Actin cores of hair-cell stereocilia support myosin motility.
Actin cores of hair-cell stereocilia support myosin motility.
复制标题
毛细胞静纤毛的肌动蛋白核心支持肌球蛋白的运动。
DOI：
10.1073/pnas.87.21.8627
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发表时间：
1990
影响因子：
11.1
通讯作者：
Block,SM
中科院分区：
文献类型：
--
作者：
Shepherd,GM;Corey,DP;Block,SM
在体外试验中，毛细胞静纤毛的肌动蛋白核心作为肌球蛋白包被的珠子运动的底物进行了测试。大量的静纤毛牛蛙球囊和半规管分离到盖玻片上的印迹和去膜，暴露其细胞骨架核心的极性肌动蛋白轨道。在ATP存在下，将涂覆有鸡骨骼肌肌球蛋白的粗丝的二氧化硅或聚苯乙烯珠加入到该核心制剂中。肌球蛋白包被的珠子可以通过单独的扩散到达一些核心，但是通过使用“光镊”（梯度力光阱）将珠子直接存款在核心上，大大提高了测定的效率和精度。以这种方式施加的珠以1-2微米/秒的速度结合并单向移动，从而逃脱捕集器的阻滞力。静纤毛内的肌动蛋白丝通过纤毛交联，但这似乎并不干扰肌球蛋白的运动。还测试了涂覆有视叶驱动蛋白的珠子的运动;当施加到基于微管的动纤毛核心时，这些珠子以0.1-0.2微米/秒的速度单向结合和移动，但当施加到基于肌动蛋白的静纤毛核心时则不然。我们的研究结果是一致的，并提供支持，毛细胞适应模型，其中分子马达，如肌球蛋白保持紧张的机械门控转导通道。光镊和视频增强差分干涉对比光学器件为肌球蛋白运动性的体外分析提供了高效率和更高的光学分辨率。
The actin cores of hair-cell stereocilia were tested as a substrate for the movement of myosin-coated beads in an in vitro assay. Large numbers of stereocilia from bullfrog sacculi and semicircular canals were isolated by blotting onto coverglasses and were demembranated to expose the polar actin tracks of their cytoskeletal cores. Silica or polystyrene beads, coated with thick filaments of chicken skeletal muscle myosin, were added to this core preparation in the presence of ATP. Myosin-coated beads could reach some of the cores by diffusion alone, but the efficiency and precision of the assay were improved considerably by the use of "optical tweezers" (a gradient-force optical trap) to deposit the beads directly on the cores. Beads applied in this fashion bound and moved unidirectionally at 1-2 microns/s, escaping the retarding force of the trap. Actin filaments within the stereocilia are cross-linked by fimbrin, but this did not appear to interfere with the motility of myosin. Beads coated with optic-lobe kinesin were also tested for movement; these bound and moved unidirectionally at 0.1-0.2 microns/s when applied to microtubule-based kinociliary cores, but not when applied to actin-based stereociliary cores. Our results are consistent with, and lend support to, a model for hair cell adaptation in which a molecular motor such as myosin maintains tension on the mechanically gated transduction channels. Optical tweezers and video-enhanced differential interference contrast optics provide high efficiency and improved optical resolution for the in vitro analysis of myosin motility.