https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=pubmed&id=31920560&retmode=xml&tool=Litmetric&email=readroberts32@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09 3192056020201001
1662-5102132019Frontiers in cellular neuroscienceFront Cell Neurosci3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.56056056010.3389/fncel.2019.00560Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features.Copyright © 2019 Triffo, Palsdottir, Song, Morgan, McDonald, Auer and Raphael.TriffoWilliam JeffreyWJMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.Department of Bioengineering, George R. Brown School of Engineering, Rice University, Houston, TX, United States.Department of Radiology, Geisinger, Danville, PA, United States.PalsdottirHildurHMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.SongJunhaJMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.MorganDavid GeneDGInterdisciplinary Center for Electron Microscopy, University of California, Davis, Davis, CA, United States.McDonaldKent LKLElectron Microscope Laboratory, University of California, Berkeley, Berkeley, CA, United States.AuerManfredMMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.RaphaelRobert MRMDepartment of Bioengineering, George R. Brown School of Engineering, Rice University, Houston, TX, United States.engJournal Article20191220
SwitzerlandFront Cell Neurosci1014779351662-5102cortical cytoskeletonelectron tomography (ET)high pressure freezing and freeze substitutionouter hair cell (OHC)subsurface cisternae2019482019124202011160202011160202011161201911epublish31920560PMC693331610.3389/fncel.2019.00560Ashmore J. F. (1987). A fast motile response in guinea-pig outer hair cells: the cellular basis of the cochlear amplifier. J. Physiol. 388, 323–347. 10.1113/jphysiol.1987.sp01661710.1113/jphysiol.1987.sp016617PMC11925513656195Ashmore J. (2008). Cochlear outer hair cell motility. Physiol. Rev. 88, 173–210. 10.1152/physrev.00044.200610.1152/physrev.00044.200618195086Brownell W. E. (1990). Outer hair cell electromotility and otoacoustic emissions. Ear Hear. 11, 82–92. 10.1097/00003446-199004000-0000310.1097/00003446-199004000-00003PMC27962342187727Brownell W. E. (2017). What is electromotility? The history of its discovery and its relevance to acoustics. Acoust. Today 13, 20–27.PMC564505329051713Brownell W. E., Bader C. R., Bertrand D., de Ribaupierre Y. (1985). Evoked mechanical responses of isolated cochlear outer hair cells. Science 227, 194–196. 10.1126/science.396615310.1126/science.39661533966153Brownell W. E., Spector A., Raphael R. M., Popel A. S. (2001). Micro- and nanomechanics of the cochlear outer hair cell. Annu. Rev. Biomed. Eng. 3, 169–194. 10.1146/annurev.bioeng.3.1.16910.1146/annurev.bioeng.3.1.16911447061Buser C., Walther P. (2008). Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around −60 degrees C. J. Microsc. 230, 268–277. 10.1111/j.1365-2818.2008.01984.x10.1111/j.1365-2818.2008.01984.x18445157Chertoff M. E., Brownell W. E. (1994). Characterization of cochlear outer hair cell turgor. Am. J. Physiol. 266, C467–C479. 10.1152/ajpcell.1994.266.2.c46710.1152/ajpcell.1994.266.2.c4678141262Dallos P., Evans B. N., Hallworth R. (1991). Nature of the motor element in electrokinetic shape changes of cochlear outer hair cells. Nature 350, 155–157. 10.1038/350155a010.1038/350155a02005965Dallos P., Hallworth R., Evans B. N. (1993). Theory of electrically driven shape changes of cochlear outer hair cells. J. Neurophysiol. 