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Rho-family G-proteins are required for the recovery of traumatized hair bundle mechanoreceptors in the sea anemone, Nematostella vectensis
Comparative Biochemistry and Physiology, Part A 242 (2020) 110637
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Rho-family G-proteins are required for the recovery of traumatized hair bundle mechanoreceptors in the sea anemone, Nematostella vectensis Shelcie S. Menard-Harvey
T
⁎,1
, Glen M. Watson
Department of Biology, University of Louisiana at Lafayette, 410 E. St. Mary Blvd., Lafayette, LA 70504, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Actin Calcium-free Rho inhibitor I Rhosin Stereocilia
Immersing anemones in calcium-free seawater disorganizes hair bundle mechanoreceptors on tentacles of sea anemones while causing a loss of vibration sensitivity. Remarkably, anemone hair bundles recover after being returned to calcium-containing seawater. Reorganization of actin in stereocilia likely follows during the recovery of normal morphology of hair bundles after such immersion. Previous studies have reported that Rho G-proteins are located in the stereocilia of hair bundles in sea anemones where they participate in polymerizing actin in stereocilia upon activation of specific chemoreceptors. We here find that immersing anemones in calcium-free seawater significantly reduces the abundance of hair bundles. A partial recovery of abundance of hair bundles occurs within 3 h post-immersion, but a full recovery of abundance does not occur even 6 h after specimens are returned to calcium-containing seawater. Anemones recovering from immersion in calcium-free seawater feature hair bundles that are significantly wider at their tips than in controls. The hair bundles subsequently narrow at their tips, becoming comparable to those of untreated controls within 6 h. Stereocilia of hair bundles are significantly longer in experimental animals than in controls at 2 h of recovery before shortening to lengths comparable to untreated controls at 6 h. In the presence of Rho inhibitors, the recovery in abundance of hair bundles through 6 h is delayed or inhibited. Likewise, in the presence of Rho inhibitors, stereocilia fail to significantly elongate within 2 h of recovery. These data suggest that Rho G-proteins participate in the normal recovery of abundance and recovery of normal morphology of experimentally damaged hair bundle mechanoreceptors.
1. Introduction Sea anemones employ hair bundle mechanoreceptors located on their tentacles to detect the rhythmic movements of nearby swimming prey (Mire-Thibodeaux and Watson, 1994; Watson and Hessinger, 1989). As in vertebrate animals possessing hair cells, anemone hair bundle mechanoreceptors are composed of dozens to hundreds of actinbased stereocilia held together by specific cadherin-based linkages. After immersion of the sensory epithelium in calcium-depleted buffers, the stereocilia of hair bundles splay apart rendering the hair bundle non-functional (Assad et al., 1991; Watson et al., 1998; Watson et al., 1997). Evidently, the cadherins forming extracellular linkages interconnecting stereocilia require calcium ions in order to maintain their structural integrity (Kazmierczak et al., 2007). After such immersion, and upon returning the sensory epithelium to calcium-containing buffers, at least some of the hair cells on anemone tentacles spontaneously
recover normal structure and function of their hair bundles over a timecourse that increases with the duration of immersion in the calciumdepleted buffer (Watson et al., 1998). As compared to hair bundles in other animals, hair bundles of sea anemones are remarkable for their ability to recover from severe damage (for these experiments caused by a 1 h immersion in calcium-free seawater; Watson et al., 1998). Presumably, under natural conditions, anemone hair bundles are frequently damaged by the vigorous movements of captured prey struggling to escape. In other animals, overstimulation of hair bundles shears linkages between stereocilia to disrupt the normal structure and function of the hair bundles (Husbands et al., 1999; Kurian et al., 2003; Pickles et al., 1987). Stereocilia are evaginations of the plasma membrane filled with tightly packed, parallel arrays of actin filaments. Stereocilia are considered to be rigid structures (reviewed: Hudspeth, 1985). Consequently, the means by which the hair cells restore order to the splayed
Abbreviations: KSW, high‑potassium seawater; PBS, phosphate-buffered saline; BSA, bovine serum albumin ⁎ Corresponding author. E-mail addresses: [email protected] (S.S. Menard-Harvey), [email protected] (G.M. Watson). 1 Present Address: Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC, 28223, USA https://doi.org/10.1016/j.cbpa.2019.110637 Received 5 September 2019; Received in revised form 26 November 2019; Accepted 13 December 2019 Available online 20 December 2019 1095-6433/ © 2019 Elsevier Inc. All rights reserved.
Comparative Biochemistry and Physiology, Part A 242 (2020) 110637
S.S. Menard-Harvey and G.M. Watson
Invitrogen, Grand Island, NY, USA. Glutaraldehyde was acquired from Sigma-Aldrich, St. Louis, MO, USA. Paraformaldehyde was acquired from Electron Microscopy Sciences, Hatfield, PA, USA. ProLong Gold Antifade Mountant was acquired from ThermoFisher Scientific, Waltham, MA, USA. Statistica software was acquired from Statsoft, Tulsa, OK, USA. ImageJ software was acquired from National Institutes of Health, Bethesda, MD, USA. MaximDL software was acquired from Diffraction Limited, Ottawa, ON, Canada. Microcal Origin 5.0 was acquired from Microcal Software, Northampton, MA, USA.
hair bundle is of interest. Lost or damaged linkages would have to be replaced or otherwise re-established. In addition, the stereocilia would have to be repositioned. After a prolonged exposure (1 h) to calciumdepleted seawater, a spontaneous recovery of hair bundles in anemones requires a minimum of 4 h to be completed. Such a recovery involves the secretion of so called ‘repair proteins’ as well as secretion of ATP. Exogenously supplied repair proteins and ATP speed the recovery of structure and function of experimentally damaged hair bundles to a matter of a few minutes (Watson et al., 1999; Watson et al., 1998). A 1 h immersion in calcium-free seawater extensively disorganizes hair bundle mechanoreceptors on the tentacles of sea anemones such that the conical form of the hair bundle is unrecognizable, suggesting a loss of linkages although the stereocilia persist (Watson et al., 1998). Within a few minutes after immersion in calcium-free seawater, levels of Factin in stereocilia significantly decrease. However, in the presence of exogenously supplied repair proteins, levels of F-actin in stereocilia increase to become comparable to those in stereocilia of untreated controls at approximately 40 mins into recovery (Watson and Mire, 2002). Thus, the events of repair include extracellular molecules (repair proteins and ATP) that somehow help to re-establish cadherin linkages between stereocilia and at the same time may help to coordinate events occurring in the cytoplasm of the damaged hair cells, including a reorganization of actin in stereocilia. In a wide variety of eukaryotic cells, Rho proteins participate in signaling cascades that culminate in changes to the actin cytoskeleton (reviewed: Etienne-Manneville and Hall, 2002). Rho proteins are small monomeric proteins that belong to the Ras family of G-proteins (reviewed: Barbacid, 1987). Previously, Rho has been implicated in the normal changes to the actin cytoskeleton that occur in stereocilia of hair bundles in response to chemoreceptor activation in hair cells of sea anemones. In anemones, hair bundle mechanoreceptors normally lengthen in response to exogenously added N-acetylated sugars including N-acetylneuraminic acid (NANA), and then shorten in response to exogenously added proline (Mire-Thibodeaux and Watson, 1994; Watson and Hessinger, 1994). Both chemoreceptor-induced responses involve a reorganization of the actin cytoskeleton in stereocilia: actin polymerization in response to NANA and actin depolymerization in response to proline (Watson and Roberts, 1995). When pharmacological activators of Rho are experimentally introduced, hair bundles elongate in a comparable fashion to the elongation normally induced by exposure to NANA but fail to shorten in response to proline (Allaire and Watson, 2013). However, when pharmacological inhibitors of Rho are experimentally introduced, hair bundles fail to elongate in response to NANA but shorten in response to proline (Allaire and Watson, 2013). Rho homologs have been identified in the genome of the sea anemone Nematostella vectensis and localized by immunocytochemistry to the stereocilia of hair bundles (Allaire and Watson, 2013). Clearly, Rho G-proteins are involved in the reorganization of the actin cytoskeleton within the stereocilia of hair bundle mechanoreceptors during elongation of the hair bundle in response to chemoreceptor activation, but it is unclear to what extent Rho G-proteins might be involved in the reorganization of the actin cytoskeleton during the repair of experimentally-damaged hair bundle mechanoreceptors. This study aims to determine whether or not repair of experimentallydamaged hair bundle mechanoreceptors occurs in the absence of Rho activity. In the event that Rho activity is required for repair to occur, this study aims to determine whether a critical period occurs during which Rho G-proteins must be active in order to facilitate repair.
