Abstract
The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. The origin of this cochlear amplification is 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. The gene of this protein has been identified and termed prestin. We, herein, present a method for observation by atomic force microscopy (AFM) of prestin expressed in the Chinese hamster ovary (CHO) cell plasma membrane. To obtain a stable sample for AFM imaging in liquid, we used as an example in the protocol provide here, CHO cells transfected with prestin or FLAG-tagged prestin, and untransfected CHO cells (1). The cells attached to a substrate were subjected to ultrasonic waves generated from a sonicator probe so that the inside-out plasma membranes remained on the substrate. Prestin was immunostained with mouse anti-FLAG primary antibody and FITC-conjugated goat anti-mouse IgG secondary antibody. The lipid of the plasma membrane was labeled with fluorescence probes. The cytoplasmic faces of the cells were then observed in liquid by the tapping mode of AFM at low and high magnifications. More particle-like structures 8–12 nm in diameter were observed in the plasma membranes of the prestin-transfected CHO cells than in those of the untransfected CHO cells. Since the difference between these two types of cells is due to the existence of prestin, such particle-like structures in the prestin-transfected CHO cells are possibly constituted by prestin.
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Acknowledgment
The authors are grateful to Professor M. Sokabe (Nagoya University, Japan), Dr. D. Furness (Keele University, U.K.), Mr. A. Yagi (Olympus, Japan), and Professor D. He (Creighton University, U.S.A.) for their helpful suggestions and comments. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas 15086202 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a Health and Labour Science Research Grant from the Ministry of Health, Labour and Welfare of Japan, and by a grant from the Human Frontier Science Program to H.W., and by a Grant-in-Aid for JSPS Fellows 19002194 from the Japan Society for the Promotion of Science and by 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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Murakoshi, M., Wada, H. (2009). Atomic Force Microscopy in Studies of the Cochlea. In: Sokolowski, B. (eds) Auditory and Vestibular Research. Methods in Molecular Biology™, vol 493. Humana Press. https://doi.org/10.1007/978-1-59745-523-7_24
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DOI: https://doi.org/10.1007/978-1-59745-523-7_24
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