Immunological evidence that anemone repair proteins include replacement linkages.

In response to damage to hair bundles caused by exposure to calcium free buffers, sea anemones secrete large protein complexes named 'repair proteins' that rapidly restore structural integrity and function to hair bundles. A specific chromatographic fraction of the repair protein mixture, named 'fraction beta', has biological activity comparable to the complete repair protein mixture (Watson et al., 1998, Hear. Res. 115, 119-128). In this study, we find that polyclonal antibodies raised against deglycosylated fraction beta specifically bind fraction beta on Western blots. Anti-fraction beta delays the normal recovery of vibration sensitivity in experimental animals (i.e., those with hair bundles damaged by calcium free buffers). Moreover, anti-fraction beta disrupts vibration sensitivity in control animals (i.e., those with healthy hair bundles). Experimentally damaged hair bundles subsequently exposed to repair protein and then processed for immunoelectron microscopy show labeled linkages interconnecting stereocilia of the hair bundle. Immunofluorescence microscopy confirms strong labeling of hair bundles treated with repair proteins and only weak labeling of tips of hair bundles from control animals. Immunofluorescence microscopy indicates stores of repair proteins in gland cells of the body column in control animals and in gland cells of the mouth in experimental animals. Repair biological activity is confirmed in column purified homogenates of these tissues. Apparently repair proteins are delivered to damaged hair bundles in mucus carried by beating cilia.

Gap junctional communication in the vibration-sensitive response of sea anemones.

Although gap junctions occur in auditory and vestibular systems, their function is unclear. Here we present evidence for gap junctional communication in transmitting mechanosensory signals in a sea anemone model system. Hair bundles on anemone tentacles are vibration-sensitive mechanoreceptors that regulate discharge of nematocyst from effector cells. We find that vibration-dependent nematocyst discharge is selectively and reversibly blocked by the gap junction uncouplers, heptanol and arachidonic acid. Epidermal cells within excised tentacles exhibit a low level of dye coupling which is significantly enhanced upon deflection of overlying hair bundles. Dye coupling is inhibited both by gap junction uncouplers and by agents that interfere with mechanotransduction, including streptomycin and elastase. Electrophysiological data suggest gap junctional communication between cells giving rise to different hair bundles. When hair bundles are stimulated with a sweep of vibrations, individual cells show responses to five to eight frequencies. The number of responsive frequencies is reduced to one or two by heptanol and essentially abolished with streptomycin treatment. Immunoreactivity to the gap junction protein, connexin 43, is abundant in the tentacle epidermis and localized to membranes at junctions between several cell types. Small areas of close membrane apposition are observed between these cell types with intermembrane clefts of 4-7 nm. Of the several membrane proteins isolated from tentacles, immunoreactivity to connexin 43 is observed in a single band with an apparent molecular weight of approximately 46 kDa.