Imaging by Atomic Force Microscopy of the Plasma Membrane of Prestin-Transfected Chinese Hamster Ovary Cells

Abstract

The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. This cochlear amplification is thought to be generated by 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. Although a fructose transporter, GLUT-5, was initially proposed to be this protein, a later study identified the gene of the motor protein distributed throughout the OHC plasma membrane. This protein has been named Bprestin.^However, although previous morphological studies by electron microscopy and atomic force microscopy (AFM) found the lateral wall of OHCs to be covered with 10-nm particles, believed to be motor proteins, it is unknown whether such particles consist only of prestin or are a complex of GLUT-5 and prestin molecules. To determine if the 10-nm particles are indeed constituted only of prestin, plasma membranes of prestin-transfected and untransfected Chinese hamster ovary (CHO) cells, which do not express GLUT-5, were observed by AFM. First, the cells attached to a substrate were sonicated so that only the plasma membrane remained on the substrate. The cytoplasmic face of the cell was observed by the tapping mode of the AFM in liquid. As a result, particle-like structures were recognized on the plasma membranes of both the prestintransfected and untransfected CHO cells. Comparison of the difference in the frequency distribution of these structures between those two cells showed approximately 75% of the particle-like structures with a diameter of 8-12 nm in the prestin-transfected CHO cells to be possibly constituted only by prestin molecules. Our data suggest that the densely packed 10-nm particles observed on the OHC lateral wall are likely to be constituted only of prestin molecules.

The Motor Protein Prestin Is a Bullet-shaped Molecule with Inner Cavities

“…This was supported by AFM observation of similar sized particles on the surface of Chinese hamster ovary cells in which prestin was heterologously expressed (24). The diameter of the particles was reported 8 -15 nm, with the highest population at 11 nm (22)(23)(24).…”

“…This was supported by AFM observation of similar sized particles on the surface of Chinese hamster ovary cells in which prestin was heterologously expressed (24). The diameter of the particles was reported 8 -15 nm, with the highest population at 11 nm (22)(23)(24). The dimensions presented in this study (77 Å in side length at the widest cytoplasmic region and 96 Å in diagonal length) agree with the previous observations; therefore, we conclude that the prestin molecules presented in this study correspond to individual particles on the OHC lateral membrane.…”

“…It has been observed as highly accumulated membrane integral proteins (2,500 particles/m 2 ) about 11 nm in diameter by using the freeze-fracture technique (22) and atomic force microscopy (AFM) (23). Recent AFM observation of Chinese hamster ovary cells expressing recombinant prestin showed similar particles of diameters between 8 and 12 nm on the cell surface (24), suggesting that reported particles on the surface of OHCs are the motor protein prestin.…”

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

“…The lateral membrane has also been observed by atomic force microscopy (AFM) and the existence of many particles with diameters of about 10 nm has also been reported [17]. Imaging by AFM of prestin-transfected Chinese hamster ovary (CHO) cells has revealed that particle-like structures 8-12 nm in diameter are possibly prestin [18]. 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.…”

Purification of the Motor Protein Prestin from Chinese Hamster Ovary Cells Stably Expressing Prestin

“…Mutational studies have suggested that prestin is a glycosylated protein (11) and is phosphorylated (12) . It has also been indicated that prestin exists as tetramers (13) , which form particles with a diameter of 8-12 nm (14) (15) .…”