as at a DMSO concentration 10 fold higher (1%). Polyclonal antibodies to HSC70/HSP70 (ADI-SPA-757-D) and to the 20S proteasome core subunits (BML-PW8155-0025; Enzo Life Sciences, NY, USA) were added to stellaSW to potentially block extracellular Hsp70 and extracellular 20S proteasome activity, respectively, during hair bundle repair. The antibodies to HSC70/ HSP70 are supplied diluted in PBS after they were affinity purified using protein A columns. The antibodies to the 20S proteasome core subunits are supplied in immune serum diluted in PBS. Healthy controls and traumatized controls (n ¼ 5 each) were assessed alongside the experimental animals that were allowed to recover from severe trauma caused by a 1 h immersion in calciumdepleted stellaSW. For the experimental animals, the appropriate antibody stock solution was diluted (1/3000) in stellaSW immediately after trauma. The experimental animals were incubated in the dilute antibody solution during the 4-h recovery period. The vibration sensitivity assay was performed on five individuals for each treatment sampled hourly over the course of a 4-h incubation. Two replicate experiments were performed. 2.10. Antibody characterization Commercial polyclonal antibodies to Hsp70 and the core subunits of the 20S proteasome were used in the study. In order to ensure the antibodies also recognize and bind to target proteins in N. vectensis, western blots were conducted. Traumatized mucus proteins were mixed with Laemmli sample buffer (10% sucrose, 2% SDS, 62.5 mM Tris [pH 6.8], 0.01% bromophenol blue, and 5% bmercaptoethanol) and boiled for 3e5 min before putting on ice. Approximately 2.5 mg of protein were loaded into a pre-cast 4e20% gel (Bio-Rad, CA, USA), and electrophoresis was performed at 100 V for ~1.5 h. Molecular weight standards (Kaleidoscope Prestained Standards from Bio-Rad) were also run on the same gel. Proteins were transferred to nitrocellulose membranes at 12 V for 1 h using the Genie Blotter (Idea Scientific, MN, USA) and blocked for 1 h in TBST with 3% BSA. The two membranes were incubated with a 1:1000 dilution of each primary antibody at RT for 1 h followed by three washes with TBST (5 min each time). A 1/30000 dilution of secondary antibody conjugated to alkaline phosphatase was incubated with the protein-laden membranes at RT for 1 h followed by three TBST washes. The signal was developed using FAST BCIP/NBT alkaline phosphatase substrate tablets (Sigma). Dot blots were also performed following the same steps as for the western blot although with shorter incubation times after having spotted 0.8 mg of trauma mucus protein directly onto the nitrocellulose membrane. 3. Results and discussion 3.1. Trauma mucus assists repair of hair bundles in N. vectensis A vibration sensitivity assay was used to examine the recovery of function to experimentally traumatized hair bundles in N. vectensis (Fig. 2). Hair bundle mechanoreceptors were severely traumatized by immersing specimens in calcium-depleted stellaSW for 1 h. After returning the specimens to normal, calciumcontaining stellaSW, tentacles were touched at specific intervals with test probes in the presence of nearby vibrations at a key frequency to stimulate maximal discharge of nematocysts into the probes. Discharged nematocysts were counted upon examining the test probes using phase contrast microscopy. Healthy controls discharged 36.0 ± 3.7 (mean ± SE) nematocysts/field of view (FOV) at 400 magnification. Traumatized anemones that were allowed to recover in stellaSW for 30 min (i.e., “traumatized



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3.2. Isolation and bioactivity of key RPs

Fig. 2. Effect of trauma mucus on the recovery of hair bundles in Nematostella vectensis. After severe trauma, vibration sensitivity was tested at 30-min intervals by touching tentacles with test probes to elicit discharge of nematocysts in the presence of nearby vibrations. Vibration sensitivity was evaluated after adding 0.1% (v/v) trauma mucus (open triangles) or 0.1% (v/v) boiled, trauma mucus (open diamonds) to the stellaSW containing anemones that had been previously traumatized by a 1-h exposure to calcium-depleted stellaSW. The mean number of nematocysts counted per field of view (±SE) is plotted for the experimental animals as well as for healthy, vibrating controls (closed squares) and traumatized controls (closed circles). aSignificant difference (Fisher's post-hoc test, p < 0.05 after one-way ANOVA) in mean nematocyst discharge between the traumatized controls (i.e., traumatized anemones recovering in stellaSW alone), and healthy controls. bSignificant difference in mean nematocyst discharge between 0.1% trauma mucus-treated animals and healthy controls. cSignificant difference in mean nematocyst discharge between 0.1% boiled trauma mucustreated animals and healthy, vibrating controls.