2.2. Animal maintenance Stock cultures of Nematostella vectensis were established from approximately 50 individuals locally collected by personnel of the Marine Biological Laboratory, Woods Hole, MA. Specimens of Nematostella vectensis were maintained in Pyrex dishes containing natural seawater diluted to 16 ppt (Allaire and Watson, 2013). The animals were maintained at 22 °C, fed brine shrimp nauplii twice weekly and cleaned after feeding. Experiments were scheduled for a minimum of 24 to 48 h after feeding (Allaire and Watson, 2013). 2.3. Effects of exposure to calcium-free high‑potassium seawater on the abundance of hair bundles at the tips of tentacles of sea anemones Sea anemones are anesthetized in formulations of artificial seawater that contain elevated levels of potassium ions and low levels of sodium ions (Watson et al., 2009). Such anesthetics are referred to as KSW. For these experiments, a combination of KSW and low levels of calcium ions was employed in order to damage hair bundles in anesthetized anemones. It was important to anesthetize the anemones so that tentacles could be excised from the anemones and fixed for microscopic examination without contracting. Sea anemones were immersed in calcium-free high‑potassium seawater (calcium-free KSW: 166 mM NaCl; 62 mM KCl; 26 mM MgCl2; 13 mM MgSO4; 4 mM EGTA, a widely-used calcium chelator; 1 mM NaHCO3) for 1 h, and hair bundles were counted at specific time points after the anemones were returned to KSW (containing calcium ions) and subsequently fixed to determine the abundance of sensory/supporting cell hair bundles. Each replicate experiment required eight animals: one untreated control and seven experimental animals. The untreated control was immersed in KSW containing normal levels of calcium ions (166.5 mM NaCl; 50 mM KCl; 13 mM MgSO4; 12 mM MgCl2; 6 mM CaCl2; 1.15 mM NaHCO3) for 1 h before having the oral disc excised and fixed in 4% paraformaldehyde and 0.025% glutaraldehyde in Millonig's buffer (as per the methods of Watson et al., 2009). The experimental animals were immersed in calcium-free KSW for a 5 min wash before being immersed in calciumfree KSW for a full hour. The 5 min wash was intended to ensure that the animals would exchange the natural seawater inside the coelenteron for the calcium-free KSW in the dish. After a 1 h immersion in calcium-free KSW, the animals were washed for 5mins in KSW, and then allowed to recover in KSW for up to 6 h. Exposure to KSW alone over 6 h does not significantly affect the abundance of hair bundles on the tentacles of sea anemones (Menard and Watson, 2017). Starting immediately after the 5 min KSW wash, the oral disc of one experimental animal (T0) was excised and fixed for one hour. At each subsequent hour (T1–T6) of recovery, the oral disc of another animal was excised and fixed for 1 h. After fixation, oral discs were washed in phosphate-buffered saline (PBS) for 5mins. Tentacles were then excised from the oral discs and prepared as wet mounts for observation using phase-contrast microscopy (40× objective; n.a. = 0.65). One tentacle was excised from each animal. Hair bundles appearing to possess a conical shape (stereocilia surrounding and leaning toward the central kinocilium) while viewed in profile were counted in three fields of view (equivalent to 0.69 mm tentacle length) on one side of the tentacle, starting at the tip of the tentacle. Hair bundles having disorganized stereocilia (splayed away from the central kinocilium and each other)
2. Materials and methods 2.1. Materials Rho Inhibitor I was acquired from Cytoskeleton, Inc., Denver, CO, USA. Rho inhibitor, Rhosin, was acquired from Millipore Sigma, Burlington, MA, USA. Rhodamine phalloidin was acquired from 2
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were not counted. This approach is intended to determine the time required for the abundance of intact hair bundles to return to untreated control levels. Previous studies performed to determine the time course of recovery of morphology and function on hair bundles damaged by exposure to calcium-depleted seawater did not consider the abundance of hair bundles (Watson et al., 1998; Watson and Mire, 2002). Microscopy was performed using a LOMO microscope (Labaroscope, LOMO America, Prospect Heights, IL, USA). Data were plotted as the mean abundance of hair bundles per 100 μm tentacle length ± SEM (n = 9 tentacles per time point). Each tentacle is excised from a different animal.
microscopy (100× objective; n.a. = 1.30). When viewing the tentacle in profile, a total of 10 hair bundles was measured, starting at the tip of the tentacle. Only certain hair bundles were measured. The entire hair bundle (from base to tip) was required to be in focus along its length to be selected for measurement. Measurements were obtained from digital photomicrographs using ImageJ. Data were pooled for the 10 hair bundles for each specimen to give a single value per specimen (tentacle). Three tentacles (each from a different animal) were obtained for each treatment. Data were plotted as mean length, mean base width, or mean tip width of hair bundles ± SEM (n = 3 tentacles per time point). Each tentacle is excised from a different animal.
2.4. The effects of Rho inhibition on recovery of abundance of hair bundles from exposure to calcium-free KSW
2.6. Lengths of rhodamine phalloidin-stained stereocilia of hair bundles following exposure to calcium-free KSW and exposure to Rhosin
Sea anemones were immersed in a Rho inhibitor during their recovery from exposure to calcium-free KSW to test effects of Rho inhibitor on the recovery of abundance of hair bundles. Each replicate experiment involved eight animals: one untreated control and seven experimental animals. The experimental protocol was the same as in Section 2.3 above, except for exposure to Rho inhibitor I or Rho inhibitor (Rhosin) beginning at the start of recovery (T0), beginning one hour into recovery (T1), or beginning two hours into recovery (T2) for Rhosin only. Rho inhibitors were reconstituted according to manufacturer's instructions. The animals were treated with Rho inhibitor I at a concentration of 1 μg/ml, as per manufacturer's instructions. In the case of Rhosin, the animals were treated with 100 μM Rhosin, as per manufacturer's instructions. The reconstituted Rho inhibitors were added directly to the KSW (to the desired final concentration of the designated inhibitor) in the dish of recovering animals at specific timepoints during recovery, allowing each animal to begin the exposure period at the same time. Once added, the Rho inhibitors were present for the remainder of the recovery period concluding at 6 h of recovery. Rho inhibitors were added at different time points to determine when during the recovery period is Rho activity critical for the normal recovery of the abundance of hair bundle mechanoreceptors. Rho inhibitor I was used to inactivate the Rho-family proteins RhoA, RhoB, and RhoC via the protein C3 transferase (Benink and Bement, 2005; Burakov et al., 2003). Rhosin was used to inactivate Rho-family proteins RhoA, RhoB, and RhoC via interference with the GEF (i.e., guanine nucleotide exchange factor) binding domain (Shang et al., 2012). The incorporation of two mechanistically different Rho inhibitors into the experimental design was intended to control for the possibility of off-effects of the Rho inhibitors on hair bundle structure. Tentacles were excised from the oral discs, fixed and processed as was described in Section 2.3 and then prepared as wet mounts for viewing under phase-contrast microscopy (40× objective; n.a. = 0.65). Hair bundles appearing to possess a conical shape while viewed in profile on one side of the tentacle were counted in three fields of view, starting at the tip of the tentacle. Data were plotted as the mean abundance of hair bundles per 100 μm tentacle length ± SEM (n = 4 tentacles per time point for Rho inhibitor I [T0]; n = 3 tentacles per time point for Rho inhibitor I [T1]; n = 6 tentacles per time point for all Rhosin experiments). Each tentacle is excised from a different animal.
Hair bundles of sea anemones consist of several small-diameter stereocilia that converge on 5–10 large-diameter stereocilia located at the center of the hair bundle (Peteya, 1975; Watson et al., 1997). Largediameter stereocilia are longer than are small-diameter stereocilia. The lengths of large-diameter stereocilia of hair bundles were measured from micrographs taken of rhodamine-phalloidin stained specimens that were fixed and processed at specific intervals through 6 h of recovery from exposure to calcium-free KSW. For these experiments the ‘control’ animals were immersed in calcium-free KSW and then allowed to spontaneously recover in KSW alone. The ‘experimental’ animals were immersed in calcium-free KSW and then allowed to recover in the presence of 100 μM Rhosin in KSW. Each replicate experiment required eight animals: one untreated specimen and seven animals that were immersed in calcium-free KSW. The experimental protocol was similar to that explained in Section 2.3. After fixation of the oral discs, the oral discs were transferred to a solution of rhodamine phalloidin and 3% bovine serum albumin (BSA) in PBS. The addition of BSA to the staining solution is intended to prevent non-specific staining. Staining in rhodamine-phalloidin continued for 8 h (overnight) with specimens rocking at 4 °C. The final dilution of the rhodamine phalloidin conjugate was prepared according to manufacturer's instructions. On the following day, the oral discs were washed in PBS. Next, one randomlyselected tentacle was excised from each oral disc and mounted in ProLong Gold overnight at 4 °C. Slides were imaged on the next day via epifluorescence microscopy (100× objective; n.a. = 1.30). Hair bundles were imaged in profile. The lengths of stereocilia were measured from digital photomicrographs of ten hair bundles per specimen using Image J. To measure the length of stereocilia, the ImageJ line tool was used to draw a straight line from the bottom of the stereocilium to the tip of the stereocilium of the hair bundle. The data were plotted as the mean length in microns of stereocilia ± SEM (n = 3 tentacles per time point). Each tentacle is excised from a different animal. 3. Statistics Statistica software (Tulsa, OK, USA) was used to perform a one-way ANOVA and post-hoc Fisher LSD analysis to compare specific groups (i.e., treatments or timepoints). Data were determined to be statistically significant at a p-value of 0.05 or less. Microcal Origin software (Northampton, MA) was used to create graphs.
2.5. Dimensions of hair bundles on tentacles of sea anemones following immersion in calcium-free KSW
4. Results 4.1. Effects of exposure to calcium-free KSW on the abundance of hair bundles at the tips of the tentacles of sea anemones
The mean length, mean base width, and mean tip width were calculated from measurements of hair bundles taken from digital photomicrographs obtained along the length of fixed tentacles. The specimens were immersed in calcium-free KSW for 1 h at room temperature as detailed in Section 2.3. Each replicate experiment required eight animals: one untreated control and seven experimental animals. A fixed tentacle from each animal was randomly selected, excised from the oral disc, and prepared as a wet mount for imaging using oblique
After a 1 h immersion in calcium-free KSW, the mean abundance of hair bundles at the tips of the tentacles of sea anemones significantly decreased (by approximately 60%) compared to untreated controls (p = .000002; Fig. 1). The mean abundance of hair bundles partially recovered 3 h after the specimens were returned to calcium containing 3
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One curve for the experiments testing Rho inhibitors beginning at T1 was unusual (i.e., different from that shown in Figs. 3a, b) in that the abundance of hair bundles did not significantly decrease relative to untreated controls until T1 (p = .02; Fig. 4a). Upon adding Rho inhibitor I at T1, the mean abundance of hair bundles became statistically comparable to both the untreated control (p = .16) and T1 (p = .24) at 2 h, then at 3 h became statistically comparable to only T1 (p = .87) through 6 h (p = .39) (Fig. 4a). Following immersion in calcium-free KSW, the mean abundance of hair bundles significantly decreased compared to untreated controls immediately on the second curve (p = .007; Fig. 4b). Following the addition of Rhosin at T1, the mean abundance of hair bundles remained comparable to that for T0 through 2 h (p = .14), and then significantly decreased compared to T0 through 4 h (p = .003), before becoming comparable to T0 at 5 h (p = .44), and finally significantly decreasing again compared to T0 at 6 h (p = .002; Fig. 4b). For this set of experiments, the Rho inhibitor was added at T2. Following immersion of anemones in calcium-free KSW, the mean abundance of hair bundles significantly decreased compared to untreated controls at T0 (p = .01; Fig. 5). Following the addition of Rhosin at T2, the mean abundance of hair bundles remained comparable to that for T0 through 6 h (p = .29; Fig. 5).
Fig. 1. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to calcium-free KSW. The abundance of hair bundles was determined in untreated controls and in experimental animals after 1 h of exposure to calcium-free KSW (experimentals only) and through 6 h of recovery in KSW by phase-contrast microscopy of fixed specimens imaged in profile at a single focal plane. Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. The mean abundance was calculated from nine animals per data point. UC = untreated control (n = 9); T0 = immediately following trauma (n = 9); T1 = 1 h recovery; (n = 9); T2 = 2 h recovery (n = 9); T3 = 3 h recovery (n = 9); T4 = 4 h recovery (n = 9); T5 = 5 h recovery (n = 9); T6 = 6 h recovery (n = 9). The letters “a, b and c” denote groups that are significantly different from each other (p ≤ .05).
4.3. Morphology of hair bundles on tentacles of sea anemones during their recovery after immersion in calcium-free KSW Tip width, bundle length, and base width were measured from digital photomicrographs of hair bundles after immersion in calcium-free KSW and through 6 h of recovery. Representative images of hair bundles are shown for untreated controls (Fig. 6a), experimental animals immediately after exposure to calcium-free KSW (Fig. 6b), experimental animals 3 h into recovery (Fig. 6c), and experimental animals 6 h into recovery (Fig. 6d). The mean tip width of hair bundles significantly increased 2 h into recovery (p = .04) and remained wider than controls through 4 h of recovery (p = .03) before returning to widths comparable to untreated controls thereafter (Fig. 7a). With respect to mean bundle length and mean base width, these dimensions remained comparable to untreated controls throughout the 6 h recovery period (Fig. 7b and c, respectively).
KSW, as the mean abundance 3 h into recovery (T3) was significantly higher than at T0 (p = .004) but, nevertheless, remained significantly lower than the mean abundance of untreated controls (p = .03; Fig. 1). The mean abundance of hair bundles remained significantly different from both untreated controls and T0 animals through the remainder of the 6 h recovery period. After the initial knockdown, the abundance of hair bundles remained fairly steady through 2 h into the recovery period, rose significantly by hour 3, and then seemed to plateau through the remainder of the 6 h recovery period. Thus, the shape of the recovery curve approximated an S-shaped curve.
4.4. Lengths of rhodamine phalloidin-stained stereocilia of hair bundles following exposure to calcium-free KSW and exposure to Rhosin
4.2. The effects of Rho inhibitors on the recovery of abundance of hair bundles after exposure to calcium-free KSW
Rhodamine-phalloidin, a F-actin stain, was used to visualize the actin cytoskeleton of stereocilia of hair bundles in controls and in experimental animals after immersion in calcium-free KSW and through 6 h of recovery. Representative images of hair bundles are shown for untreated controls (Fig. 8a), experimental animals immediately following exposure to calcium-free KSW (Fig. 8b), experimental animals 3 h into recovery (Fig. 8c), and experimental animals 6 h into recovery (Fig. 8d). Following exposure to calcium-free KSW, the mean lengths of the stereocilia significantly increased relative to untreated controls at 2 through 3 h of recovery (p = .01 and p = .02, respectively) before shortening to lengths comparable to untreated controls thereafter (Fig. 9). Representative images of hair bundles are shown for untreated controls (Fig. 10a), experimental animals immediately following exposure to calcium-free KSW (Fig. 10b), experimental animals 3 h into recovery (Fig. 10c), and experimental animals 6 h into recovery (Fig. 10d). After exposure to Rhosin, the lengths of the stereocilia were comparable to untreated controls until they significantly decreased in length 5 h into recovery (p = .02) before returning to lengths comparable to untreated controls 6 h into recovery (Fig. 11).
Two Rho inhibitors (Rho Inhibitor I and Rhosin) were tested in anemones after immersing anemones in calcium-free KSW for 1 h. Experiments were performed such that inhibitors were added immediately after trauma (T0), 1 h after trauma (T1), or 2 h after trauma (T2) in order to attempt to determine whether Rho activity was needed throughout the recovery period or only during specific intervals during the recovery period. As a control, Rho inhibitor I alone had no significant adverse effects on the mean abundance of hair bundles on the tentacles of sea anemones through six hours of exposure (p = .71; Fig. 2a). Likewise, Rhosin alone had no adverse effects on the mean abundance of hair bundles on the tentacles of sea anemones through 6 h of exposure (p = .96; Fig. 2b). The mean abundance of hair bundles decreased significantly at T0 compared to untreated controls following exposure to calcium-free KSW (p = .003; Fig. 3a; and p = .000049; Fig. 3b). When administered immediately after the immersion in calcium-free KSW, both Rho inhibitor I and Rhosin prevented the recovery normally observed 3 h after trauma (T3) such that the abundance of hair bundles remained comparable to that for T0 through 6 h (p = .31–0.58; Fig. 3). 4
Comparative Biochemistry and Physiology, Part A 242 (2020) 110637
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Fig. 2. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to Rho inhibitors. The abundance of hair bundles was determined through 6 h of exposure to either Rho Inhibitor I or Rhosin by phase-contrast microscopy of fixed specimens imaged in profile. (a) Exposure to Rho Inhibitor I (n = 3 tentacles per time point). (b) Exposure to Rhosin (n = 6 tentacles per time point). Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. UC = untreated control; T0 = immediately following trauma; T1 = 1 h exposure; T2 = 2 h exposure; T3 = 3 h exposure; T4 = 4 h exposure; T5 = 5 h exposure; T6 = 6 h exposure.
5. Discussion
terminates other linkages between the stereocilia of the hair bundle (reviewed: Fettiplace and Hackney, 2006), as cadherin-23 (and likely protocadherin-15) is present in the kinociliary linkages and transient lateral linkages of the developing hair cells of mammals (Lagziel et al., 2005; Michel et al., 2005; reviewed: Müller, 2008; Rzadzinska et al., 2005; Siemens et al., 2004;). Similar consequences of exposure to calcium-free seawater are expected in the hair bundles of sea anemones, as sea anemones also have lateral linkages and a homolog of cadherin-23 in tip links of their hair bundles (Watson et al., 2008; Watson et al., 1997). In line with such expectations, a 1 h exposure to calcium-free seawater causes a majority of the hair bundles of sea anemones to become unrecognizable as hair bundles. This disorganization is such that stereocilia persist (Watson and Mire, 2002). If the specimens are
Although it has been known for some time that prolonged immersion in calcium-free seawater (i.e., 1 h) severely disorganizes hair bundle mechanoreceptors located on tentacles of sea anemones (Watson et al., 1998), nothing was known about the effects of such trauma on the abundance of hair bundles on the tentacle epithelium. In vertebrate animals, exposure to calcium-free buffer causes a disorganization of the hair bundle because the calcium-dependent proteins, cadherin-23 and protocadherin-15, that make up the tip links between stereocilia no longer maintain tip link structure (and as a result, mechanotransduction) in the absence of extracellular calcium (Assad et al., 1991; Lelli et al., 2010). Removal of calcium also
Fig. 3. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to calcium-free KSW followed by exposure to Rho inhibitors immediately after trauma. The abundance of hair bundles was determined after 1 h of exposure to calcium-free KSW followed immediately by administration of either Rho Inhibitor I or Rhosin and through 6 h of recovery by phase-contrast microscopy of fixed specimens imaged in profile. (a) Recovery after exposure to Rho Inhibitor I (n = 4 tentacles per time point). (b) Recovery after exposure to Rhosin (n = 6 tentacles per time point). Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
5
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Fig. 4. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to calcium-free KSW followed by exposure to Rho inhibitors beginning at 1 h after calcium-free KSW. The abundance of hair bundles was determined after 1 h of exposure to calcium-free KSW followed by administration of either Rho Inhibitor I or Rhosin (1 h into recovery) and through 6 h of recovery by phase-contrast microscopy of fixed specimens imaged in profile. (a) Recovery after exposure to Rho Inhibitor I (n = 3 tentacles per time point). (b) Recovery after exposure to Rhosin (n = 6 tentacles per time point). Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. The letters “a, b and c” denote groups that are significantly different from each other (p ≤ .05). The letters “ab” denote groups that are statistically comparable to groups labeled “a” and groups labeled “b”.
Fig. 5. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to calcium-free KSW followed by exposure to Rhosin beginning at 2 h after trauma. The abundance of hair bundles was determined after 1 h of exposure to calcium-free KSW followed by administration of Rhosin (2 h into recovery) and through 6 h of recovery by phase-contrast microscopy of fixed specimens imaged in profile. Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. The mean abundance was calculated from six animals per data point. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
Fig. 6. Hair bundles after exposure to calcium-free KSW imaged by oblique microscopy. Representative hair bundles are shown as follows: (a) untreated controls; (b) immediately following calcium-free trauma; (c) 3 h into recovery; (d) 6 h into recovery. hb = hair bundle; bl = bundle length; tw = tip width; bw = base width. Scale bar = 10 μm.
