|ARO 1999: Abstract #359, Date 2/15/99, Session K5, Poster (B106)|
|High resolution imaging of the outer hair cell lateral wall using atomic force microscopy|
|*C. Le Grimellec (INSERM Unit 414, Montpellier, France); M. Vater (University of Potsdam, Germany); M. Lenoir (INSERM Unit 254, Univ. Montpellier 1, France); M.C. Giocondi (INSERM Unit 414, Montpellier, France); R. Pujol (INSERM Unit 254, Univ. Montpellier 1, France)|
We have investigated the unique molecular organization of the outer hair cell (OHC) lateral wall using atomic force microscopy. The results are compared with electron microscopic data (Forge, Cell Tissue Res., 1991, 265:473-483), and with another proposed model of organization (Kalinec et al., PNAS, 1992, 89:8761-8675).
OHCs were mechanically dissociated from the organ of Corti of young (2 week old) rats and deposited in Hank's medium onto polylysine coated coverslips. They were fixed for 15 min in 0.8% glutaraldehyde, washed in the buffer and examined under an atomic force microscope (Bioscope, Digital Instruments, Santa Barbara, CA). Silicon nitride cantilevers with 0.01 N/m spring constant were used, and OHCs were examined with the tapping mode (TMAFM).
At low magnification, TMAFM allowed to recognize OHC morphological characteristics such as their cylindrical shape and their cuticular plate. Strikingly, when compared to other cell types (Le Grimellec et al., C.R. Acad. Sci. Paris, Life Sci., 1997, 320:637-643), the deflection signals did not reveal the classical loose network of submembraneous cytoskeletal proteins (mostly actin). With increasing the magnification, a very regular linear pattern of structures was observed on the lateral wall. This structural organization appeared to be strictly dependent on the presence of calcium in the medium. At the highest resolution (better than 5 nm), the external surface of the lateral plasma membrane appeared as covered with globular particles, likely protein or lipo-protein complexes, some of them forming channel-like structures. The internal surface of the membrane showed a regular alignment of tightly packed structures which could correspond to the pillars as seen under transmission electron microscope.
In conclusion from this preliminary study, TMAFM appears to be a promising technique to investigate the structure-function relationships of the OHC lateral wall.