Tectorial Membrane Material Properties in Tecta[superscript Y1870C+] Heterozygous Mice The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Masaki, Kinuko, Roozbeh Ghaffari, Jianwen Wendy Gu, Guy P. Richardson, Dennis M. Freeman, and A.J. Aranyosi. “Tectorial Membrane Material Properties in Tecta[superscript Y1870C+] Heterozygous Mice.” Biophysical Journal 99, no. 10 (November 2010): 3274–3281. © 2010 Biophysical Society As Published http://dx.doi.org/10.1016/j.bpj.2010.09.033 Publisher Elsevier Version Final published version Citable link http://hdl.handle.net/1721.1/96042 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. 3274 Biophysical Journal Volume 99 November 2010 3274–3281 Tectorial Membrane Material Properties in TectaY1870C/þ Heterozygous Mice Kinuko Masaki,†‡{ Roozbeh Ghaffari,†‡{ Jianwen Wendy Gu,†‡{ Guy P. Richardson,k Dennis M. Freeman,†‡§{ and A. J. Aranyosi‡* †Harvard-MIT Division of Health Sciences and Technology, ‡Research Laboratory of Electronics, and §Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts; {Eaton-Peabody Laboratory of Auditory Physiology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; and kSchool of Life Sciences, University of Sussex, Falmer, United Kingdom ABSTRACT The solid component of the tectorial membrane (TM) is a porous matrix made up of the radial collagen fibers and the striated sheet matrix. The striated sheet matrix is believed to contribute to shear impedance in both the radial and longitudinal directions, but the molecular mechanisms involved have not been determined. A missense mutation in Tecta, a gene that encodes for the a-tectorin protein in the striated sheet matrix, causes a 60-dB threshold shift in mice with relatively little reduction in outer hair cell amplification. Here, we show that this threshold shift is coupled to changes in shear impedance, response to osmotic pressure, and concentration of fixed charge of the TM. In TectaY1870C/þ mice, the tectorin content of the TM was reduced, as was the content of glycoconjugates reacting with the lectin wheat germ agglutinin. Charge measurements showed Y C þ a decrease in fixed charge concentration from À6:451:4 mmol/L in wild-types to À2:150:7 mmol/L in Tecta 1870 / TMs. TMs from TectaY1870C/þ mice showed little volume change in response to osmotic pressure compared to those of wild-type mice. The magnitude of both radial and longitudinal TM shear impedance was reduced by 1051:6 dB in TectaY1870C/þ mice. However, the phase of shear impedance was unchanged. These changes are consistent with an increase in the porosity of the TM and a cor- responding decrease of the solid fraction. Mechanisms by which these changes can affect the coupling between outer and inner hair cells are discussed. INTRODUCTION The tectorial membrane (TM) is an acellular gelatinous elevation in compound action potential (CAP) threshold, structure that contacts the hair cells in the organ of Corti but only an 8-dB reduction in basilar membrane (BM) and which contains multiple glycoproteins that are highly displacement (1). Taken together, these results indicate expressed only in the inner ear (1). Sound-induced vibra- that OHC amplification is only slightly affected in tions of the organ drive radial shearing displacement of TectaY1870C/þmice, but the amplified motion does not effec- the TM relative to the reticular lamina (RL) (2). This radial tively drive IHC bundle deflection. Here, we report motion is believed to deflect outer hair cell (OHC) bundles measurements of bulk compressibility, fixed charge concen- directly and inner hair cell (IHC) bundles through fluid tration, and shear impedance of TMs from wild-type and interactions (3). The deflection of OHC bundles drives elec- TectaY1870C/þ mice (hereafter referred to as wild-type and tromechanical processes in both cell body (4) and hair TectaY1870C/þ TMs). These measurements illustrate the crit- bundles (5), one or both of which deflections are believed ical importance of the striated-sheet matrix to the mechan- to underlie cochlear amplification. The amplified motion ical, material, and electrical properties of the TM. increases IHC bundle deflection, which provides the Moreover, they provide some insight into how the molecular majority of auditory input to the central nervous system. structure of the TM contributes to its two roles in cochlear Consequently, the TM plays at least two mechanical roles function. in hearing: driving OHC motility and coupling the increased motion to IHCs. METHODS A recent study (1) provides evidence that these two roles of the TM are separable. That study presented a mouse with Preparation of the isolated TM a missense mutation in Tecta that caused a Y1870C amino þ TectaY1870C/ transgenic and wild-type mice 6–10 weeks old were asphyx- acid substitution in a-tectorin, a protein that is specific to iated with CO2 and then decapitated. The pinnae and surrounding tissues the TM in adult cochlea (6). In humans, a similar mutation were removed and the temporal bone was isolated. The temporal bone causes a dominant 50- to 80-dB hearing loss. Mice hetero- was chipped away with a scalpel to isolate the cochlea, using a dissecting zygous for this mutation (TectaY1870C/þ mice) had a 55-dB microscope for observation. The cochlea was placed in an artificial-endo- lymph (AE) solution containing (in mmol/L) 174 KCl, 2 NaCl, 0.02 CaCl2, and 5 HEPES, with pH adjusted to 7.3. The cochlea was widely opened to allow access to the organ of Corti. The TM was isolated from Submitted May 9, 2010, and accepted for publication September 20, 2010. the rest of the organ by probing the organ with an eyelash. Individual pieces *Correspondence: [email protected] of TM, primarily from the midfrequency region of the cochlea (apical and Kinuko Masaki’s present address is Advanced Bionics Corporation, 28515 middle turns), were located and transferred via pipette to a glass slide. This Westinghouse Pl., Valencia, CA 91355. slide was coated with 0.3 mL of Cell-Tak bioadhesive (BD Biosciences, Editor: Denis Wirtz. Ó 2010 by the Biophysical Society 0006-3495/10/11/3274/8 $2.00 doi: 10.1016/j.bpj.2010.09.033 TM Material Properties in Tecta Mice 3275 Bedford, MA) to immobilize the TM on the slide surface. This immobiliza- potential between baths was measured with Ag/AgCl electrodes, and tion served three purposes: 1), it kept the TM from being carried out with coupled through an amplifier (DAM60-G Differential Amplifier, World the effluent as various fluids were perfused; 2), it allowed microfabricated Precision Instruments, Sarasota, FL) to a multimeter (TX3 True RMS Mul- probes to exert shearing forces rather than displace the bulk of the TM; 3), it timeter, Tektronix, Portland, OR) connected to a computer. DC and AC allowed TM volume changes to be calculated by tracking the positions of potentials were read from the multimeter by the computer every 2–3 s. beads on the TM and the surrounding glass slide. Measurements with AC potentials as large as the DC ones indicated the presence of electrical noise and were discarded. Each test bath was perfused for 10–30 min at a time, and each bath was perfused at least twice over the Analysis of tectorial membrane proteins course of an experiment. Tectorial membranes were collected from cochleas of wild-type and Y1870C/þ Tecta mice at 3 weeks of age, as described previously (7). In brief, Measuring stress-strain relation mice were killed by exposure to CO2, the labyrinths were placed in cold phosphate-buffered saline (PBS), and the bony capsules surrounding the The methods used to measure the stress-strain relation of the TM are as pub- cochlea and the lateral wall of the cochlear duct were removed by dissec- lished previously (9,13). Briefly, TMs from TectaY1870C/þ and wild-type tion. The samples were briefly stained with 1% Alcian Blue for mice were isolated and affixed to a glass slide. The TM was immersed in 15 min to lightly stain the upper surface of the TM and therefore aid visu- AE containing various concentrations of polyethylene glycol (PEG) with alization and subsequent collection. The lightly stained TMs were then a molecular weight (MW) of 511 kDa. The applied osmotic pressure for teased away from the surface of the spiral limbus with a fine dissecting nee- each solution ranged from 0 to 10 kPa and was computed from the concen- dle, collected in cold PBS containing 0.1% TX-100, pelleted in a microfuge tration and molecular weight of PEG as described previously (13). The tube, and solubilized by heating to 100 C for 4 min in reducing SDS PAGE surface of the TM was decorated with fluorescent microspheres (i.e., sample buffer. The solubilized TM proteins from a roughly equivalent beads), and sets of 100 images of the TM at focal depths separated by number of TMs were separated on 8.25% acrylamide gels and visualized 1 mm were taken once per minute. The positions of beads on the surface by one of three methods. One gel was stained with Coomassie Brilliant were tracked to determine changes in TM height with an accuracy of blue to make all protein bands visible. Two others were electrophoretically 0.1 mm. The z-component of the strain, 3z, is given by transferred to polyvinylidene fluoride membranes that were then stained with 1), a mixture of polyclonal sera raised to chicken Tecta and chicken 3z ¼ 1 À vz; (2) Tectb (R9 and R7) (8); or 2), biotinylated wheat germ agglutinin (WGA, Vector, Burlingame, CA).