abundance of hair bundles occurring from 3 to 6 h of recovery. We stress that after 6 h of recovery, the abundance of hair bundles on the tentacle epithelium remains significantly smaller than in untreated controls at approximately 2/3 of control levels. Thus, there may be at least two phases of hair bundle recovery with one occurring at approximately 3 h of recovery and a second, slower recovery occurring sometime after 6 h. However, within 24 h after exposure to calciumdepleted seawater, the abundance of hair bundles recovers to control levels (data not shown). Such a significant, protracted reduction in
returned to calcium-containing seawater, at least some of the hair bundles on the tentacle epithelium recover their normal structure and function in 4 h (Watson et al., 1998). Here, we find that the abundance of hair bundles at the tips of tentacles of sea anemones is significantly decreased after a 1 h immersion in calcium-free KSW. However, we find that the recovery of abundance of hair bundles follows a S-shaped function with a dramatic increase in abundance occurring at approximately 3 h into recovery, but with only very modest increases in the 6
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Fig. 7. Mean dimensions of hair bundles after exposure to calcium-free KSW. The mean tip width, bundle length, and base width of hair bundles were determined after 1 h of exposure to calcium-free KSW and through 6 h of recovery by oblique microscopy of fixed specimens. Images taken via oblique microscopy were analyzed using ImageJ. (a) Mean tip widths of hair bundles. Data are presented as mean tip width in microns ± SEM. (b) Mean bundle lengths of hair bundles. Data are presented as mean bundle length in microns ± SEM. (c) Mean base widths of hair bundles. Data are presented as mean base width in microns ± SEM. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. The same hair bundles were measured for all three dimensions. Mean dimensions were calculated from three animals per data point. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
Fig. 9. Mean lengths of stereocilia after exposure to calcium-free KSW. The mean lengths of stereocilia were determined after 1 h of exposure to calciumfree KSW and through 6 h of recovery by epifluorescence microscopy of fixed specimens. Lengths of stereocilia were measured using ImageJ. Data are presented as the mean length of stereocilia in microns ± SEM. The mean length of stereocilia was calculated from three animals per data point. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
Fig. 8. Phalloidin cytochemistry of hair bundles after exposure to calcium-free KSW. Representative hair bundles are shown as follows: (a) untreated controls; (b) immediately following immersion in calcium-free KSW; (c) 3 h into recovery; (d) 6 h into recovery. hb = hair bundle; sc = stereocilia; ca = cytoplasmic actin. Scale bar = 10 μm.
remains comparable to the abundance of hair bundles at T0 or significantly decreases compared to the abundance of hair bundles at T0. The exception lies in Fig. 4a, where the effects (decreased abundance of hair bundles) of calcium depletion were somehow delayed by 1 h. As a result, the addition of Rho Inhibitor I at T1 is likened to adding in the inhibitor at T0 (immediately after decrease in abundance). Even with the delayed decrease in abundance of hair bundles, the overall result of Rho inhibition is consistent with that of the other experiments: no significant recovery without Rho activity. These results indicate that
abundance of hair bundles might be expected to significantly impair the sea anemone's ability to capture prey. Further experimentation would be necessary to test such an effect on prey capture in animals allowed to recover from extensive trauma for 6 h. Rho inhibitors were tested to investigate whether Rho is involved in the recovery of structural organization of hair bundles normally occurring 3 h after exposure to calcium-free KSW. Regardless of the onset of Rho inhibition between T0 and T2, the abundance of hair bundles 7
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fully blocked. Here it is important to recall that the Rho inhibitors evidently do not destabilize existing hair bundles (Fig. 2). Thus, Rho likely participates in reorganizing the actin cytoskeleton throughout the early hours of recovery, certainly during the most dramatic phase of recovery of hair bundle abundance occurring between hours 2 and 3 of recovery. Next, the tip widths of the bundles significantly increase compared to untreated controls at hours 2 through 4 of recovery. The widening at the tips of the hair bundles coincides with the significant lengthening of the stereocilia that occurs at hours 2 and 3 of recovery compared to untreated controls. These results indicate that the length of actin bundles is dynamically regulated at a time during which the tips of hair bundles are wide. During repair from exposure to calcium-free KSW, the tip links and other linkages interconnecting stereocilia must be replaced. It is likely that as the linkages are being replaced, Rho G-proteins are actively reorganizing the actin cytoskeletons of the stereocilia of the hair bundles. The stereocilia return to control lengths within 4 h of recovery, just before the tips of the hair bundles return to control widths at 5 h of recovery. Perhaps regulatory processes allow the Rho G-proteins to normalize the length of stereocilia as the linkages are installed. In the presence of Rhosin, elongation of the stereocilia does not occur during recovery. In fact, exposure to calcium-free KSW followed by exposure to Rhosin results in stereocilia significantly shortening 5 h into recovery before becoming comparable to untreated controls again 6 h into recovery. The failure of stereocilia to elongate and the subsequent shortening of the stereocilia in the presence of Rhosin are consistent with the possibility that Rho G-proteins are actively involved in the reorganization of the actin cytoskeleton in stereocilia during the recovery of hair bundles from exposure to calcium-free seawater.
Fig. 10. Phalloidin cytochemistry of hair bundles after exposure to calcium-free KSW followed by exposure to Rhosin. Representative hair bundles are shown as follows: (a) untreated controls; (b) immediately following trauma; (c) 3 h into recovery; (d) 6 h into recovery. hb = hair bundle; sc = stereocilia; ca = cytoplasmic actin. Scale bar = 10 μm.
6. Conclusions Previous research has established that exposure to calcium-depleted seawater disorganizes the stereocilia of hair bundle mechanoreceptors in sea anemones, and damaged hair bundles regain typical morphology and function within 4 h of recovery (Watson et al., 1998). It has also been found that F-actin levels in hair bundles decrease with exposure to calcium-depleted seawater but increase over time during recovery, indicating a reorganization of actin in hair bundles after trauma caused by calcium-depleted seawater (Watson and Mire, 2002). Such findings predict that Rho G-proteins might be involved in the repair process. The findings of the present study contribute new details to understanding both the repair process and the participation of Rho G-proteins in the repair process. Whereas previous research finds that recovery of morphology and function occurs over 4 h in individual hair bundles damaged by calcium-depleted seawater, this study finds that a partial recovery of abundance of intact hair bundles at the tip of the tentacle occurs within 3 h but that a full recovery of abundance requires > 6 h and is completed within 24 h. These findings suggest that recovery of damaged hair bundles may occur via multiple pathways or over multiple periods of time. This study also finds that important steps of the repair process occurring between hours 2 and 3 of the recovery period do not occur in the absence of Rho activity, as there is no partial recovery of abundance of intact hair bundles or lengthening of stereocilia in hair bundles without Rho activity. As a result, Rho G-proteins are required to be active 2 - 3 h into the recovery period. These findings indicate that not only are Rho G-proteins involved in the repair process but that they are required for repair to occur. Finally, there may be a Rho-dependent, dynamic regulation of stereocilium length occurring during the installation of replacement linkages located near the tips of stereocilia.
Fig. 11. Mean lengths of stereocilia after exposure to calcium-free KSW followed by exposure to Rhosin. The mean lengths of stereocilia were determined after 1 h of exposure to calcium-free KSW followed by administration of Rhosin and through 6 h of recovery by epifluorescence microscopy of fixed specimens. Lengths of stereocilia were measured using ImageJ. Data are presented as the mean length of stereocilia in microns ± SEM. The mean length of stereocilia was calculated from three animals per data point. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
active Rho proteins may be required soon after the damage caused by a 1 h immersion in calcium-free seawater and are certainly necessary for the increase in abundance of hair bundles otherwise observed between 2 and 3 h of recovery. As long as the Rho inhibitor is applied before this increase in abundance of hair bundles normally occurs (i.e., beginning at 2 h into recovery), then the recovery of abundance of hair bundles is
Funding This research did not receive any specific grant from funding 8
Comparative Biochemistry and Physiology, Part A 242 (2020) 110637
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agencies in the public, commercial, or not-for-profit sectors.
regeneration of sensory transduction in auditory cells requires functional interaction between cadherin-23 and Protocadherin-15. J. Neurosci. 30, 11259–11269. Menard, S.S., Watson, G.M., 2017. Evidence for two populations of hair bundles in the sea anemone, Nematostella vectensis. Comp. Biochem. Physiol. A 208, 14–23. Michel, V., Goodyear, R.J., Weil, D., Marcotti, W., Perfettini, I., Wolfrum, U., Kros, C.J., Richardson, G.P., Petit, C., 2005. Cadherin 23 is a component of the transient lateral links in the developing hair bundles of cochlear sensory cells. Dev. Biol. 280, 281–294. Mire-Thibodeaux, P., Watson, G.M., 1994. Morphodynamic hair bundles arising from sensory cell/supporting cell complexes frequency-tune nematocyst discharge in sea anemones. J. Exp. Zool. 268, 282–292. Müller, U., 2008. Cadherins and mechanotransduction by hair cells. Curr. Opin. Cell Biol. 20, 557–566. Peteya, D.J., 1975. The ciliary-cone sensory cell of anemones and cerianthids. Tissue Cell 7, 243–252. Pickles, J.O., Osborne, M.P., Comis, S.D., 1987. Vulnerability of tip links between stereocilia to acoustic trauma in the Guinea pig. Hear. Res. 25, 173–183. Rzadzinska, A.K., Derr, A., Kachar, B., Noben-Trauth, K., 2005. Sustained cadherin 23 expression in young and adult cochlea of normal and hearing-impaired mice. Hear. Res. 208, 114–121. Shang, X., Marchioni, F., Sipes, N., Evelyn, C.R., Jerabek-Willemsen, M., Duhr, S., Seibel, W., Wortman, M., Zheng, Y., 2012. Rational design of small molecule inhibitors targeting RhoA subfamily rho GTPases. Chem. Biol. 19, 699–710. Siemens, J., Lillo, C., Dumont, R.A., Reynolds, A., Williams, D.S., Gillespie, P.G., Müller, U., 2004. Cadherin 23 is a component of the tip link in hair-cell stereocilia. Nature 428, 950–955. Watson, G.M., Hessinger, D.A., 1989. Cnidocyte mechanoreceptors are tuned to the movements of swimming prey by chemoreceptors. Science 243, 1589–1591. Watson, G.M., Hessinger, D.A., 1994. Antagonistic frequency tuning of hair bundles by different chemoreceptors regulates nematocyst discharge. J. Exp. Biol. 187, 57–73. Watson, G.M., Mire, P., 2002. Reorganization of actin during repair of hair bundle mechanoreceptors. J. Neurocytol. 30, 895–906. Watson, G.M., Roberts, J., 1995. Chemoreceptor-mediated polymerization and depolymerization of actin in hair bundles of sea anemones. Cell Motil. Cytoskeleton 30, 208–220. Watson, G.M., Mire, P., Hudson, R.R., 1997. Hair bundles of sea anemones as a model system for vertebrate hair bundles. Hear. Res. 107, 53–66. Watson, G.M., Mire, P., Hudson, R.R., 1998. Repair of hair bundles in sea anemones by secreted proteins. Hear. Res. 115, 119–128. Watson, G.M., Venable, S., Hudson, R.R., Repass, J.J., 1999. ATP enhances repair of hair bundles in sea anemones. Hear. Res. 136, 1–12. Watson, G.M., Pham, L., Graugnard, E.M., Mire, P., 2008. Cadherin 23-like polypeptide in hair bundle mechanoreceptors of sea anemones. J. Comp. Physiol. A. 194, 811–820. Watson, G.M., Mire, P., Kinler, K.M., 2009. Mechanosensitivity in the model sea anemone Nematostella vectensis. Mar. Biol. 156, 2129–2137.