controls”) discharged only 8.4 ± 2.5 nematocysts/FOV, ~approximately 1/4 as many nematocysts/FOV as healthy controls. Without exogenously supplied RPs, levels of nematocyst discharge (23.0 ± 4.7 nematocysts/FOV) spontaneously recovered to become comparable to healthy controls (34.9 ± 4.6 nematocysts/FOV) beginning at 4 h in stellaSW (p ¼ 0.065) and continuing throughout the remainder of the experiment (Fig. 2). However, if stellaSW was enriched with the addition of 0.1% trauma mucus (v/ v) at time zero (immediately after trauma), traumatized anemones attained levels of nematocyst discharge (36.8 ± 4.0 nematocysts/FOV) comparable to the healthy controls (35.8 ± 4.3 nematocysts/FOV) beginning at 2.5 h (p ¼ 0.874) and continuing to the end of the experiment. Evidently, trauma mucus enhanced the recovery rate. On the other hand, traumatized anemones that were allowed to recover in the presence of boiled trauma mucus, intended to denature proteins in the trauma mucus, discharged fewer nematocysts than healthy controls until the 4th h of recovery (p ¼ 0.071), after which, levels of discharge were comparable to those of healthy controls (Fig. 2). Because the time course of recovery for traumatized specimens treated with boiled trauma mucus was similar to that of traumatized controls allowed to spontaneously recover in stellaSW alone, it appears that trauma mucus from N. vectensis demonstrates repair protein activity that is sensitive to boiling. Trauma mucus collected from another anemone, Haliplanella luciae also possesses repair activity (Watson et al., 1998) that may be more potent than that of N. vectensis because it more dramatically shortened the time course of recovery. Nevertheless, a clear advantage of using trauma mucus from N. vectensis is that the genome of N. vectensis is fully sequenced (Putnam et al., 2007). Therefore, trauma mucus isolated from N. vectensis is amenable to proteomic analyses.

Mucus contains a variety of proteins, sugars, and glycoproteins (Fountain, 1982; Caincattin et al., 1992; Blomberg et al., 1993; Berne et al., 2003; Leroy et al., 2006). In cnidarians, mucus may participate in non-self recognition, defense, and immunity (Edmundsa et al., 1976; Lubbock, 1980; Krupp, 1985; Sauer et al., 1986; Brown and Bythell, 2005). Therefore, mucus is likely to contain a variety of molecules unrelated to repair of hair cells. A comparative approach was employed in order to isolate RPs from mucus. Watson et al. (1998) showed that RPs likely occur in large complexes. Blue native PAGE was employed to compare native protein complexes occurring in control mucus to those found in trauma mucus. Blue native PAGE was developed to maintain the native state of proteins and protein complexes during separation €gger and von Jagow, 1991) and has been used in many (Scha studies, including those aimed at isolating mitochondrial and membrane protein complexes (reviewed in Nijtmans et al., 2002; Peng et al., 2011). Because control mucus lacks repair activity, key RPs were hypothesized to be enriched in trauma mucus, or exclusively present in trauma mucus. A total of four bands were enriched in, or unique to, trauma mucus based on blue native gel electrophoresis of three independently collected batches of trauma and control mucus (Fig. 1). SYTOX Green assays were performed to test the possibility that proteins isolated from mucus samples originated from lysed cells. Positive control anemones permeabilized with Triton X-100 showed labeling with SYTOX Green stain (Supplementary Fig. 1A). However, no staining was observed in trauma or control mucus samples, nor was staining observed in tissues of healthy anemones or in tissues of anemones traumatized in calcium-depleted stellaSW (Supplementary Figs. 1B, C). These results suggest that polypeptides isolated from mucus samples were unlikely to have originated from lysed cells and, instead, are likely to have been secreted by living anemone cells. The repair activity of the polypeptides eluted from RP bands was tested using the vibration sensitivity bioassay (Fig. 3). It was assumed a priori that polypeptides eluted from RP bands would be less abundant than in intact trauma mucus and, accordingly, would exhibit less repair activity than trauma mucus. Therefore, a more moderate trauma regime (only 15 min incubation in calciumdepleted stellaSW) was used in these experiments. It should be noted that a 15-min incubation in calcium-depleted stellaSW decreased mean nematocyst discharge in the presence of vibrations less dramatically than a 1-h incubation in calcium-depleted stellaSW (e.g., compare Figs. 2 and 3.). Moderately traumatized anemones were allowed to recover in stellaSW fortified with polypeptides eluted from RP bands or those eluted from control bands. An additional traumatized control group was allowed to recover in stellaSW with supernatant from protein-free gel pieces alone. Throughout the 3-h experimental period, moderately traumatized anemones that were allowed to recover in stellaSW alone (traumatized controls) discharged significantly fewer nematocysts than healthy controls (Fig. 3). In contrast, moderately traumatized anemones that recovered in the presence of polypeptides eluted from RP bands discharged as many nematocysts (33.16 ± 1.6 nematocysts/FOV) as healthy vibrating controls (32.1 ± 2.2 nematocysts/FOV) beginning at 60 min of recovery (p ¼ 0.240) and continuing through the remainder of the experiment. Finally, moderately traumatized anemones that recovered in the presence of polypeptides eluted from control bands failed to recover vibration sensitivity even after 3 h of recovery. At this time point, these anemones discharged 29.4 ± 1.6 nematocysts/FOV, significantly fewer (p ¼ 0.040) than healthy vibrating controls (35.7 ± 1.9 nematocysts/FOV) sampled at the same time. Taken together, these


