Structure for a Physiologic Model: Electron Tomography of the Outer Hair Cell Lateral Wall

William Triffo, Rice University

Photo of William Triffo

In the mammalian cochlea, the outer hair cell (OHC) is capable of generating axial deformations in response to variations in transmembrane potential. A complete understanding of electromechanical transduction in the OHC depends on precise knowledge of intracellular structure, and efforts to model OHC physiology are likewise limited to the fidelity of known intracellular geometry. The cortex of the OHC, referred to as the lateral wall, can be viewed as a trilaminate composite made up of (1) the plasma membrane, (2) a network of actin and spectrin termed the cortical lattice, and (3) lamellar stacks known as the subsurface cisternae (SSC).

Previous studies of the cortical lattice using conventional TEM and AFM techniques relied on protocols that removed the lattice from its native environment. Moreover, depending on the exact fixation protocols, conflicting depictions of SSC ultrastructure have been reported; this uncertainty carries large implications when implementing electrophysiological models of OHC motility. We have therefore evaluated a variety of sample preparation methods, including conventional fixation protocols as well as high-pressure freezing (HPF) and freeze-substitution (FS). Using HPF/FS cochlear samples from mouse and guinea pig, we have employed Electron Tomography (ET) to study the cortical lattice and its structural relationship to the plasma membrane and SSC, resolving the pillar proteins known to span the extracisternal space between the plasma membrane and cortical lattice. ET utilizes a series of TEM projections from a tilted sample to reconstruct a volume density map, allowing us to visualize macromolecular assemblies in their native cellular context. We discuss our findings, the choice of reconstruction and post-processing algorithms, and the impact of these results on physical models of OHC motility.

Abstract Author(s): William J. Triffo (1,3), Kent L. McDonald (2), Manfred Auer (1), and Robert M. Raphael (3)<br /><br />(1) Lawrence Berkeley National Laboratory and (2) University of California at Berkeley, Berkeley, CA, (3) Rice University, Houston, TX