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Immune atomic force microscopy of prestin-transfected CHO cells using quantum dots

  • Sensory Physiology
  • Published:
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Abstract

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.

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Acknowledgements

This work was supported by Grant-in-Aid for Scientific Research on Priority Areas 15086202 from the Ministry of Education, Cultures, Sports, Science and Technology of Japan, Grant-in-Aid for Scientific Research (B) 18390455 from the Japan Society for the Promotion of Science, a Health and Labour Science Research Grant from the Ministry of Health, Labour and Welfare of Japan, Grant-in-Aid for Exploratory Research 18659495 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a grant from the Human Frontier Science Program, a grant from the Iketani Science and Technology Foundation and a grant from the Daiwa Securities Health Foundation to H.W., Grant-in-aid for JSPS Fellows 19002194 from the Japan Society for the Promotion of Science and Special Research Grants 11170012 and 11180001 from the Tohoku University 21st Century COE Program of the “Future Medical Engineering Based on Bio-nanotechnology” to M.M.

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  1. Department of Bioengineering and Robotics, Tohoku University, 6-6-01 Aoba-yama, Sendai, 980-8579, Japan

    Michio Murakoshi, Koji Iida, Shun Kumano & Hiroshi Wada

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  1. Michio Murakoshi
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  2. Koji Iida
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  3. Shun Kumano
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  4. Hiroshi Wada
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Correspondence to Hiroshi Wada.

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Murakoshi, M., Iida, K., Kumano, S. et al. Immune atomic force microscopy of prestin-transfected CHO cells using quantum dots. Pflugers Arch - Eur J Physiol 457, 885–898 (2009). https://doi.org/10.1007/s00424-008-0560-z

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  • DOI: https://doi.org/10.1007/s00424-008-0560-z

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