García J A, Yee A G, Gillespie P G, Corey D P
Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Neurosci. 1998 Nov 1;18(21):8637-47. doi: 10.1523/JNEUROSCI.18-21-08637.1998.
Current evidence suggests that the adaptation motor of mechanoelectrical transduction in vertebrate hair cells is myosin-Ibeta. Previously, confocal and electron microscopy of bullfrog saccular hair cells using an anti-myosin-Ibeta antibody labeled the tips of stereocilia. We have now done quantitative immunoelectron microscopy to test whether myosin-Ibeta is enriched at or near the side plaques of tip links, the proposed sites of adaptation, using hair bundles that were serially sectioned parallel to the macular surface. The highest particle density occurred at stereocilia bases, close to the cuticular plate. Also, stereocilia of differing lengths had approximately the same number of total particles, suggesting equal targeting of myosin-Ibeta to all stereocilia. Finally, particles tended to clump in clusters of two to five particles in the distal two-thirds of stereocilia, suggesting a tendency for self-assembly of myosin-Ibeta. As expected from fluorescence microscopy, particle density was high in the distal 1 micrometer of stereocilia. If myosin-Ibeta is the adaptation motor, a difference should exist in particle density between regions containing the side plaque and those excluding it. Averaging of particle distributions revealed two regions with approximately twice the average density: at the upper ends of tip links in a 700-nm-long region centered approximately 100 nm above the side plaque, and at the lower ends of tip links within the tip plaques. 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-Ibeta at both ends of tip links supports its role as the adaptation motor.
目前的证据表明，脊椎动物毛细胞中机械电转导的适应马达是肌球蛋白-Iβ。此前，使用抗肌球蛋白-Iβ抗体对牛蛙球囊毛细胞进行共聚焦和电子显微镜观察时，标记了静纤毛的尖端。我们现在进行了定量免疫电子显微镜检查，以测试肌球蛋白-Iβ是否在与黄斑表面平行连续切片的毛束的顶端连接侧斑处或其附近富集，而侧斑被认为是适应发生的部位。最高的颗粒密度出现在靠近角质板的静纤毛基部。此外，不同长度的静纤毛的总颗粒数大致相同，这表明肌球蛋白-Iβ对所有静纤毛的靶向作用相同。最后，颗粒倾向于在静纤毛远端三分之二处聚集成两到五个颗粒的簇，这表明肌球蛋白-Iβ有自我组装的趋势。正如荧光显微镜所预期的那样，静纤毛远端1微米处的颗粒密度很高。如果肌球蛋白-Iβ是适应马达，那么包含侧斑的区域和不包含侧斑的区域之间的颗粒密度应该存在差异。颗粒分布的平均结果显示有两个区域的密度约为平均密度的两倍：在侧斑上方约100纳米处为中心的700纳米长区域内顶端连接的上端，以及顶端斑内顶端连接的下端。对照显示没有这种增加。最短的静纤毛没有侧斑，其侧面没有浓度升高。因此，肌球蛋白-Iβ在顶端连接两端的特异性定位支持了它作为适应马达的作用。