Declaration of Competing Interest None. Acknowledgements We recognize the University of Louisiana at Lafayette Graduate Student Organization for their contribution to funding this research. This organization made no other contributions to the completion of either the research or the manuscript. References Allaire, K.M., Watson, G.M., 2013. Rho participates in chemoreceptor-induced changes in morphology to hair bundle mechanoreceptors of the sea anemone, Nematostella vectensis. Comp. Biochem. Physiol. A 165, 139–148. Assad, J.A., Shepherd, G.M.G., Corey, D.P., 1991. Tip-link integrity and mechanical transduction in vertebrate hair cells. Neuron 7, 985–994. Barbacid, M., 1987. ras genes. Annu. Rev. Biochem. 56, 779–827. Benink, H.A., Bement, W.M., 2005. Concentric zones of active RhoA and Cdc42 around single cell wounds. J. Cell Biol. 168, 429–439. Burakov, A., Nadezhdina, E., Slepchenko, B., Rodionov, V., 2003. Centrosome positioning in interphase cells. J. Cell Biol. 162, 963–969. Etienne-Manneville, S., Hall, A., 2002. Rho GTPases in cell biology. Nature 420, 629–635. Fettiplace, R., Hackney, C.M., 2006. The sensory and motor roles of auditory hair cells. Nat. Rev. Neurosci. 7, 19–29. Hudspeth, A.J., 1985. The cellular basis of hearing: the biophysics of hair cells. Science 230, 745–752. Husbands, J.M., Steinberg, S.A., Kurian, R., Saunders, J.C., 1999. Tip link integrity on chick tall hair cell stereocilia following intense sound exposure. Hear. Res. 135, 135–145. Kazmierczak, P., Sakaguchi, H., Tokita, J., Wilson-Kubalek, E.M., Milligan, R.A., Müller, U., Kachar, B., 2007. Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells. Nature 449, 87–92. Kurian, R., Krupp, N.L., Saunders, J.C., 2003. Tip link loss and recovery on chick short hair cells following intense exposure to sound. Hear. Res. 181, 40–50. Lagziel, A., Ahmed, Z.M., Schultz, J.M., Morell, R.J., Belyantseva, I.A., Friedman, T.B., 2005. Spatiotemporal pattern and isoforms of cadherin 23 in wild type and Waltzer mice during inner ear hair cell development. Dev. Biol. 280, 295–306. Lelli, A., Kazmierczak, P., Kawashima, Y., Müller, U., Holt, J.R., 2010. Development and
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Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Rho-family G-proteins are required for the recovery of traumatized hair bundle mechanoreceptors in the sea anemone, Nematostella vectensis Shelcie S. Menard-Harvey
T
⁎,1
, Glen M. Watson
Department of Biology, University of Louisiana at Lafayette, 410 E. St. Mary Blvd., Lafayette, LA 70504, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Actin Calcium-free Rho inhibitor I Rhosin Stereocilia
Immersing anemones in calcium-free seawater disorganizes hair bundle mechanoreceptors on tentacles of sea anemones while causing a loss of vibration sensitivity. Remarkably, anemone hair bundles recover after being returned to calcium-containing seawater. Reorganization of actin in stereocilia likely follows during the recovery of normal morphology of hair bundles after such immersion. Previous studies have reported that Rho G-proteins are located in the stereocilia of hair bundles in sea anemones where they participate in polymerizing actin in stereocilia upon activation of specific chemoreceptors. We here find that immersing anemones in calcium-free seawater significantly reduces the abundance of hair bundles. A partial recovery of abundance of hair bundles occurs within 3 h post-immersion, but a full recovery of abundance does not occur even 6 h after specimens are returned to calcium-containing seawater. Anemones recovering from immersion in calcium-free seawater feature hair bundles that are significantly wider at their tips than in controls. The hair bundles subsequently narrow at their tips, becoming comparable to those of untreated controls within 6 h. Stereocilia of hair bundles are significantly longer in experimental animals than in controls at 2 h of recovery before shortening to lengths comparable to untreated controls at 6 h. In the presence of Rho inhibitors, the recovery in abundance of hair bundles through 6 h is delayed or inhibited. Likewise, in the presence of Rho inhibitors, stereocilia fail to significantly elongate within 2 h of recovery. These data suggest that Rho G-proteins participate in the normal recovery of abundance and recovery of normal morphology of experimentally damaged hair bundle mechanoreceptors.
1. Introduction Sea anemones employ hair bundle mechanoreceptors located on their tentacles to detect the rhythmic movements of nearby swimming prey (Mire-Thibodeaux and Watson, 1994; Watson and Hessinger, 1989). As in vertebrate animals possessing hair cells, anemone hair bundle mechanoreceptors are composed of dozens to hundreds of actinbased stereocilia held together by specific cadherin-based linkages. After immersion of the sensory epithelium in calcium-depleted buffers, the stereocilia of hair bundles splay apart rendering the hair bundle non-functional (Assad et al., 1991; Watson et al., 1998; Watson et al., 1997). Evidently, the cadherins forming extracellular linkages interconnecting stereocilia require calcium ions in order to maintain their structural integrity (Kazmierczak et al., 2007). After such immersion, and upon returning the sensory epithelium to calcium-containing buffers, at least some of the hair cells on anemone tentacles spontaneously
recover normal structure and function of their hair bundles over a timecourse that increases with the duration of immersion in the calciumdepleted buffer (Watson et al., 1998). As compared to hair bundles in other animals, hair bundles of sea anemones are remarkable for their ability to recover from severe damage (for these experiments caused by a 1 h immersion in calcium-free seawater; Watson et al., 1998). Presumably, under natural conditions, anemone hair bundles are frequently damaged by the vigorous movements of captured prey struggling to escape. In other animals, overstimulation of hair bundles shears linkages between stereocilia to disrupt the normal structure and function of the hair bundles (Husbands et al., 1999; Kurian et al., 2003; Pickles et al., 1987). Stereocilia are evaginations of the plasma membrane filled with tightly packed, parallel arrays of actin filaments. Stereocilia are considered to be rigid structures (reviewed: Hudspeth, 1985). Consequently, the means by which the hair cells restore order to the splayed
Abbreviations: KSW, high‑potassium seawater; PBS, phosphate-buffered saline; BSA, bovine serum albumin ⁎ Corresponding author. E-mail addresses: [email protected] (S.S. Menard-Harvey), [email protected] (G.M. Watson). 1 Present Address: Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC, 28223, USA https://doi.org/10.1016/j.cbpa.2019.110637 Received 5 September 2019; Received in revised form 26 November 2019; Accepted 13 December 2019 Available online 20 December 2019 1095-6433/ © 2019 Elsevier Inc. All rights reserved.
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Invitrogen, Grand Island, NY, USA. Glutaraldehyde was acquired from Sigma-Aldrich, St. Louis, MO, USA. Paraformaldehyde was acquired from Electron Microscopy Sciences, Hatfield, PA, USA. ProLong Gold Antifade Mountant was acquired from ThermoFisher Scientific, Waltham, MA, USA. Statistica software was acquired from Statsoft, Tulsa, OK, USA. ImageJ software was acquired from National Institutes of Health, Bethesda, MD, USA. MaximDL software was acquired from Diffraction Limited, Ottawa, ON, Canada. Microcal Origin 5.0 was acquired from Microcal Software, Northampton, MA, USA.
hair bundle is of interest. Lost or damaged linkages would have to be replaced or otherwise re-established. In addition, the stereocilia would have to be repositioned. After a prolonged exposure (1 h) to calciumdepleted seawater, a spontaneous recovery of hair bundles in anemones requires a minimum of 4 h to be completed. Such a recovery involves the secretion of so called ‘repair proteins’ as well as secretion of ATP. Exogenously supplied repair proteins and ATP speed the recovery of structure and function of experimentally damaged hair bundles to a matter of a few minutes (Watson et al., 1999; Watson et al., 1998). A 1 h immersion in calcium-free seawater extensively disorganizes hair bundle mechanoreceptors on the tentacles of sea anemones such that the conical form of the hair bundle is unrecognizable, suggesting a loss of linkages although the stereocilia persist (Watson et al., 1998). Within a few minutes after immersion in calcium-free seawater, levels of Factin in stereocilia significantly decrease. However, in the presence of exogenously supplied repair proteins, levels of F-actin in stereocilia increase to become comparable to those in stereocilia of untreated controls at approximately 40 mins into recovery (Watson and Mire, 2002). Thus, the events of repair include extracellular molecules (repair proteins and ATP) that somehow help to re-establish cadherin linkages between stereocilia and at the same time may help to coordinate events occurring in the cytoplasm of the damaged hair cells, including a reorganization of actin in stereocilia. In a wide variety of eukaryotic cells, Rho proteins participate in signaling cascades that culminate in changes to the actin cytoskeleton (reviewed: Etienne-Manneville and Hall, 2002). Rho proteins are small monomeric proteins that belong to the Ras family of G-proteins (reviewed: Barbacid, 1987). Previously, Rho has been implicated in the normal changes to the actin cytoskeleton that occur in stereocilia of hair bundles in response to chemoreceptor activation in hair cells of sea anemones. In anemones, hair bundle mechanoreceptors normally lengthen in response to exogenously added N-acetylated sugars including N-acetylneuraminic acid (NANA), and then shorten in response to exogenously added proline (Mire-Thibodeaux and Watson, 1994; Watson and Hessinger, 1994). Both chemoreceptor-induced responses involve a reorganization of the actin cytoskeleton in stereocilia: actin polymerization in response to NANA and actin depolymerization in response to proline (Watson and Roberts, 1995). When pharmacological activators of Rho are experimentally introduced, hair bundles elongate in a comparable fashion to the elongation normally induced by exposure to NANA but fail to shorten in response to proline (Allaire and Watson, 2013). However, when pharmacological inhibitors of Rho are experimentally introduced, hair bundles fail to elongate in response to NANA but shorten in response to proline (Allaire and Watson, 2013). Rho homologs have been identified in the genome of the sea anemone Nematostella vectensis and localized by immunocytochemistry to the stereocilia of hair bundles (Allaire and Watson, 2013). Clearly, Rho G-proteins are involved in the reorganization of the actin cytoskeleton within the stereocilia of hair bundle mechanoreceptors during elongation of the hair bundle in response to chemoreceptor activation, but it is unclear to what extent Rho G-proteins might be involved in the reorganization of the actin cytoskeleton during the repair of experimentally-damaged hair bundle mechanoreceptors. This study aims to determine whether or not repair of experimentallydamaged hair bundle mechanoreceptors occurs in the absence of Rho activity. In the event that Rho activity is required for repair to occur, this study aims to determine whether a critical period occurs during which Rho G-proteins must be active in order to facilitate repair.