70, 299–323. 10.1152/jn.1993.70.1.29910.1152/jn.1993.70.1.2998395582Dallos P., Wu X., Cheatham M. A., Gao J., Zheng J., Anderson C. T., et al. . (2008). Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification. Neuron 58, 333–339. 10.1016/j.neuron.2008.02.02810.1016/j.neuron.2008.02.028PMC243506518466744Dieler R., Shehata-Dieler W. E., Brownell W. E. (1991). Concomitant salicylate-induced alterations of outer hair cell subsurface cisternae and electromotility. J. Neurocytol. 20, 637–653. 10.1007/bf0118706610.1007/bf011870661940979Duret G., Pereira F. A., Raphael R. M. (2017). Diflunisal inhibits prestin by chloride-dependent mechanism. PLoS One 12:e0183046. 10.1371/journal.pone.018304610.1371/journal.pone.0183046PMC556073428817613Flock A., Flock B., Ulfendahl M. (1986). Mechanisms of movement in outer hair cells and a possible structural basis. Arch. Otorhinolaryngol. 243, 83–90. 10.1007/bf0045375510.1007/bf004537553521564Forge A. (1991). Structural features of the lateral walls in mammalian cochlear outer hair cells. Cell Tissue Res. 265, 473–483. 10.1007/bf0034087010.1007/bf003408701786594Frolenkov G. I. (2006). Regulation of electromotility in the cochlear outer hair cell. J. Physiol. 576, 43–48. 10.1113/jphysiol.2006.11497510.1113/jphysiol.2006.114975PMC199562316887876Frolenkov G. I., Mammano F., Kachar B. (2001). Action of 2,3-butanedione monoxime on capacitance and electromotility of guinea-pig cochlear outer hair cells. J. Physiol. 531, 667–676. 10.1111/j.1469-7793.2001.0667h.x10.1111/j.1469-7793.2001.0667h.xPMC227849211251049Furness D. N., Hackney C. M. (1990). Comparative ultrastructure of subsurface cisternae in inner and outer hair cells of the guinea pig cochlea. Eur. Arch. Otorhinolaryngol. 247, 12–15. 10.1007/bf0024094110.1007/bf002409412310542Gao J., Wang X., Wu X., Aguinaga S., Huynh K., Jia S., et al. . (2007). Prestin-based outer hair cell electromotility in knockin mice does not appear to adjust the operating point of a cilia-based amplifier. Proc. Natl. Acad. Sci. U S A 104, 12542–12547. 10.1073/pnas.070035610410.1073/pnas.0700356104PMC194150517640919Goddard T. D., Huang C. C., Ferrin T. E. (2007). Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287. 10.1016/j.jsb.2006.06.01010.1016/j.jsb.2006.06.01016963278Grant L., Slapnick S., Kennedy H., Hackney C. (2006). Ryanodine receptor localisation in the mammalian cochlea: an ultrastructural study. Hear. Res. 219, 101–109. 10.1016/j.heares.2006.06.00210.1016/j.heares.2006.06.00216889917Greeson J. N., Raphael R. M. (2009). Amphipath-induced nanoscale changes in outer hair cell plasma membrane curvature. Biophys. J. 96, 510–520. 10.1016/j.bpj.2008.09.01610.1016/j.bpj.2008.09.016PMC271646419167301Hallworth R., Evans B. N., Dallos P. (1993). The location and mechanism of electromotility in guinea pig outer hair cells. J. Neurophysiol. 70, 549–558. 10.1152/jn.1993.70.2.54910.1152/jn.1993.70.2.5498410156Halter J. A., Kruger R. P., Yium M. J., Brownell W. E. (1997). The influence of the subsurface cisterna on the electrical properties of the outer hair cell. Neuroreport 8, 2517–2521. 10.1097/00001756-199707280-0002010.1097/00001756-199707280-000209261819He D. Z., Zheng J., Kalinec F., Kakehata S., Santos-Sacchi J. (2006). Tuning in to the amazing outer hair cell: membrane wizardry with a twist and shout. J. Membr. Biol. 209, 119–134. 