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Fig. 3. The effect of eluted repair proteins (RP) on hair bundle recovery. After moderate trauma caused by a 15 min immersion in calcium-depleted stellaSW, vibration sensitivity was tested at specific intervals in anemones incubated in eluted RPs (open triangles) or proteins eluted from control bands (open diamonds) in stellaSW. The mean number of nematocysts counted per field of view (FOV) at 400 is plotted, and error bars represent ±SE. Data also are plotted for healthy controls (closed squares) and traumatized controls (closed circles). aSignificant difference (Fisher's post-hoc test, p < 0.05 after one-way ANOVA) in mean nematocyst discharge between the traumatized controls, (i.e., traumatized anemones recovering in stellaSW alone) and healthy controls. bSignificant difference in mean nematocyst discharge between animals recovering in the presence of proteins eluted from RP bands and healthy controls. c Significant difference in mean nematocyst discharge between animals recovering in the presence of proteins eluted from control bands and healthy controls.

results indicate that polypeptides eluted from RP bands possess repair activity. 3.3. Localization of biotinylated-RP polypeptides isolated from specific bands To test whether proteins eluted from the RP bands directly interact with traumatized hair bundles, we biotinylated polypeptides in trauma mucus and then separated native proteins or protein complexes using blue native PAGE. Animals were severely traumatized and then transferred to potassium-enriched stellaSW fortified with the polypeptides eluted from the biotinylated-RP bands, where they remained for 1 h. Immunocytochemistry localized biotinylated polypeptides to hair bundles of traumatized anemone tentacles (Fig. 4A), including small- and large-diameter stereocilia (Fig. 4B). No significant immuno-labeling of biotinylated-RPs was observed on hair bundles of healthy controls (Fig. 4C). 3.4. RP identification Fifty-one polypeptides were identified from a proteomic analysis of RP bands on the basis of having at least two unique peptides detected. An additional 41 polypeptides were identified on the basis of having only one unique peptide detected, bringing the total number of identified polypeptides to 92. Those polypeptides with only one identified peptide are listed in Supplementary Table 1. Herein, we discuss only those polypeptides for which at least two unique peptides were sequenced. Of these 51 polypeptides, four were redundant, giving a total of 47 polypeptides. Of these 47 polypeptides, seven possessed no identifiable domains, and so conjectures could not be made about their function. Three

