Structural analysis of the motor protein prestin

本项目的目的是阐明运动蛋白普雷斯廷，其构象变化已被证明是诱导内耳外毛细胞的伸长和收缩。为此，首先，尝试观察质膜prestin转染的中国仓鼠卵巢（CHO）细胞和未转染的CHO细胞的原子力显微镜（AFM）。结果，颗粒状结构可能表明膜蛋白被确认在两个细胞的质膜，虽然有没有明显的差异。对观察到的结构的形状和大小的分析表明，在转染prestin的CHO细胞的质膜中比在未转染的CHO细胞的质膜中有更多的直径为8-12 nm的颗粒状结构，第二个奋进是更特异性地检测普雷斯廷质膜中的普雷斯廷普雷斯廷分子，转染CHO细胞以更详细地阐明普雷斯廷的结构。为此，结合原子力显微镜与抗普雷斯廷抗体与量子点（Qdots），用作地形表面标记，进行检测单个普雷斯廷分子的实验方法。由于量子点是直径为8 nm的硬材料，因此它们可以很容易地通过AFM看到。另外，由于普雷斯廷可以存在于Qdot附近，因此可以进行普雷斯廷分子的检测和观察。通过AFM对用Qdot标记的Prestin转染的CHO细胞进行成像的结果是，在Qdot附近观察到方形环状结构，每个结构在其中心具有四个峰和一个谷，这表明普雷斯廷在Prestin转染的CHO细胞的质膜中形成四聚体。

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.