Structural and Molecular Basis of Transduction in Auditory Sensory Organs

National Institute On Deafness And Other Communication Disorders

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Abstract

We previously observed that MYO3A and MYO7A, two stereocilia myosins essential for hearing, transport components of the mechanoelectrical transduction (MET) complex to stereocilia tips and to the upper tip link density location with the potential for both selectivity and complementary function and/or redundancy. In our efforts to identify regulatory pathways for these motor and cargo transport proteins we investigated whether CALML4, calmodulin-like protein 4, that binds and regulates MYO7B in intestinal microvilli, would bind and co-localize with MYO7A in stereocilia. We used COS7 cells as a heterologous expression model system to examine the interactions of CALML4 with stereocilia myosins. We observed that CALML4 co-translocates with MYO7A along filopodial actin protrusions and co-accumulates at the filopodia tips. This co-translocation is selective for MYO7A and is not observed for other stereocilia myosins including MYO3A and MYOXVA. Using immunofluorescence, we observed CALML4 enrichment at the same stereocilia compartments where MYO7A is localized including the tip and tapered base, as well as around the cuticular plate region. Surprisingly we could not detect CALML4 at the stereocilia upper tip link density. We hypothesize CALML4 functions as a site-specific light chain regulator for MYO7A isoforms in hair cells. Early work from our lab showed that during hair cell development the actin filaments that form the stereocilia actin core elongate by incorporation of monomers at stereocilia tips and the actin core undergoes turnover by treadmilling. The actin incorporation is length-dependent and slows down significantly as the bundle matures. More recent reports show that actin turnover occurs only at stereocilia tips. Most of the studies of actin dynamics in stereocilia involve the use of large expressable fluorophore-tags that can interfere with native actin dynamics. Our new experiments using tags connected to actin by flexible linkers as well as smaller tags confirm our early findings that actin is turning over at fast rates during bundle development. We are also examining and quantifying the distribution of actin monomers along the stereocilia and how they are actively transported to stereocilia tips during stereocilia development, maturation, and aging. An important question in hair cell biology and function is how myosins traffic and transport actin-regulatory proteins and components of the MET complex to stereocilia tips. A major shortcoming of filopodia as a model system to track and quantify this bidirectional traffic, involving multiple myosins is that they have a variable native structure and molecular composition. Thus, in our quest for a better model we came across a specialized actin protrusion induced by the Ebola virus matrix protein VP40. This much simpler actin protrusions show a remarkably regular actin core and a tightly enwrapped plasma membrane forming a cylindrical structure 100 nm in diameter and several micrometers long, reminiscent of the long vestibular hair cell stereocilia. We are now using this regular structure as a treadmilling actin railway to investigate the traffic and cargo transport function of stereocilia myosins unhindered by the complexity of filopodia and the challenges of experimenting with hair cells. MYOXVA and MYO3A traffic and form the characteristic comet-like tip-to-base distribution, in a length-dependent manner, as we previously reported to occur in native stereocilia. We are currently using this high-yield model system to investigate the mechanism of transport and assembly of components of the mechanotransduction complex. Because of their narrow diameter the VP40-induced actin protrusions are amenable not only for quantitative single molecule TIRF imaging as they can be entirely excited within the evanescent field, but are also optimal for cryo-electron tomography as they can be frozen in a uniformly thin ice layer. Outer hair cells (OHCs) contribute to cochlear amplification through voltage-dependent somatic length changes that can operate at acoustic frequencies. This unique form of motility is driven by prestin, a member of the solute carrier 26 family of anion transporters that is highly expressed along the OHC lateral plasma membrane. The lateral plasma membrane is supported by a cortical actin-spectrin lattice and by a smooth ER system to form a regular layered structure along the OHC lateral wall. We combined cryogenic sample preparation methods and electron tomography to further examine the detailed structure of the OHC lateral wall. We show that the lateral plasma membrane contains closely tiled microdomains of orthogonally packed putative prestin protein complexes spaced at 12nm center-to-center. The cortical lattice connects the plasma membrane to the adjacent lateral cisternae through two independent cross-bridging components. The lateral cisternae are in turn integrated through inter and intra-cisternal cross bridging systems. Finally, mitochondria are attached to the lateral cisternae through another set of linker elements. By quantifying the dimensions of each of these components from the tomograms we provide a detailed blueprint of the nano-architecture of the OHC electromotile apparatus. We argue that the cohesiveness of the structure enables transmission of force generated by prestin within the lateral plasma membrane to the rest of the cell. Our observation that prestin forms higher order orthogonally packed structures in the membrane is important considering recent CryoEM reports that prestin forms dimers when purified from heterologous expression systems. The dense regular orthogonal packing suggests formation of prestin tetramers (or dimer of dimers) and cooperativity within the microdomains during voltage-dependent conformation changes of the individual prestin oligomers. These findings provide new insights into how molecular packing of prestin and the stratified structure of the OHC are integrated to drive somatic motility at acoustic frequency. The role of the lateral cisternae in electromotility remains unclear, however, this ER organization has been suggested to be involved in OHC homeostasis and age-related hearing loss. Screening for the localization of proteins that are selectively enriched in OHCs, we observed that aquaporin-11 (AQP11) localizes in the lateral cisternae. AQP11 has been shown to be a poor water permeant, but interestingly it can permeate H2O2 across the ER membrane. AQP11s ability to facilitate the movement of H2O2 suggests that it could be involved in redox homeostasis and signaling. To gain further insight into the AQP11 pore structure and permeability properties we ran comparative Molecular Dynamics simulations of AQP11 and AQP1 in water and water/H2O2 mixtures. Consistent with experimentally observed permeability data, our simulations show that both aquaporins conduct water, and AQP11 can also transport H2O2. AQP11 water pore/channel showed a bottleneck of radius 0.8 compared to 2.5 for APQ1. We have recently obtained AQP11 purified to 95% and we are currently attempting to determine its structure experimentally by CryoEM. We are also generating a mouse line with hair cell specific AQP11 knockout to investigate the role of AQP11 in OHC function and homeostasis.