polypeptides appeared to be of prokaryotic origin after BLASTp analysis of the NCBI database. Thus, 37 polypeptides were of anemone origin and of known function (Table 1). Putative functions of these proteins in hair bundle repair are described in Table 1. Two heat shock proteins, Hsp70 and stress-70, were isolated from the 144-kDa band. Two additional polypeptides, cyclophilin and enolase, may also be involved in protein folding (Ou et al., 2001; reviewed in Pancholi, 2001). Ten out of the thirty-seven polypeptides were subunits of the 20S proteasome. All of the subunits of the 20S proteasome were isolated from the 696-kDa band, similar to the estimated mass of the 20S proteasome (~700 kDa; Coux et al., 1996). In addition to the 20S proteasome, four aminopeptidases were identified: a puromycin-sensitive aminopeptidase known to digest certain peptides released by the proteasome (Bhutan et al., 2007); dipeptidylpetidase II, a major peptidase of the acrosome of guinea pig sperm (Talbot and Dicarlantonio, 1985; Dicarlantonio and Talbot, 1988); prolidase, an Xaa-proline dipeptidase known to digest collagen (Erbaǧ;ci et al., 2002); and, finally, a putative aminopeptidase. An additional two polypeptides may have “moonlighting” functions that include proteolysis: dihydrolipoamide dehydrogenase and phosphogluconate dehydrogenase (Huberts and van der Klei, 2010; Jeffery, 2011; Karkowska-Kuleta and Kozik, 2014). Beta-N-acetylhexosaminidase was present in a RP band, and this enzyme is known to occur in the acrosomes of sperm. Prior to fertilization, beta-N-acetylhexosaminidase trims glycans from glycoproteins as a critical step leading to fertilization (Farooqui and Srivastave, 1980). Tryparedoxin peroxidase was also identified as an RP (Table 1), and it is considered to be similar in function to peroxiredoxins (Montemartin et al., 1998). On the basis of a single sequenced peptide, two isoforms of peroxiredoxin were tentatively identified in the RP samples along with an additional two chaperones and an additional subunit of the 20S proteasome (Supplementary Table 1). Several proteins identified in the RP bands are known to function in glycolysis and gluconeogenesis, including phosphoenolpyruvate carboxykinase, 6-phosphogluconate dehydrogenase, and enolase (Table 1). However, some of these proteins have been described as “moonlighting,” or multifunctional, proteins (Jeffery, 1999). In the past few decades, many highly conserved glycolytic proteins have been discovered to possess one or two additional functions beyond their canonical cellular roles (reviewed in Copley, 2003; Jeffery, 2011). This moonlighting phenomenon may explain why proteins that are not inherently involved in cell repair and protein turnover were uncovered in the MS-based analysis. These metabolism-targeted proteins might be serving alternative roles once secreted from the cells. Their function in the traumatized anemone mucus, thus, demands further attention. Of the 37 polypeptides identified in RP samples, 29 (~78%) have been reported in exosomes (Table 1). Exosomes are small vesicles that transport molecules or release molecules after lysis (Johnstone ry, 2011). Enolase, et al., 1987; Jansen et al., 2009; reviewed in The actin, pyruvate kinase, cyclophilin, adolase, and Hsp70 are commonly found in exosomes (Mathivanan and Simpson, 2009; Mathivanan et al., 2010). Exosomes arise from invaginations of membrane into late stage endosomes to form multivesicular bodies. They are released into the extracellular fluid upon exocytotic fusion of multivesicular bodies with the plasma membrane (Johnstone, 1992). Thus, some of the polypeptides identified in the RP samples, particularly those that normally function in glycolysis, might have been detected because they are common constituents of exosomes, rather than because they directly function in repair of hair cells. In sea anemones, multivesicular bodies were observed in hair cells and sensory neurons (Watson and Hessinger, 1987; Watson et al., 1997). The SYTOX assay discussed in Section 3.2



7

indicated that RPs are likely to be secreted, extracellular proteins, instead of diffusing from the cytoplasm of lysed cells. We therefore propose that exosomes serve as a delivery mechanism of RPs to the secreted mucus and thus to their sites of activity in the extracellular fluid near hair bundles (i.e., damaged hair cells). 3.5. The effects of inhibiting 20S proteasomes or Hsp70s on hair bundle recovery Hsp70s and polypeptide subunits of the 20S proteasome were among the polypeptides identified from the sequenced RP bands. To test whether these candidate polypeptides are involved in repair of hair bundles, pharmacological inhibitors and polyclonal antibodies were employed in time course experiments. The spontaneous recovery of vibration sensitivity after severe trauma required 3.5e4 h in stellaSW alone (Figs. 5 and 6). To test whether Hsp70s are involved in repair of hair bundles, traumatized anemones were allowed to recover from severe trauma in stellaSW supplemented with 10 mM VER155008, an inhibitor of Hsp70s (Fig. 5A). The mean number of nematocysts discharged by traumatized anemones that were allowed to recover in the presence of VER155008 (15.0 ± 1.7 nematocysts/FOV) was significantly lower than healthy controls (34.3 ± 1.2 nematocysts/FOV, p < 0.001) even after 6 h. VER155008 inhibits chaperone activity of Hsp70s by binding to the ATPase pocket of Hsp70s (Massey et al., 2010; Goloudina et al., 2012). Hsp70 and stress-70 were identified in RP and each of these polypeptides possesses an N-terminal ATPase pocket. Thus, it is likely that VER155008 inhibited the activity of Hsp70s in RPs to significantly delay or arrest the recovery of vibration sensitivity. A commercial polyclonal antibody to Hsp70 was diluted in stellaSW to possibly interfere with extracellular Hsp70 activity during hair bundle repair. The specificity of the Hsp70 antibody to N. vectensis proteins in trauma mucus was verified by western blot insofar as an immunopositive band was observed at approximately 70 kDa (Fig. 6A). The recovery of vibration sensitivity from severe trauma was delayed or inhibited in stellaSW containing a 1/3000 dilution of this antibody. At 4 h recovery, experimental animals discharged (17.5 ± 2.1 nematocysts/FOV) significantly fewer nematocysts than healthy controls (33.9 ± 1.7 nematocysts/FOV, p < 0.001) and significantly fewer nematocysts than traumatized controls (30.1 ± 2.3 nematocysts/FOV, p < 0.001) allowed to recover in stellaSW alone (Fig. 6C). Taken together, these data suggest that biologically active Hsp70s are required in the extracellular fluid surrounding traumatized hair bundles in order for repair to occur. Lim et al. (1993) showed that Hsp70 expression in the cochlea increases several hours after exposure to acoustic trauma. Furthermore, murine cochleae are protected from a permanent threshold shift following acoustic overstimulation if they are exposed to heat-stress prior to acoustic overstimulation (Yoshida et al., 1999). Secreted Hsp70 by supporting cells protects hair cells from aminoglycoside toxicity in murine utricles (May et al., 2013). Hence, extracellular Hsp70s may play an important role in survival of hair cells following trauma. Previously, extracellular Hsp60 was implicated in repair of anemone hair bundles (Nag and Watson, 2007).

