Immune atomic force microscopy of prestin-transfected CHO cells using quantum dots
Immune atomic force microscopy of prestin-transfected CHO cells using quantum dots
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DOI：
10.1007/s00424-008-0560-z
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发表时间：
2009-02
期刊：
影响因子：
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通讯作者：
M. Murakoshi;Koji Iida;S. Kumano;H. Wada
中科院分区：
文献类型：
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作者：
M. Murakoshi;Koji Iida;S. Kumano;H. Wada
普雷斯廷是外毛细胞（OHC）的一种膜蛋白，是驱动OHC体细胞电活动的马达。电子显微镜研究表明，外毛细胞的侧膜上密集地覆盖着10 nm的颗粒，它们被认为是一种运动蛋白。通过原子力显微镜（AFM）成像prestin转染的中国仓鼠卵巢（CHO）细胞显示8- 12-nm的颗粒状结构可能是普雷斯廷。然而，由于在OHC和CHO细胞的质膜中存在除普雷斯廷之外的多种内在膜蛋白，因此不可能明确在这些膜中观察到的结构是普雷斯廷。在本研究中，结合原子力显微镜与量子点（Qdots），作为地形表面标记，进行了检测单个普雷斯廷分子的实验方法。从prestin转染和未转染的CHO细胞中分离由内而外的质膜。然后将这种膜与抗prestin一抗和Qdot缀合的二抗孵育。证实了Prestin转染的CHO细胞的荧光标记，但未转染的CHO细胞的荧光标记未被证实。随后通过AFM扫描膜，并且在prestin转染的CHO细胞中清楚地看到Qdot。在Qdot附近观察到环状结构，每个在其中心具有四个峰和一个谷，表明这些结构是在prestin转染的CHO细胞的质膜中表达的普雷斯廷。
Prestin, a membrane protein of the outer hair cells (OHCs), is known to be the motor which drives OHC somatic electromotility. Electron microscopic studies showed the lateral membrane of the OHCs to be densely covered with 10-nm particles, they being believed to be a motor protein. Imaging by atomic force microscopy (AFM) of prestin-transfected Chinese hamster ovary (CHO) cells revealed 8- to 12-nm particle-like structures to possibly be prestin. However, since there are many kinds of intrinsic membrane proteins other than prestin in the plasma membranes of OHCs and CHO cells, it was impossible to clarify which structures observed in such membranes were prestin. In the present study, an experimental approach combining AFM with quantum dots (Qdots), used as topographic surface markers, was carried out to detect individual prestin molecules. The inside-out plasma membranes were isolated from the prestin-transfected and untransfected CHO cells. Such membranes were then incubated with antiprestin primary antibodies and Qdot-conjugated secondary antibodies. Fluorescence labeling of the prestin-transfected CHO cells but not of the untransfected CHO cells was confirmed. The membranes were subsequently scanned by AFM, and Qdots were clearly seen in the prestin-transfected CHO cells. Ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, suggesting that these structures are prestin expressed in the plasma membranes of the prestin-transfected CHO cells.