This project will investigate the mechanism by which hair bundle mechanoreceptors located on tentacles of sea anemones are 'tuned' by activated chemoreceptors. The hair bundle mechanoreceptors are involved in prey capture by detecting prey movements both before the prey contacts the tentacle and then afterwards, once the prey has been stung by anemone nematocysts (stinging organelles). The chemoreceptors bind two prey derived compounds: (i) N-acetylated sugars that signal proximity of potential prey and (ii) amino compounds (from the hemolymph) that indicate that the prey (zooplankton) is successfully wounded. Each of the chemosensitizers 'tunes' (i.e., frequency and amplitude shifts) the hair bundles. Tuning involves significant changes to the structure of the hair bundle. The research will examine chemosensitizer effects on three aspects of hair bundles: linkages, actin, and motors. Linkages: Electrophysiological recordings will be conducted to whether linkages interconnecting stereocilia remain functional during tuning. A bioassay based on counting nematocysts discharged into vibrating test probes will determine if animals remain vibration sensitive during tuning. Anemones fixed at increments during tuning will be examined by electron microscopy to determine how linkages are altered during tuning. Applications of a commercial pan-cadherin antibody will be used to investigate a hypothesis that linkage cycling occurs in resting hair cells and is up regulated during tuning. Genes coding for anemone linkages will also be isolated by RT-PCR. Actin and actin binding proteins: Stereocilia in vertebrate hair cells contain beta and gamma actin. These genes will be fully sequenced and isoform specific antibodies will be generated and used to determine whether structural dynamics occurring during tuning are isoform specific. Motors: Anemone hair bundles are immunopositive for myosin VIIA. Inhibiting myosins using vanadate mimics tuning by N-acetylated sugars. However, electrophysiology suggests that the vanadate treated hair bundles are unresponsive. This phenomenon will be examined in greater detail.
This research will be conducted by the only team of researchers in the world currently studying hair bundles in anemones. Consequently, funding of this project directly impacts this field of knowledge. The Co-PI will recruit undergraduate honor's students into the project (as REUs') as freshmen or sophomores; such early exposure to experimentation may steer these students into research careers. The PI will mentor undergraduate seniors engaged in independent research for course credit; these students are ethnically diverse and will include students from underrepresented groups. Finally, funding will support a graduate student as a research assistant. Thus, this project will impact students at all levels of academic training.