Fig. 4. Immunocytochemistry of biotinylated-RP bands in Nematostella vectensis. (A). Biotinylated polypeptides eluted from RP bands localized to stereocilia of hair bundles

(arrow heads) recovering from severe trauma. (B) A magnified image demonstrating localization of biotinylated RPs to a recovering hair bundle (HB). Scale bar ¼ 2 mm. For panels (A) and (B), the merged image of a hair bundle is shown in transmitted light by oblique contrast (green) and epifluorescence microscopy for biotinylated-RP immunocytochemistry (red). (C) No obvious labeling by biotinylated RPs was observed in healthy control anemone tissues. For panels (A) and (C), scale bar ¼ 5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


8


Table 1 Proteins identified in the RP bands. Band numbers refer to molecular weights (kDa). Predicted repair protein

NCBI accession#

Band 144

heat shock protein 70a Enolasea stress-70 proteina fructose-1,6-bisphosphate aldolasea Cyclophilina 20S proteasome, alpha type 6b Xaa-Pro dipeptidasea 20S proteasome, alpha type 7b 20S proteasome, alpha type 3b 20S proteasome, beta type 5b 20S proteasome, beta type 6b 20S proteasome, alpha type 1b 20S proteasome, beta type 4b 20S proteasome, alpha type 5b dihydrolipoamide dehydrogenasea dipeptidylpeptidase II puromycin-sensitive aminopeptidasea 20S proteasome, alpha type 2b 20S proteasome, beta type 2b 6-phosphogluconate dehydrogenasea putative aminopeptidase alpha-aminoadipic semialdehyde dehydrogenasea Fumarylacetoacetasea dihydropyrimidine dehydrogenase tryparedoxin peroxidasea beta-N-acetylhexosaminidasea aspartate aminotransferasea

gij156351241 gij156383570 gij156402816 gij156374279 gij156355365 gij156405260 gij156407139 gij156401589 gij156371095 gij156363343 gij156387836 gij156371281 gij156382256 gij156379569 gij156408155 gij156403949 gij156394423 gij156387508 gij156359803 gij156367416 gij156372710 gij156356011

x x x

gij156391233 gij156369958 gij156404129 gij156408528 gij156364446

x

actin, betaa fumarate hydratase phosphoenolpyruvate carboxykinasea aldehyde dehydrogenase 1a

gij156375400 gij156354249 gij156385552 gij156369971

x

malate dehydrogenasea

gij156350422

x

triosephosphate isomerasea succinyl-CoA synthetasea

gij156385194 gij156314281

x

acid alpha-glucosidasea histonea pyruvate kinasea

gij156386347 gij156322163 gij156408764

a b

Band 281

Band 407

Band 696

x x x x x x x x x x x x x

x

x x x x x x

x x x x x x

x

x x

x

x x x

MW (kDa)

# of unique peptides

Total % of protein coverage

Possible function in hair bundle repair Chaperone Heat shock protein Stress response, Chaperone Scaffold protein, Adhesion Protein folding Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Amino acid degradation, reduces NADþ Amino acid degradation Pyrimidine degradation? H2O2 degradation Trims glycan Amino acid synthesis/ degradation Cytoskeleton? TCA cycle? gluconeogenesis? aldehyde oxidation, reduces NADþ malate oxidation, reduces NADþ glycolysis? nucleoside triphosphate synthesis? glycogen degradation? DNA binding? glycolysis?

71 47 70 39 17 27 56 28 28 31 21 26 29 27 55 49 97 26 22 53 55 59

5 2 2 7 3 5 5 4 4 4 3 3 3 3 3 3 3 2 2 2 4 3

7 6 3 23 17 23 12 17 16 14 18 17 15 14 6 6 3 10 10 6 10 8

46 118 22 62 46

2 2 7 6 5

15 3 43 12 11

42 54 70 50

9 6 10 5

28 15 18 12

36

4

13

27 23

3 3

17 15

89 20 62

3 2 2

3 11 4

Protein reported in exosome (Mathivanan and Simpson, 2009). Proteasomes can be secreted in exosomes (Jansen et al., 2009; Carayon et al., 2011).

