Imaging by atomic force microscopy of the plasma membrane of prestin-transfected Chinese hamster ovary cells
Imaging by atomic force microscopy of the plasma membrane of prestin-transfected Chinese hamster ovary cells
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DOI：
10.1007/s10162-006-0041-z
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发表时间：
2006-09-01
影响因子：
2.4
通讯作者：
Wada, Hiroshi
中科院分区：
文献类型：
--
作者：
Murakoshi, Michio;Gomi, Takashi;Wada, Hiroshi
哺乳动物听力的高灵敏度是通过放大耳蜗区的运动来实现的。这种耳蜗放大被认为是由外毛细胞(OHC)对声音刺激的反应而伸长和收缩产生的。这种运动是由嵌入在毛细胞侧膜中的膜蛋白实现的。虽然果糖转运蛋白GLUT-5最初被认为是这种蛋白，但后来的一项研究发现，这种马达蛋白的基因分布在OHC质膜上。这种蛋白质被命名为“prestin”。然而，尽管之前的电子显微镜和原子力显微镜(AFM)的形态研究发现，OHC的侧壁覆盖着10 nm的颗粒，据信是马达蛋白，但目前尚不清楚这种颗粒是只由prestin组成，还是由Glut-5和prestin分子组成的复合体。用原子力显微镜观察了不表达GLUT-5的中国仓鼠卵巢(CHO)细胞的细胞膜，以确定10 nm的颗粒是否真的只由prestin组成。首先，对附着在底物上的细胞进行超声波处理，以便只在底物上保留质膜。用原子力显微镜在液体中敲击的方式观察细胞的胞质表面。结果表明，在未转染组和转染组CHO细胞的质膜上均可观察到颗粒状结构。比较这些结构在两种细胞中的频率分布差异，发现在转染prestin的CHO细胞中，大约75%的直径为8-12 nm的颗粒状结构可能只由prestin分子组成。我们的数据表明，在OHC侧壁上观察到的致密堆积的10 nm粒子很可能只由prestin分子组成。
The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. This cochlear amplification is thought to be generated by the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. Although a fructose transporter, GLUT-5, was initially proposed to be this protein, a later study identified the gene of the motor protein distribute throughout the OHC plasma membrane. This protein has been named "prestin." However, although previous morphological studies by electron microscopy and atomic force microscopy (AFM) found the lateral wall of OHCs to be covered with 10-nm particles, believed to be motor proteins, it is unknown whether such particles consist only of prestin or are a complex of GLUT-5 and prestin molecules. To determine if the 10-nm particles are indeed constituted only of prestin, plasma membranes of prestin-transfected and untransfected Chinese hamster ovary (CHO) cells, which do not express GLUT-5, were observed by AFM. First, the cells attached to a substrate were sonicated so that only the plasma membrane remained on the substrate. The cytoplasmic face of the cell was observed by the tapping mode of the AFM in liquid. As a result, particle-like structures were recognized on the plasma membranes of both the prestin-transfected and untransfected CHO cells. Comparison of the difference in the frequency distribution of these structures between those two cells showed approximately 75% of the particle-like structures with a diameter of 8-12 nm in the prestin-transfected CHO cells to be possibly constituted only by prestin molecules. Our data suggest that the densely packed 10-nm particles observed on the OHC lateral wall are likely to be constituted only of prestin molecules.