Problems in Cochlear Micromechanics

据推测，运动蛋白prestin密集分布在哺乳动物耳蜗外毛细胞的质膜上。较老的电子显微镜(EM)研究表明，存在致密的颗粒阵列，直径约为10 nm，尺寸均匀。据推测，这些一定是马达蛋白，但没有进行任何鉴定。在这里，开发了一种新的无细胞成像外毛细胞天然质膜的方法。我们使用原子力显微镜(AFM)对在生理条件下附着在云母底物上的质膜的细胞内域进行成像。成像显示存在致密的颗粒阵列，其大小与EM观察到的一致。此外，颗粒是以特定的模式组织起来的，而不是随机分布的。这种模式允许推测原位蛋白质的寡聚体状态。通过引入抗Prestin抗体来明确识别这些颗粒，这些抗体结合到蛋白质颗粒上，同时保持其组织模式。

The motor protein prestin is presumed to densely populate the plasma membrane of the outer hair cells in the mammalian cochlea. Older electron microscopy (EM) studies indicated the presence of a dense array of particles with uniform size of about 10nm in diameter. It was presumed that these must be the motor proteins, but no identification was ever performed. Here, a novel cell-free protocol for imaging outer hair cell native plasma membranes was developed. We used an atomic force microscope (AFM) to image the intracellular domain of a plasma membrane that was attached to a mica substrate under physiological conditions. Imaging revealed the presence of a dense array of particles whose size was consistent with those observed by EM. In addition, the particles were organized in a specific pattern, rather than a random distribution. The pattern allowed speculation as to the oligomeric state of the protein in-situ. The particles were unambiguously identified by the introduction of anti-prestin antibodies that bound to the protein particles while maintaining their organizational patterns.