Incubation of severely traumatized anemones in stellaSW supplemented with 1 mM epoxomicin, an inhibitor of the proteasome complex, significantly delayed the recovery of vibration sensitivity (Fig. 5B). In the presence of epoxomicin, a trend of slow recovery in vibration sensitivity was apparent. Nevertheless, in the presence of epoxomicin, levels of nematocyst discharge in traumatized anemones (27.6 ± 2.1 nematocysts/FOV) remained significantly lower than healthy controls (34.2 ± 1.5 nematocysts/FOV, p ¼ 0.034) throughout the 6-h experiment. A commercial polyclonal antibody to the core subunits of the 20S proteasome was diluted in stellaSW to possibly interfere with the biological activity of extracellular proteasomes during hair bundle repair. Following severe trauma, anemones were allowed to recover in stellaSW augmented with the proteasome antibodies. In the presence of the dilute antibody solution, levels of nematocyst discharge (19.4 ± 1.8 nematocysts/FOV) were significantly lower in experimental animals than in healthy controls (33.9 ± 1.7 nematocysts/FOV, p < 0.001) or in traumatized controls (30.1 ± 2.3 nematocysts/FOV, p < 0.001) at the 4th h of recovery (Fig. 6C). Thus, polyclonal antibodies to proteasomes delay or inhibit the recovery of vibration sensitivity. Unfortunately, western blots of trauma mucus protein failed to detect any immunopositive bands using the

proteasome antibodies (data not-shown). Reasoning that the antibodies might fail to significantly bind denatured polypeptide subunits of the anemone proteasomes, we performed a dot-blot on trauma mucus protein and confirmed immunoreactivity of the antibodies to polypeptides in trauma mucus protein with BSA serving as a negative control (Fig. 6B). At this point, the specificity of the antibodies to core polypeptides of anemone proteasomes remains unconfirmed. Non-immune, rabbit serum diluted in seawater does not delay the recovery of vibration sensitivity in traumatized anemones (Nag and Watson, 2007; Watson et al., 2007). Taken together, the results of these experiments suggest that extracellular 20S proteasome activity may be required for the repair of traumatized hair bundles. On the other hand, a pharmacological inhibition of proteasomes protects hair cells of zebrafish from aminoglycoside-induced cell death (Coffin et al., 2013). Perhaps these different observed effects of proteasome inhibition on hair cells might indicate different functions of proteasomes in damaged hair cells in space and time. In this study, extracellular proteasomes are likely to be important in the repair of damaged hair cells soon after trauma. In the zebrafish study, intracellular proteasomes possibly degrade pro-survival Bcl2 polypeptides as a late cellular



Fig. 5. Effect of specific pharmacological inhibitors on the recovery of vibration sensitivity in Nematostella vectensis. (A). Vibration sensitivity was tested in severely traumatized anemones recovering in the presence of 10 mM VER155008, an inhibitor of Hsp70 (open triangles). (B) Vibration sensitivity was tested in severely traumatized anemones recovering in the presence of 1 mM epoxomicin, an inhibitor of the 20S proteasome protein complex (open triangles). In each panel, the mean number of nematocysts counted per field of view (FOV ± SE) is plotted for the experimental animals as well as for healthy controls (closed squares) and traumatized controls (closed circles). aSignificant difference (Fisher's post-hoc test, p < 0.05 after one-way ANOVA) in mean nematocyst discharge between the traumatized controls, (i.e., traumatized anemones recovering in stellaSW alone), and healthy controls. bSignificant difference in mean nematocyst discharge between specimens allowed to recover in the presence of the inhibitor and healthy controls.

response to trauma leading to cell death (Coffin et al., 2013). Thus, proteasomes likely participate in hair cell responses to trauma in several different ways. The 20S proteasome is involved in several different proteolytic pathways, including ubiquitin-dependent, ubiquitin-independent, and ATP-independent pathways (Coux et al., 1996; Kisselev et al., 1999; Shringarpure et al., 2001). ADP-ribosylation can promote proteasome activity by regulating the biological activity of P131, a

9

Fig. 6. Effect of specific antibodies on the recovery of vibration sensitivity in Nematostella vectensis. (A) Western blot of trauma mucus proteins to polyclonal antibodies to heat shock protein-70 (Hsp70). A conspicuous band appears at approximately 70 kDa (B) Dot blot of trauma mucus to polyclonal antibodies to the 20S proteasome. Trauma mucus is immunopositive. BSA serves as a negative control. (C) Vibration sensitivity was tested in severely traumatized anemones recovering in the presence of antibodies diluted (1:3000) in stellaSW. Antibodies to Hsp70 (open triangles) and the 20S proteasome (open diamonds) were tested in separate experiments. The mean number of nematocysts counted per field of view (FOV ± SE) is plotted for the experimentally traumatized animals as well as for healthy controls (closed squares) and traumatized controls (closed circles). aSignificant difference (Fisher's post-hoc test, p < 0.05 after one-way ANOVA) in mean nematocyst discharge between the traumatized controls (i.e., traumatized anemones recovering in stellaSW alone) and healthy controls. bSignificant difference in mean nematocyst discharge between specimens allowed to recover in the presence of the Hsp70 antibody and healthy controls. cSignificant difference in mean nematocyst discharge between specimens allowed to recover in the presence of the antibody to 20S proteasome and healthy controls.


