Based on the paper Cadherin-23 May Be Dynamic in Hair Bundles of the Model Sea Anemone Nematostella vectensis by Pei-Ciao Tang and Glen M. Watson
Mechanotransduction, the conversion of a mechanical stimulus into a chemical activity, is necessary in humans to undergo processes such as hearing and balancing. In humans and other vertebrates, tip links of hair cells possess the protein Cadherin-23 (CDH23), which is necessary for mechanotransduction in the inner ear (Tang and Watson, 2014). In the sea anemone Nematostella vectensis (Figure 1A), mechanoreceptors are located in hair bundles of hair cells and allow the organism to sense movement and odor of prey (Tang and Watson, 2014). Interestingly, the invertebrate N. vectensis has a homolog of CDH23 in its hair bundles. Therefore, the experimenters in this study used N. vectensis as a model organism to discover how this CDH23 specifically affects mechanotransduction.
Figure 1. A. The sea anemone, Nematostella vectensis (http://www.nidcd.nih.gov/news/releases/13/Pages/071913.aspx). B. Mechanosensory hair bundle cells of the mouse inner ear shown in a scanning electron microscopy image (http://www.anemoneresearch.org/Current-Projects.html).
Understanding Hair Bundles and Mechanotransduction
Hair Cells and Bundles
In vertebrates, a hair bundle is a group of stereocilia that extend from a hair cell (Figure 2). The stereocilia are aligned in a step-like manner, growing from shortest to tallest. At the top of each stereocilium there resides a tip link, which is the location of several proteins including CDH23. The deflection of hair bundles in either direction is the mechanism that allows mechanotransduction pathways to open and close and therefore is necessary for a chemical signal to be transmitted. Though this hair cell system is typical of vertebrates, it also occurs in sea anemones including N. vectensis.
Figure 2. General layout of a hair bundle. Grey = inactive. Green = active. Hair bundle protrudes from hair cell (semicircular base) and is composed of stereocilia with tip links shown in purple. CDH23 is located in tip links .
The Gating-Spring Model: A proposed model for mechanotransduction
Several models have been suggested for the mechanism behind mechanotransduction, namely the gating-spring model. This model has been formulated in an effort to explain the relationship between hair bundles and the current that is associated with mechanotransduction. In this model, if the hair bundle is swayed in a positive rather than negative direction, the channels open quickly and allow an influx of positive ions (Tang and Watson, 2014). The movement of the hair bundle in the positive direction would be the movement of shorter stereocilia towards taller stereocilia. The gating-spring model suggests that it is energetically favorable when the mechanotransduction channels are open.
N. vectensis Mechanotransduction
The sea anemone N. vectensis exhibits normal mechanotransduction in the sense that the mechanism can be blocked reversibly by particular antibiotics and inhibited if exposed even briefly to seawater that lacks calcium (Tang and Watson, 2014). However, N. vectensis differs from vertebrates in that the responsiveness of its hair bundles is in a sense adjustable. When hair cells are stimulated, the anemone enters an excited state and any object that touches the tentacles are stung by nematocysts at maximum strength (Tang and Watson, 2014). However at rest, the nematocysts are only about halfway charged. In this regard, the nematocysts are vibration-dependent, a factor that is manipulated and tested during this study. Through the use of N. vectensis as a model, the main focus of this study is to determine how the absence of CDH23 effects hair bundle function and structure.
Procedures and Results
Investigating CDH23 Antibody
A CDH23 affinity-purified antibody was prepared and added to seawater that contained intact anemones or isolated tentacles to observe how the antibody effects hair bundles. First, using immunocytochemistry, the experimenters localized binding of CDH23 antibodies to the distal portion of hair bundles. They next tested how CDH23 antibodies effect the vibration-dependent charge and discharge of nematocysts and hair bundle morphology (Tang and Watson, 2014). Compared to the healthy control anemones, the nematocyst discharge significantly decreased when in the presence of CDH23 antibody (Figure 3A). CDH23 specificity was confirmed by conducting the same procedure using another antibody, TRPA1. Figure 3B shows the results of this TRPA1 antibody study, yielding no significant difference in nematocyst discharge between the control anemones and the TRPA1 antibody-treated anemones (Tang and Watson, 2014). Treatment of hair bundles with CDH23 antibody caused the morphology of the bundles to “splay”, making them cylindrical rather than the typical conical shape. Additionally, CDH23 antibody significantly decreased mean hair bundle abundance in comparison to healthy controls.
Figure 3. Effect of CDH23 antibody on nematocyst discharge. Exposure time (x-axis) versus mean nematocysts discharged (y-axis). (A) Shows the CHD23 antibody-treated anemones (open circles) have significantly less nematocysts discharged in comparison to healthy controls (solid squares). (B) Shows TRPA1 antibody-treated anemones do not differ from healthy controls (Tang and Watson, 2014).
Investigating FITC-conjugated CDH23 Peptide
Similar to the CDH23 antibody procedure, a CDH23 peptide was prepared and added to seawater that contained intact anemones or isolated tentacles to observe how the peptide effects hair bundles. The results of the peptide study were extremely similar to the results of the antibody study. First, the experimenters localized the peptide to stereocilia using immunochemistry techniques. In this procedure, two different concentrations of CDH23 peptide were used (0.1 and 10 nM) and the exposure time was altered. Treatment with 10 nM CDH23 peptide significantly decreased nematocyst discharge at both 5 and 10 minutes of exposure, whereas treatment with 0.1 nM CDH23 peptide only significantly decreased discharge at 10 minutes of exposure. CDH23 peptide treatment also changes the morphology of hair bundles and decreases their mean abundance similarly to the CDH23 antibody treatment (Figure 4). In the peptide study the experimenters additionally tested the effects of the CDH23 peptide on the mechanotransduction current and F-actin in stereocilia. Treatment with CDH23 peptide was found to decrease the mechanotransduction current, and also decrease the amount of F-actin in stereocilia. These findings are significant because F-actin is required for the specific movement and elongation of hair bundles in sea anemones.
Figure 4. Effect of CDH23 peptide on nematocyst discharge. Exposure time (x-axis) versus mean nematocysts discharged (y-axis). (A) 0.1 nM CDH23 peptide (open triangles) and 10 nM CDH23 peptide (open circles). Shows significant decrease of nematocyst discharge in comparison to healthy controls for 10 nM treatment from 5 minutes onward, and for 0.1 nM treatment from 10 minutes onward (Tang and Watson, 2014).
The overall results suggest that CDH23 is involved in specific protein-protein interactions on stereocilia, and without CDH23 the lack of these interactions leads to abnormal hair cell function and morphology (Tang and Watson, 2014). This study has proven that, in the model organism Nematostella vectensis, CDH23 is required for proper mechanotransduction signaling.
A weakness of this study is that the experimenters faced difficulties in localizing cadherin-binding proteins (CBPs) when investigating the specific interactions that CDH23 participates in on stereocilia. Also, they were unable to label the FITC-conjugated CDH23 peptide on their own because of technical difficulties, and therefore had to rely on results from a previous study. However, a major strength of this study is that it provides invaluable insight into the mechanism behind CDH23 function, and suggests that mutations in CDH23 can significantly disrupt hair bundle function and morphology, and consequently mechanotransduction. Ultimately, Tang and Watson prove that in N. vectensis, CDH23 is required for the occurrence of normal mechanotransduction and also for normal hair bundle morphology and structure. These findings create a model system in N. vectensis that can be used to learn more about mechanotransduction in vertebrates. Further research into CDH23’s role in mechanotransduction in sea anemones can be found here.