2.2. Animal maintenance Stock cultures of Nematostella vectensis were established from approximately 50 individuals locally collected by personnel of the Marine Biological Laboratory, Woods Hole, MA. Specimens of Nematostella vectensis were maintained in Pyrex dishes containing natural seawater diluted to 16 ppt (Allaire and Watson, 2013). The animals were maintained at 22 °C, fed brine shrimp nauplii twice weekly and cleaned after feeding. Experiments were scheduled for a minimum of 24 to 48 h after feeding (Allaire and Watson, 2013). 2.3. Effects of exposure to calcium-free high‑potassium seawater on the abundance of hair bundles at the tips of tentacles of sea anemones Sea anemones are anesthetized in formulations of artificial seawater that contain elevated levels of potassium ions and low levels of sodium ions (Watson et al., 2009). Such anesthetics are referred to as KSW. For these experiments, a combination of KSW and low levels of calcium ions was employed in order to damage hair bundles in anesthetized anemones. It was important to anesthetize the anemones so that tentacles could be excised from the anemones and fixed for microscopic examination without contracting. Sea anemones were immersed in calcium-free high‑potassium seawater (calcium-free KSW: 166 mM NaCl; 62 mM KCl; 26 mM MgCl2; 13 mM MgSO4; 4 mM EGTA, a widely-used calcium chelator; 1 mM NaHCO3) for 1 h, and hair bundles were counted at specific time points after the anemones were returned to KSW (containing calcium ions) and subsequently fixed to determine the abundance of sensory/supporting cell hair bundles. Each replicate experiment required eight animals: one untreated control and seven experimental animals. The untreated control was immersed in KSW containing normal levels of calcium ions (166.5 mM NaCl; 50 mM KCl; 13 mM MgSO4; 12 mM MgCl2; 6 mM CaCl2; 1.15 mM NaHCO3) for 1 h before having the oral disc excised and fixed in 4% paraformaldehyde and 0.025% glutaraldehyde in Millonig's buffer (as per the methods of Watson et al., 2009). The experimental animals were immersed in calcium-free KSW for a 5 min wash before being immersed in calciumfree KSW for a full hour. The 5 min wash was intended to ensure that the animals would exchange the natural seawater inside the coelenteron for the calcium-free KSW in the dish. After a 1 h immersion in calcium-free KSW, the animals were washed for 5mins in KSW, and then allowed to recover in KSW for up to 6 h. Exposure to KSW alone over 6 h does not significantly affect the abundance of hair bundles on the tentacles of sea anemones (Menard and Watson, 2017). Starting immediately after the 5 min KSW wash, the oral disc of one experimental animal (T0) was excised and fixed for one hour. At each subsequent hour (T1–T6) of recovery, the oral disc of another animal was excised and fixed for 1 h. After fixation, oral discs were washed in phosphate-buffered saline (PBS) for 5mins. Tentacles were then excised from the oral discs and prepared as wet mounts for observation using phase-contrast microscopy (40× objective; n.a. = 0.65). One tentacle was excised from each animal. Hair bundles appearing to possess a conical shape (stereocilia surrounding and leaning toward the central kinocilium) while viewed in profile were counted in three fields of view (equivalent to 0.69 mm tentacle length) on one side of the tentacle, starting at the tip of the tentacle. Hair bundles having disorganized stereocilia (splayed away from the central kinocilium and each other)
2. Materials and methods 2.1. Materials Rho Inhibitor I was acquired from Cytoskeleton, Inc., Denver, CO, USA. Rho inhibitor, Rhosin, was acquired from Millipore Sigma, Burlington, MA, USA. Rhodamine phalloidin was acquired from 2
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were not counted. This approach is intended to determine the time required for the abundance of intact hair bundles to return to untreated control levels. Previous studies performed to determine the time course of recovery of morphology and function on hair bundles damaged by exposure to calcium-depleted seawater did not consider the abundance of hair bundles (Watson et al., 1998; Watson and Mire, 2002). Microscopy was performed using a LOMO microscope (Labaroscope, LOMO America, Prospect Heights, IL, USA). Data were plotted as the mean abundance of hair bundles per 100 μm tentacle length ± SEM (n = 9 tentacles per time point). Each tentacle is excised from a different animal.
microscopy (100× objective; n.a. = 1.30). When viewing the tentacle in profile, a total of 10 hair bundles was measured, starting at the tip of the tentacle. Only certain hair bundles were measured. The entire hair bundle (from base to tip) was required to be in focus along its length to be selected for measurement. Measurements were obtained from digital photomicrographs using ImageJ. Data were pooled for the 10 hair bundles for each specimen to give a single value per specimen (tentacle). Three tentacles (each from a different animal) were obtained for each treatment. Data were plotted as mean length, mean base width, or mean tip width of hair bundles ± SEM (n = 3 tentacles per time point). Each tentacle is excised from a different animal.
2.4. The effects of Rho inhibition on recovery of abundance of hair bundles from exposure to calcium-free KSW
2.6. Lengths of rhodamine phalloidin-stained stereocilia of hair bundles following exposure to calcium-free KSW and exposure to Rhosin
Sea anemones were immersed in a Rho inhibitor during their recovery from exposure to calcium-free KSW to test effects of Rho inhibitor on the recovery of abundance of hair bundles. Each replicate experiment involved eight animals: one untreated control and seven experimental animals. The experimental protocol was the same as in Section 2.3 above, except for exposure to Rho inhibitor I or Rho inhibitor (Rhosin) beginning at the start of recovery (T0), beginning one hour into recovery (T1), or beginning two hours into recovery (T2) for Rhosin only. Rho inhibitors were reconstituted according to manufacturer's instructions. The animals were treated with Rho inhibitor I at a concentration of 1 μg/ml, as per manufacturer's instructions. In the case of Rhosin, the animals were treated with 100 μM Rhosin, as per manufacturer's instructions. The reconstituted Rho inhibitors were added directly to the KSW (to the desired final concentration of the designated inhibitor) in the dish of recovering animals at specific timepoints during recovery, allowing each animal to begin the exposure period at the same time. Once added, the Rho inhibitors were present for the remainder of the recovery period concluding at 6 h of recovery. Rho inhibitors were added at different time points to determine when during the recovery period is Rho activity critical for the normal recovery of the abundance of hair bundle mechanoreceptors. Rho inhibitor I was used to inactivate the Rho-family proteins RhoA, RhoB, and RhoC via the protein C3 transferase (Benink and Bement, 2005; Burakov et al., 2003). Rhosin was used to inactivate Rho-family proteins RhoA, RhoB, and RhoC via interference with the GEF (i.e., guanine nucleotide exchange factor) binding domain (Shang et al., 2012). The incorporation of two mechanistically different Rho inhibitors into the experimental design was intended to control for the possibility of off-effects of the Rho inhibitors on hair bundle structure. Tentacles were excised from the oral discs, fixed and processed as was described in Section 2.3 and then prepared as wet mounts for viewing under phase-contrast microscopy (40× objective; n.a. = 0.65). Hair bundles appearing to possess a conical shape while viewed in profile on one side of the tentacle were counted in three fields of view, starting at the tip of the tentacle. Data were plotted as the mean abundance of hair bundles per 100 μm tentacle length ± SEM (n = 4 tentacles per time point for Rho inhibitor I [T0]; n = 3 tentacles per time point for Rho inhibitor I [T1]; n = 6 tentacles per time point for all Rhosin experiments). Each tentacle is excised from a different animal.
Hair bundles of sea anemones consist of several small-diameter stereocilia that converge on 5–10 large-diameter stereocilia located at the center of the hair bundle (Peteya, 1975; Watson et al., 1997). Largediameter stereocilia are longer than are small-diameter stereocilia. The lengths of large-diameter stereocilia of hair bundles were measured from micrographs taken of rhodamine-phalloidin stained specimens that were fixed and processed at specific intervals through 6 h of recovery from exposure to calcium-free KSW. For these experiments the ‘control’ animals were immersed in calcium-free KSW and then allowed to spontaneously recover in KSW alone. The ‘experimental’ animals were immersed in calcium-free KSW and then allowed to recover in the presence of 100 μM Rhosin in KSW. Each replicate experiment required eight animals: one untreated specimen and seven animals that were immersed in calcium-free KSW. The experimental protocol was similar to that explained in Section 2.3. After fixation of the oral discs, the oral discs were transferred to a solution of rhodamine phalloidin and 3% bovine serum albumin (BSA) in PBS. The addition of BSA to the staining solution is intended to prevent non-specific staining. Staining in rhodamine-phalloidin continued for 8 h (overnight) with specimens rocking at 4 °C. The final dilution of the rhodamine phalloidin conjugate was prepared according to manufacturer's instructions. On the following day, the oral discs were washed in PBS. Next, one randomlyselected tentacle was excised from each oral disc and mounted in ProLong Gold overnight at 4 °C. Slides were imaged on the next day via epifluorescence microscopy (100× objective; n.a. = 1.30). Hair bundles were imaged in profile. The lengths of stereocilia were measured from digital photomicrographs of ten hair bundles per specimen using Image J. To measure the length of stereocilia, the ImageJ line tool was used to draw a straight line from the bottom of the stereocilium to the tip of the stereocilium of the hair bundle. The data were plotted as the mean length in microns of stereocilia ± SEM (n = 3 tentacles per time point). Each tentacle is excised from a different animal. 3. Statistics Statistica software (Tulsa, OK, USA) was used to perform a one-way ANOVA and post-hoc Fisher LSD analysis to compare specific groups (i.e., treatments or timepoints). Data were determined to be statistically significant at a p-value of 0.05 or less. Microcal Origin software (Northampton, MA) was used to create graphs.
