Atomic force microscopy in studies of the cochlea.
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. 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.
Department of Bioengineering and Robotics, Graduate School of Engineering Tohoku University, Sendai, Japan.
Michio
M
Murakoshi
Hiroshi
H
Wada
D000818:Animals; D027321:Anion Transport Proteins / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D003051:Cochlea / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D018625:Microscopy, Atomic Force / Q000379:methods*; D008856:Microscopy, Fluorescence
D027321:Anion Transport Proteins