Structural analysis of the motor protein prestin

这个项目的目的是阐明运动蛋白prestin，它的构象变化已被证明诱导内耳外毛细胞的伸长和收缩。为此，首先尝试用原子力显微镜(AFM)观察经prestin基因转染的中国仓鼠卵巢(CHO)细胞和未转染CHO细胞的细胞膜。结果，两种细胞的质膜中都识别出可能代表膜蛋白的颗粒状结构，尽管两者之间没有明显的差异。对观察到的结构的形状和大小的分析表明，与未转染的CHO细胞相比，转prestin基因的CHO细胞质膜中有更多直径为8-12 nm的颗粒状结构，这表明这些颗粒状结构可能是prestin分子。第二项工作是更特异地检测转prestin基因CHO细胞质膜中的prestin分子，以更详细地阐明prestin的结构。为此，利用原子力显微镜和量子点(Qdots)标记的抗prestin抗体相结合的实验方法，检测单个prestin分子。由于Q点是直径为8 nm的坚硬材料，因此很容易被AFM观察到。此外，由于在Q点附近可能存在prestin，因此可以对prestin分子进行检测和观察。用原子力显微镜对标记有Q点的CHO细胞进行AFM成像后，在Q点附近观察到四峰一谷的方形环状结构，表明Prestin在细胞膜上形成了四聚体。

The aim of this project was to clarify the motor protein prestin, whose conformational change has been indicated to induce an elongation and contraction of outer hair cells in the inner ear. To this end, first, an attempt was made to observe the plasma membrane of prestin-transfected Chinese hamster ovary (CHO) cells and that of untransfected CHO cells by atomic force microscopy (AFM). As a result, particle-like structures probably indicating membrane proteins were recognized in the plasma membranes of both cells, although there were no distinctive differences between the two. Analysis of the shape and size of the observed structures showed that there were more particle-like structures with a diameter of 8-12 nm in the plasma membrane of the prestin-transfected CHO cells than in that of the untransfected CHO cells, suggesting that these particle-like structures are possibly prestin molecules.The second endeavor was to more specifically detect prestin molecules in the plasma membrane of the prestin-transfected CHO cells to clarify the structure of prestin in more detail. For this purpose, an experimental approach combining AFM with anti-prestin antibody conjugated with quantum dots (Qdots), used as topographic surface markers, was carried out to detect individual prestin molecules. As Qdots are hard material with a diameter of 8 nm, they can be easily seen by AFM. In addition, as prestin may exist near Qdots, detection and observation of prestin molecules can be performed. As a result of imaging by AFM of the prestin-transfected CHO cells labeled with Qdots, squarish ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, indicating that prestin forms a tetramer in the plasma membranes of the prestin-transfected CHO cells.