2.5. Dimensions of hair bundles on tentacles of sea anemones following immersion in calcium-free KSW
4. Results 4.1. Effects of exposure to calcium-free KSW on the abundance of hair bundles at the tips of the tentacles of sea anemones
The mean length, mean base width, and mean tip width were calculated from measurements of hair bundles taken from digital photomicrographs obtained along the length of fixed tentacles. The specimens were immersed in calcium-free KSW for 1 h at room temperature as detailed in Section 2.3. Each replicate experiment required eight animals: one untreated control and seven experimental animals. A fixed tentacle from each animal was randomly selected, excised from the oral disc, and prepared as a wet mount for imaging using oblique
After a 1 h immersion in calcium-free KSW, the mean abundance of hair bundles at the tips of the tentacles of sea anemones significantly decreased (by approximately 60%) compared to untreated controls (p = .000002; Fig. 1). The mean abundance of hair bundles partially recovered 3 h after the specimens were returned to calcium containing 3
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One curve for the experiments testing Rho inhibitors beginning at T1 was unusual (i.e., different from that shown in Figs. 3a, b) in that the abundance of hair bundles did not significantly decrease relative to untreated controls until T1 (p = .02; Fig. 4a). Upon adding Rho inhibitor I at T1, the mean abundance of hair bundles became statistically comparable to both the untreated control (p = .16) and T1 (p = .24) at 2 h, then at 3 h became statistically comparable to only T1 (p = .87) through 6 h (p = .39) (Fig. 4a). Following immersion in calcium-free KSW, the mean abundance of hair bundles significantly decreased compared to untreated controls immediately on the second curve (p = .007; Fig. 4b). Following the addition of Rhosin at T1, the mean abundance of hair bundles remained comparable to that for T0 through 2 h (p = .14), and then significantly decreased compared to T0 through 4 h (p = .003), before becoming comparable to T0 at 5 h (p = .44), and finally significantly decreasing again compared to T0 at 6 h (p = .002; Fig. 4b). For this set of experiments, the Rho inhibitor was added at T2. Following immersion of anemones in calcium-free KSW, the mean abundance of hair bundles significantly decreased compared to untreated controls at T0 (p = .01; Fig. 5). Following the addition of Rhosin at T2, the mean abundance of hair bundles remained comparable to that for T0 through 6 h (p = .29; Fig. 5).
Fig. 1. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to calcium-free KSW. The abundance of hair bundles was determined in untreated controls and in experimental animals after 1 h of exposure to calcium-free KSW (experimentals only) and through 6 h of recovery in KSW by phase-contrast microscopy of fixed specimens imaged in profile at a single focal plane. Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. The mean abundance was calculated from nine animals per data point. UC = untreated control (n = 9); T0 = immediately following trauma (n = 9); T1 = 1 h recovery; (n = 9); T2 = 2 h recovery (n = 9); T3 = 3 h recovery (n = 9); T4 = 4 h recovery (n = 9); T5 = 5 h recovery (n = 9); T6 = 6 h recovery (n = 9). The letters “a, b and c” denote groups that are significantly different from each other (p ≤ .05).
4.3. Morphology of hair bundles on tentacles of sea anemones during their recovery after immersion in calcium-free KSW Tip width, bundle length, and base width were measured from digital photomicrographs of hair bundles after immersion in calcium-free KSW and through 6 h of recovery. Representative images of hair bundles are shown for untreated controls (Fig. 6a), experimental animals immediately after exposure to calcium-free KSW (Fig. 6b), experimental animals 3 h into recovery (Fig. 6c), and experimental animals 6 h into recovery (Fig. 6d). The mean tip width of hair bundles significantly increased 2 h into recovery (p = .04) and remained wider than controls through 4 h of recovery (p = .03) before returning to widths comparable to untreated controls thereafter (Fig. 7a). With respect to mean bundle length and mean base width, these dimensions remained comparable to untreated controls throughout the 6 h recovery period (Fig. 7b and c, respectively).
KSW, as the mean abundance 3 h into recovery (T3) was significantly higher than at T0 (p = .004) but, nevertheless, remained significantly lower than the mean abundance of untreated controls (p = .03; Fig. 1). The mean abundance of hair bundles remained significantly different from both untreated controls and T0 animals through the remainder of the 6 h recovery period. After the initial knockdown, the abundance of hair bundles remained fairly steady through 2 h into the recovery period, rose significantly by hour 3, and then seemed to plateau through the remainder of the 6 h recovery period. Thus, the shape of the recovery curve approximated an S-shaped curve.
4.4. Lengths of rhodamine phalloidin-stained stereocilia of hair bundles following exposure to calcium-free KSW and exposure to Rhosin
4.2. The effects of Rho inhibitors on the recovery of abundance of hair bundles after exposure to calcium-free KSW
Rhodamine-phalloidin, a F-actin stain, was used to visualize the actin cytoskeleton of stereocilia of hair bundles in controls and in experimental animals after immersion in calcium-free KSW and through 6 h of recovery. Representative images of hair bundles are shown for untreated controls (Fig. 8a), experimental animals immediately following exposure to calcium-free KSW (Fig. 8b), experimental animals 3 h into recovery (Fig. 8c), and experimental animals 6 h into recovery (Fig. 8d). Following exposure to calcium-free KSW, the mean lengths of the stereocilia significantly increased relative to untreated controls at 2 through 3 h of recovery (p = .01 and p = .02, respectively) before shortening to lengths comparable to untreated controls thereafter (Fig. 9). Representative images of hair bundles are shown for untreated controls (Fig. 10a), experimental animals immediately following exposure to calcium-free KSW (Fig. 10b), experimental animals 3 h into recovery (Fig. 10c), and experimental animals 6 h into recovery (Fig. 10d). After exposure to Rhosin, the lengths of the stereocilia were comparable to untreated controls until they significantly decreased in length 5 h into recovery (p = .02) before returning to lengths comparable to untreated controls 6 h into recovery (Fig. 11).
Two Rho inhibitors (Rho Inhibitor I and Rhosin) were tested in anemones after immersing anemones in calcium-free KSW for 1 h. Experiments were performed such that inhibitors were added immediately after trauma (T0), 1 h after trauma (T1), or 2 h after trauma (T2) in order to attempt to determine whether Rho activity was needed throughout the recovery period or only during specific intervals during the recovery period. As a control, Rho inhibitor I alone had no significant adverse effects on the mean abundance of hair bundles on the tentacles of sea anemones through six hours of exposure (p = .71; Fig. 2a). Likewise, Rhosin alone had no adverse effects on the mean abundance of hair bundles on the tentacles of sea anemones through 6 h of exposure (p = .96; Fig. 2b). The mean abundance of hair bundles decreased significantly at T0 compared to untreated controls following exposure to calcium-free KSW (p = .003; Fig. 3a; and p = .000049; Fig. 3b). When administered immediately after the immersion in calcium-free KSW, both Rho inhibitor I and Rhosin prevented the recovery normally observed 3 h after trauma (T3) such that the abundance of hair bundles remained comparable to that for T0 through 6 h (p = .31–0.58; Fig. 3). 4
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Fig. 2. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to Rho inhibitors. The abundance of hair bundles was determined through 6 h of exposure to either Rho Inhibitor I or Rhosin by phase-contrast microscopy of fixed specimens imaged in profile. (a) Exposure to Rho Inhibitor I (n = 3 tentacles per time point). (b) Exposure to Rhosin (n = 6 tentacles per time point). Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. UC = untreated control; T0 = immediately following trauma; T1 = 1 h exposure; T2 = 2 h exposure; T3 = 3 h exposure; T4 = 4 h exposure; T5 = 5 h exposure; T6 = 6 h exposure.
5. Discussion
terminates other linkages between the stereocilia of the hair bundle (reviewed: Fettiplace and Hackney, 2006), as cadherin-23 (and likely protocadherin-15) is present in the kinociliary linkages and transient lateral linkages of the developing hair cells of mammals (Lagziel et al., 2005; Michel et al., 2005; reviewed: Müller, 2008; Rzadzinska et al., 2005; Siemens et al., 2004;). Similar consequences of exposure to calcium-free seawater are expected in the hair bundles of sea anemones, as sea anemones also have lateral linkages and a homolog of cadherin-23 in tip links of their hair bundles (Watson et al., 2008; Watson et al., 1997). In line with such expectations, a 1 h exposure to calcium-free seawater causes a majority of the hair bundles of sea anemones to become unrecognizable as hair bundles. This disorganization is such that stereocilia persist (Watson and Mire, 2002). If the specimens are
Although it has been known for some time that prolonged immersion in calcium-free seawater (i.e., 1 h) severely disorganizes hair bundle mechanoreceptors located on tentacles of sea anemones (Watson et al., 1998), nothing was known about the effects of such trauma on the abundance of hair bundles on the tentacle epithelium. In vertebrate animals, exposure to calcium-free buffer causes a disorganization of the hair bundle because the calcium-dependent proteins, cadherin-23 and protocadherin-15, that make up the tip links between stereocilia no longer maintain tip link structure (and as a result, mechanotransduction) in the absence of extracellular calcium (Assad et al., 1991; Lelli et al., 2010). Removal of calcium also
Fig. 3. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to calcium-free KSW followed by exposure to Rho inhibitors immediately after trauma. The abundance of hair bundles was determined after 1 h of exposure to calcium-free KSW followed immediately by administration of either Rho Inhibitor I or Rhosin and through 6 h of recovery by phase-contrast microscopy of fixed specimens imaged in profile. (a) Recovery after exposure to Rho Inhibitor I (n = 4 tentacles per time point). (b) Recovery after exposure to Rhosin (n = 6 tentacles per time point). Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
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Fig. 4. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to calcium-free KSW followed by exposure to Rho inhibitors beginning at 1 h after calcium-free KSW. The abundance of hair bundles was determined after 1 h of exposure to calcium-free KSW followed by administration of either Rho Inhibitor I or Rhosin (1 h into recovery) and through 6 h of recovery by phase-contrast microscopy of fixed specimens imaged in profile. (a) Recovery after exposure to Rho Inhibitor I (n = 3 tentacles per time point). (b) Recovery after exposure to Rhosin (n = 6 tentacles per time point). Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. The letters “a, b and c” denote groups that are significantly different from each other (p ≤ .05). The letters “ab” denote groups that are statistically comparable to groups labeled “a” and groups labeled “b”.
