Atomic force microscopic studies of inner ear structure and mechanics

Abstract: The transduction processes occurring in the inner ear, from sound induced vibration or head movements to nerve impulses sent to the brain, are to a large part dependent on the mechanical properties of the different components of this organ, especially the sensory hair cells. The function of the hearing organ is for example dependent on the unique mechanoelectric transduction properties of the cochlear outer hair cells (OHCs), which are able to respond to electrophysiological and mechanical stimulations by changing their shape and their mechanical properties. The study of these cells requires the development of nanomechanics tools that are enough sensitive to probe the cells with high resolution. The aim of this thesis was to develop and apply atomic force microscopy (AFM) for the characterisation of structural and mechanical properties of different structures from the inner ear. The work was articulated in five different projects: (1) In a first study, a novel AFM technique was applied to non-biological materials. The interphase of composite materials was characterized with the modified Scanning Local Acceleration Microscopy (SLAM). Using this technique the "contact stiffness" between the tip and surface, related to the elastic modulus of the sample, was detected. (2) The morphology of otoconia crystals from the vestibular system was investigated with AFM in air. The nanostructure of crystals from both the saccula and utricle of the guinea pig were imaged and compared. The surface of single otoconia exhibited a dense packing of round units whose characteristic dimensions were analyzed. (3) In order to explore the role of the protein prestin in outer hair cell electromotility, the AFM was used in combination with the Patch Clamp technique. It was reported to augment voltage-dependent movement when expressed in HEK-293 cells. We measured the differences between control and transfected cells. The study showed that prestin transfected cells exhibited an electromotile response of similar magnitude but opposite polarity to control cells. This phase change was removed when intracellular C1- was substituted with F-. (4) By scanning cells over the surface and acquiring force curves as a function of lateral position (the so-called force volume mapping), we evaluated local elastic properties at different positions on the OHC membrane. (5) AFM was used to investigate the mechanical responses of isolated OHCs to indentation by the AFM tip. Indentation curves showed a break at the contact point, a feature characteristic of an indentation of a stiff membrane surrounding a softer elastic medium (core-shell organization of the cell). Further, we showed that the responses of the OHC lateral wall are highly nonhysteretic at deformation rates of more than 50 µm/s. This suggests OHCs are highly elastic structures with little viscous dissipation compared to other cell types. In summary, atomic force microscopy provides unique possibilities to investigate the morphology and biomechanics of structures from the inner ear, especially the sensory outer hair cells. This technique allows one to image the sample at nanometer resolution, both on dry preparations and in fluid (which is most important for the study of living cells). The findings on the OHCs highlight the importance of the mechanical properties of these cells for hearing, and have consequences for the very fast motility that these cells are believed to undergo in vivo.