10


polypeptide involved in the assembly of functional 26S proteasomes (Cho-Park and Steller, 2013). Interestingly, Arl-5b, an ADPribosylation factor, was previously implicated in anemone hair bundle repair (Watson et al., 2007). Although Arl-5b was not identified in our proteomic analysis of the RP bands, Arl-5b was detected in the 696-kDa RP band by a western blot after twodimensional blue native/SDS-PAGE (Supplementary Fig. 3). Recently, extracellular active 20S proteasomes were documented (Mueller et al., 2012; Sixt et al., 2009), and these protein complexes are involved in the degradation, modification, or activation of cosecreted proteins (Sixt et al., 2009; Sixt and Peters, 2010). In anemone hair bundle repair, extracellular 20S proteasomes might be regulated by ADP-ribosylation and then may be involved in the removal or modification of damaged proteins. The ubiquitin-proteasome system (UPS) is known to degrade proteins not only under normal conditions but also in response to cellular stress (Goldberg, 2003; reviewed in Berthon et al., 2007). If a chaperone bound-misfolded protein is not efficiently refolded by chaperones, Hsp70s (among other chaperones) recruit E3 ubiquitin ligases to the misfolded protein so that it can be ubiquitinated (Petrucelli et al., 2004; Pratt et al., 2010). Polyubiquitinated polypeptides are targeted for degradation by the proteasome. Extracellular UPS is involved in fertilization of sea urchins (reviewed in Sawada et al., 2014) and mammals (Sutovsky et al., 2004). Both VER155008 and epoxomicin must be dissolved in DMSO. The final concentration of DMSO in our experiments was 0.1%. As controls, possible effects of DMSO on the recovery of vibration sensitivity were tested. Traumatized anemones that were incubated in 0.1% DMSO recovered vibration sensitivity (33.9 ± 2.5 nematocysts/FOV) along a similar time course as traumatized controls tested in stellaSW alone (Supplementary Fig. 2A). Traumatized anemones that were incubated in 1% DMSO delayed the recovery of vibration sensitivity until the 4th hour of recovery (29.4 ± 2.7 nematocysts/FOV). Results of these experiments suggest that exposure to 0.1% DMSO is unlikely to affect the recovery of hair bundles. Healthy anemones were incubated in the inhibitors as controls to test whether the inhibitors decrease vibration sensitivity in healthy anemones (Supplementary Fig. 2B). Notably, significant differences were observed between the responses of healthy controls (23.6 ± 2.9 nematocysts/FOV) and inhibitor-treated specimens (10 mM VER155008: 32.9 ± 1.5 nematocysts/FOV; 1 mM epoxomicin: 32.8 ± 1.7 nematocysts/FOV) at 2 h, although this result may have been due to an unusually low average number of nematocysts discharged in the healthy controls at this time point. Occasionally, unexpected levels of nematocyst discharge occur. However, the inhibitors did not significantly decrease vibration sensitivity in healthy anemones throughout the experiment (Supplementary Fig. 2B). 3.6. A model for repair of damaged hair cells Disruption of hair cell tip links and splaying of stereocilia are among the first responses of hair cells to noise trauma (Engstrom et al., 1983; Pickles et al., 1987a,b; Osborne and Comis, 1990; Clark and Pickles, 1996), and hair cells may die if the damage is severe. Cadherin 23, a component of tip links, is known to bind calcium, a phenomenon that is considered to influence its rigidity (Sotomayor et al., 2010). Calcium chelators disrupt tip links (Assad et al., 1991), possibly by destabilizing the cadherins so that they readily denature. Hsp70s are associated with responses to hair cell trauma as described in Section 3.5. The secreted Hsp70s may function as anti-inflammatory signaling molecules (Borges et al., 2012), and/or attempt to refold denatured polypeptides in the extracellular fluid. In this context, it is noteworthy that ATP is