Fig. 5. Mean abundance of hair bundles at the tips of tentacles of sea anemones after exposure to calcium-free KSW followed by exposure to Rhosin beginning at 2 h after trauma. The abundance of hair bundles was determined after 1 h of exposure to calcium-free KSW followed by administration of Rhosin (2 h into recovery) and through 6 h of recovery by phase-contrast microscopy of fixed specimens imaged in profile. Data are presented as the mean abundance of hair bundles per 100 μm tentacle length ± SEM. The mean abundance was calculated from six animals per data point. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
Fig. 6. Hair bundles after exposure to calcium-free KSW imaged by oblique microscopy. Representative hair bundles are shown as follows: (a) untreated controls; (b) immediately following calcium-free trauma; (c) 3 h into recovery; (d) 6 h into recovery. hb = hair bundle; bl = bundle length; tw = tip width; bw = base width. Scale bar = 10 μm.
abundance of hair bundles occurring from 3 to 6 h of recovery. We stress that after 6 h of recovery, the abundance of hair bundles on the tentacle epithelium remains significantly smaller than in untreated controls at approximately 2/3 of control levels. Thus, there may be at least two phases of hair bundle recovery with one occurring at approximately 3 h of recovery and a second, slower recovery occurring sometime after 6 h. However, within 24 h after exposure to calciumdepleted seawater, the abundance of hair bundles recovers to control levels (data not shown). Such a significant, protracted reduction in
returned to calcium-containing seawater, at least some of the hair bundles on the tentacle epithelium recover their normal structure and function in 4 h (Watson et al., 1998). Here, we find that the abundance of hair bundles at the tips of tentacles of sea anemones is significantly decreased after a 1 h immersion in calcium-free KSW. However, we find that the recovery of abundance of hair bundles follows a S-shaped function with a dramatic increase in abundance occurring at approximately 3 h into recovery, but with only very modest increases in the 6
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Fig. 7. Mean dimensions of hair bundles after exposure to calcium-free KSW. The mean tip width, bundle length, and base width of hair bundles were determined after 1 h of exposure to calcium-free KSW and through 6 h of recovery by oblique microscopy of fixed specimens. Images taken via oblique microscopy were analyzed using ImageJ. (a) Mean tip widths of hair bundles. Data are presented as mean tip width in microns ± SEM. (b) Mean bundle lengths of hair bundles. Data are presented as mean bundle length in microns ± SEM. (c) Mean base widths of hair bundles. Data are presented as mean base width in microns ± SEM. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. The same hair bundles were measured for all three dimensions. Mean dimensions were calculated from three animals per data point. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
Fig. 9. Mean lengths of stereocilia after exposure to calcium-free KSW. The mean lengths of stereocilia were determined after 1 h of exposure to calciumfree KSW and through 6 h of recovery by epifluorescence microscopy of fixed specimens. Lengths of stereocilia were measured using ImageJ. Data are presented as the mean length of stereocilia in microns ± SEM. The mean length of stereocilia was calculated from three animals per data point. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
Fig. 8. Phalloidin cytochemistry of hair bundles after exposure to calcium-free KSW. Representative hair bundles are shown as follows: (a) untreated controls; (b) immediately following immersion in calcium-free KSW; (c) 3 h into recovery; (d) 6 h into recovery. hb = hair bundle; sc = stereocilia; ca = cytoplasmic actin. Scale bar = 10 μm.
remains comparable to the abundance of hair bundles at T0 or significantly decreases compared to the abundance of hair bundles at T0. The exception lies in Fig. 4a, where the effects (decreased abundance of hair bundles) of calcium depletion were somehow delayed by 1 h. As a result, the addition of Rho Inhibitor I at T1 is likened to adding in the inhibitor at T0 (immediately after decrease in abundance). Even with the delayed decrease in abundance of hair bundles, the overall result of Rho inhibition is consistent with that of the other experiments: no significant recovery without Rho activity. These results indicate that
abundance of hair bundles might be expected to significantly impair the sea anemone's ability to capture prey. Further experimentation would be necessary to test such an effect on prey capture in animals allowed to recover from extensive trauma for 6 h. Rho inhibitors were tested to investigate whether Rho is involved in the recovery of structural organization of hair bundles normally occurring 3 h after exposure to calcium-free KSW. Regardless of the onset of Rho inhibition between T0 and T2, the abundance of hair bundles 7
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fully blocked. Here it is important to recall that the Rho inhibitors evidently do not destabilize existing hair bundles (Fig. 2). Thus, Rho likely participates in reorganizing the actin cytoskeleton throughout the early hours of recovery, certainly during the most dramatic phase of recovery of hair bundle abundance occurring between hours 2 and 3 of recovery. Next, the tip widths of the bundles significantly increase compared to untreated controls at hours 2 through 4 of recovery. The widening at the tips of the hair bundles coincides with the significant lengthening of the stereocilia that occurs at hours 2 and 3 of recovery compared to untreated controls. These results indicate that the length of actin bundles is dynamically regulated at a time during which the tips of hair bundles are wide. During repair from exposure to calcium-free KSW, the tip links and other linkages interconnecting stereocilia must be replaced. It is likely that as the linkages are being replaced, Rho G-proteins are actively reorganizing the actin cytoskeletons of the stereocilia of the hair bundles. The stereocilia return to control lengths within 4 h of recovery, just before the tips of the hair bundles return to control widths at 5 h of recovery. Perhaps regulatory processes allow the Rho G-proteins to normalize the length of stereocilia as the linkages are installed. In the presence of Rhosin, elongation of the stereocilia does not occur during recovery. In fact, exposure to calcium-free KSW followed by exposure to Rhosin results in stereocilia significantly shortening 5 h into recovery before becoming comparable to untreated controls again 6 h into recovery. The failure of stereocilia to elongate and the subsequent shortening of the stereocilia in the presence of Rhosin are consistent with the possibility that Rho G-proteins are actively involved in the reorganization of the actin cytoskeleton in stereocilia during the recovery of hair bundles from exposure to calcium-free seawater.
Fig. 10. Phalloidin cytochemistry of hair bundles after exposure to calcium-free KSW followed by exposure to Rhosin. Representative hair bundles are shown as follows: (a) untreated controls; (b) immediately following trauma; (c) 3 h into recovery; (d) 6 h into recovery. hb = hair bundle; sc = stereocilia; ca = cytoplasmic actin. Scale bar = 10 μm.
6. Conclusions Previous research has established that exposure to calcium-depleted seawater disorganizes the stereocilia of hair bundle mechanoreceptors in sea anemones, and damaged hair bundles regain typical morphology and function within 4 h of recovery (Watson et al., 1998). It has also been found that F-actin levels in hair bundles decrease with exposure to calcium-depleted seawater but increase over time during recovery, indicating a reorganization of actin in hair bundles after trauma caused by calcium-depleted seawater (Watson and Mire, 2002). Such findings predict that Rho G-proteins might be involved in the repair process. The findings of the present study contribute new details to understanding both the repair process and the participation of Rho G-proteins in the repair process. Whereas previous research finds that recovery of morphology and function occurs over 4 h in individual hair bundles damaged by calcium-depleted seawater, this study finds that a partial recovery of abundance of intact hair bundles at the tip of the tentacle occurs within 3 h but that a full recovery of abundance requires > 6 h and is completed within 24 h. These findings suggest that recovery of damaged hair bundles may occur via multiple pathways or over multiple periods of time. This study also finds that important steps of the repair process occurring between hours 2 and 3 of the recovery period do not occur in the absence of Rho activity, as there is no partial recovery of abundance of intact hair bundles or lengthening of stereocilia in hair bundles without Rho activity. As a result, Rho G-proteins are required to be active 2 - 3 h into the recovery period. These findings indicate that not only are Rho G-proteins involved in the repair process but that they are required for repair to occur. Finally, there may be a Rho-dependent, dynamic regulation of stereocilium length occurring during the installation of replacement linkages located near the tips of stereocilia.
Fig. 11. Mean lengths of stereocilia after exposure to calcium-free KSW followed by exposure to Rhosin. The mean lengths of stereocilia were determined after 1 h of exposure to calcium-free KSW followed by administration of Rhosin and through 6 h of recovery by epifluorescence microscopy of fixed specimens. Lengths of stereocilia were measured using ImageJ. Data are presented as the mean length of stereocilia in microns ± SEM. The mean length of stereocilia was calculated from three animals per data point. UC = untreated control; T0 = immediately following trauma; T1 = 1 h recovery; T2 = 2 h recovery; T3 = 3 h recovery; T4 = 4 h recovery; T5 = 5 h recovery; T6 = 6 h recovery. An asterisk denotes a significant difference from untreated controls (p ≤ .05).
active Rho proteins may be required soon after the damage caused by a 1 h immersion in calcium-free seawater and are certainly necessary for the increase in abundance of hair bundles otherwise observed between 2 and 3 h of recovery. As long as the Rho inhibitor is applied before this increase in abundance of hair bundles normally occurs (i.e., beginning at 2 h into recovery), then the recovery of abundance of hair bundles is
Funding This research did not receive any specific grant from funding 8
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agencies in the public, commercial, or not-for-profit sectors.
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