LOCALIZATION OF MYOSIN-1-BETA NEAR BOTH ENDS OF TIP LINKS IN FROG SACCULAR HAIR-CELLS

Current evidence suggests that the adaptation motor of mechanoelectric al transduction in vertebrate hair cells is myosin-I beta. Previously, confocal and electron microscopy df bullfrog saccular hair cells usin g an anti-myosin-I beta antibody labeled the tips of stereocilia. We h ave now done quantitative immunoelectron microscopy to test whether my osin-I beta is enriched at or near the side plaques of tip links, the proposed sites of adaptation, using hair bundles that were serially se ctioned parallel to the macular surface. The highest particle density occurred at stereocilia bases, close to the cuticular plate. Also, ste reocilia of differing lengths had approximately the same number of tot al particles, suggesting equal targeting of myosin-I beta to all stere ocilia. Finally, particles tended to clump in clusters of two to five particles in the distal two-thirds of stereocilia, suggesting a tenden cy for self-assembly of myosin-I beta. As expected from fluorescence m icroscopy, particle density was high in the distal 1 mu m of stereocil ia. If myosin-I beta is the adaptation motor, a difference should exis t in particle density between regions containing the side plaque and t hose excluding it. Averaging of particle distributions revealed two re gions with approximately twice the average density: at the upper ends of tip links in a 700-nm-long region centered similar to 100 nm above the side plaque, and at the lower ends of tip links within the tip pla ques. Controls demonstrated no such increase. The shortest stereocilia , which lack side plaques, showed no concentration rise on their sides . Thus, the specific localization of myosin-I beta at both ends of tip links supports its role as the adaptation motor.