secreted following hair cell trauma (Watson et al., 1999; Gale et al., 2004; Lahne and Gale, 2010), and ATP is required for the chaperone function of Hsp70s. In addition, secreted ATP might provide energy to ATP-dependent enzymes in the RP mixture (Watson et al., 1999) or activate purinoceptors as part of a signaling cascade (Housley, 2000; Gale et al., 2004). Increased respiration following trauma (Henderson et al., 2006) may be necessary to generate sufficient secreted ATP, and elevated metabolic rates would generate higher levels of H2O2 among other ROS. Thus, the peroxiredoxins identified in the RP suite might be involved in the removal of excess H2O2. Exogenously added H2O2 disrupts normal morphology of outer hair cells (Clerici et al., 1995). Secreted peroxiredoxins occur in mammals (Kang et al., 1998). Furthermore, experimentally upregulated peroxiredoxins protect mice from permanent threshold shifts following acoustic trauma (Ahn et al., 2013). Several other antioxidant proteins are upregulated following noise trauma (Jacono et al., 1998). If the hair cell damage is so severe as to damage proteins beyond their capacity to be refolded by chaperones, Hsp70 may assist in the ubiquitination of the damaged polypeptides (Petrucelli et al., 2004; Pratt et al., 2010). The polyubiquitinated polypeptides would then be digested by proteolytic activity residing within the secreted RP proteasomes. Thus, proteasomes and their associated peptidases might function to selectively remove permanently damaged proteins from hair cells. In birds, acoustic trauma results in an increase in transcription of a gene encoding a specific E3 ubiquitin-ligase (Lomax et al., 2000), suggesting that proteasome activity might be elevated following acoustic trauma. Selective protein degradation might explain the temporary loss of cadherin 23 from tip links of hair cells recovering from trauma (Indzhykulian et al., 2013). 3.7. Conclusions This study identified 37 polypeptides that might be involved in repair of hair bundles in sea anemones. Inhibitors of Hsp70s and the 20S proteasome delayed or arrested recovery of hair bundle function, supporting the possibility of an involvement of Hsp70s and proteasomes in repair of hair bundles. Clearly, additional research is needed to test the proposed model as well as to more fully elucidate the molecular and cellular mechanisms underlying the repair of hair cells following trauma. 4. Conflicts of interest Neither author has a conflict of interest related to the research presented in this article. Acknowledgments The authors thank Dr. A.B. Mayfield for critical reviews of this manuscript. In addition, the authors thank two anonymous reviewers for their helpful suggestions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.heares.2015.07.005. References Ahn, J.H., Shin, J.-E., Chung, B.Y., Lee, H.M., Kang, H.H., Chung, J.W., Pak, J.H., 2013. Involvement of retinoic acid-induced perioredoxin 6 expression in recovery of noise-induced temporary hearing threshold shifts. Environ. Toxicol. Pharmacol. 36, 463e471. Assad, J.A., Shepherd, G.M., Corey, D.P., 1991. Tip-link integrity and mechanical transduction in vertebrate hair cells. Neuron 7, 985e994.


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

Integrins of the starlet sea anemone Nematostella vectensis.

Continuous drug release by sea anemone Nematostella vectensis stinging microcapsules.

Sex-specific and developmental expression of Dmrt genes in the starlet sea anemone, Nematostella vectensis.

Functional characterisation of a TRPM2 orthologue from the sea anemone Nematostella vectensis in human cells.

Fast neurotransmission related genes are expressed in non nervous endoderm in the sea anemone Nematostella vectensis.

Histological study on maturation, fertilization and the state of gonadal region following spawning in the model sea anemone, Nematostella vectensis.

PaxA, but not PaxC, is required for cnidocyte development in the sea anemone Nematostella vectensis.

Characterization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis.

Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis.

RGM regulates BMP-mediated secondary axis formation in the sea anemone Nematostella vectensis.

The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate development and regeneration.

Do novel genes drive morphological novelty? An investigation of the nematosomes in the sea anemone Nematostella vectensis.

Developmental and light-entrained expression of melatonin and its relationship to the circadian clock in the sea anemone Nematostella vectensis.

ADP-Ribose Activates the TRPM2 Channel from the Sea Anemone Nematostella vectensis Independently of the NUDT9H Domain.

Characterization of the Cadherin-Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell-Cell Adhesion.

MAPK signaling is necessary for neurogenesis in Nematostella vectensis.

HSPG2 gene and its activation in regenerating Nematostella vectensis.

Initiating a regenerative response; cellular and molecular features of wound healing in the cnidarian Nematostella vectensis.

Sequential actions of β-catenin and Bmp pattern the oral nerve net in Nematostella vectensis.

Antagonistic BMP-cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm.

Proteomic identification of differentially expressed proteins in sea cucumber Apostichopus japonicus coelomocytes after Vibrio splendidus infection.

Response of bacterial colonization in Nematostella vectensis to development, environment and biogeography.

Molecular characterization of the apical organ of the anthozoan Nematostella vectensis.