1) Tip link structure: In the gating-spring model of transduction, forces are applied to transducer channels by increased tension in a protein filament (the tip link) that spans the distance between two adjacent stereocilia. Using freeze- etching electron microscopy (EM) we have observed that the tip link is made of an 8 to 11 nm right-handed coiled (with a 60 nm pitch) double-filament that ends in multiple insertion points on the membrane of the stereocilia. This indicates that there may be more than one channel per tip-link working in parallel, and also in series if transducer channels are indeed present at either end of the link. Coiled protein filaments are known to be particularly stable because their internal structure can balance compression and tension. Actin filaments, for example, have a very high flexural and torsional rigidity. Given the short length of the tip-link and the similarity in structure with the actin filament, we postulate that the tip-link may also have a very high flexural and torsional rigidity. Such properties are likely to be fundamental in the control of the gating of the transducer channel especially under dynamic conditions at acoustic frequencies. 2) Molecular organization of the membrane-based mechanism of outer hair cell (OHC) electromotility. We have previously shown that electromotility depends on a mechanism incorporated in the structure of the lateral plasma membrane. Using freeze-etching EM we have observed that the surface of the lateral plasma membrane of the OHC is covered with plaques consisting of a dense packing of protein particles. Image processing of these plaques revealed a highly organized orthogonal array of particles with a center-to-center distance of 13nm. Neighboring plaques show slightly different angles of orientation of these protein lattices. The mosaic or tile organization of the plaques appears to match our previous observation that the underlying cortical cytoskeleton has a lattice organization. The exoplasmic domain of the protein particles on the external surface of the cell is shallow and does not appear to have a glycocalyx. The cytoplasmic domain of each particle appears larger than the exoplasmic one and has a globular shape. In addition to the orthogonally packed particles, the lateral plasma membrane contains linear arrays of particles that match the pattern of pillar structures that physically couple the plasma membrane to the underlying cortical cytoskeleton. The plaques of orthogonally packed proteins likely correspond to units of organization of the electromotility mechanism. Voltage driven conformation changes in these arrays of proteins would produce the area changes that drive outer hair cell electromotility.
Showing the most recent 10 out of 74 publications