10.1007/s00232-005-0833-910.1007/s00232-005-0833-916773497Holley M. C., Ashmore J. F. (1988a). On the mechanism of a high-frequency force generator in outer hair cells isolated from the guinea pig cochlea. Proc. R. Soc. Lond. B Biol. Sci. 232, 413–429. 10.1098/rspb.1988.000410.1098/rspb.1988.00042895478Holley M. C., Ashmore J. F. (1988b). A cytoskeletal spring in cochlear outer hair cells. Nature 335, 635–637. 10.1038/335635a010.1038/335635a03173482Holley M. C., Ashmore J. F. (1990). Spectrin, actin and the structure of the cortical lattice in mammalian cochlear outer hair cells. J. Cell Sci. 96, 283–291.2211870Holley M. C., Kalinec F., Kachar B. (1992). Structure of the cortical cytoskeleton in mammalian outer hair cells. J. Cell Sci. 102, 569–580.1506434Huang G., Santos-Sacchi J. (1994). Motility voltage sensor of the outer hair cell resides within the lateral plasma membrane. Proc. Natl. Acad. Sci. U S A 91, 12268–12272. 10.1073/pnas.91.25.1226810.1073/pnas.91.25.12268PMC454187991617Hudspeth A. J. (2014). Integrating the active process of hair cells with cochlear function. Nat. Rev. Neurosci. 15, 600–614. 10.1038/nrn378610.1038/nrn378625096182Iwasa K. H. (1994). A membrane motor model for the fast motility of the outer hair cell. J. Acoust. Soc. Am. 96, 2216–2224. 10.1121/1.41009410.1121/1.4100947963034Kachar B., Brownell W. E., Altschuler R., Fex J. (1986). Electrokinetic shape changes of cochlear outer hair cells. Nature 322, 365–368. 10.1038/322365a010.1038/322365a03736662Kakehata S., Dallos P., Brownell W. E., Iwasa K. H., Kachar B., Kalinec F., et al. . (2000). Current concept of outer hair cell motility. Auris Nasus Larynx 27, 349–355. 10.1016/s0385-8146(00)00081-x10.1016/s0385-8146(00)00081-x10996495Kalinec F., Holley M. C., Iwasa K. H., Lim D. J., Kachar B. (1992). A membrane-based force generation mechanism in auditory sensory cells. Proc. Natl. Acad. Sci. U S A 89, 8671–8675. 10.1073/pnas.89.18.867110.1073/pnas.89.18.8671PMC499821528879Kremer J. R., Mastronarde D. N., McIntosh J. R. (1996). Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76. 10.1006/jsbi.1996.001310.1006/jsbi.1996.00138742726Liberman M. C., Gao J., He D. Z., Wu X., Jia S., Zuo J. (2002). Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 419, 300–304. 10.1038/nature0105910.1038/nature0105912239568Lutz C., Schweitzer L. (1995). Longitudinal and radial differences in the subsurface cisternal system in the gerbil cochlea. Hear. Res. 84, 12–18. 10.1016/0378-5955(95)00008-r10.1016/0378-5955(95)00008-r7642445Mastronarde D. N. (2005). Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51. 10.1016/j.jsb.2005.07.00710.1016/j.jsb.2005.07.00716182563McDonald K. (2007). Cryopreparation methods for electron microscopy of selected model systems. Methods Cell Biol. 79, 23–56. 10.1016/s0091-679x(06)79002-110.1016/s0091-679x(06)79002-117327151McDonald K. L., Auer M. (2006). High-pressure freezing, cellular tomography, and structural cell biology. Biotechniques 41, 137, 139,, 141 passim. 10.2144/00011222610.2144/00011222616925014McDonald K., Müller-Reichert T. (2002). Cryomethods for thin section electron microscopy. Methods Enzymol. 351, 96–123. 10.1016/s0076-6879(02)51843-710.1016/s0076-6879(02)51843-712073378Morimoto N., Raphael R. M., Brownell W. E. (2002). Excess plasma membrane and effects of ionic amphipaths on mechanics of outer hair cell lateral wall. Am. J. Physiol. Cell Physiol. 282, C1076–C1086. 10.1152/ajpcell.00210.200110.1152/ajpcell.00210.200111940523Müller-Reichert T., Hohenberg H., O’Toole E. T., McDonald K. (2003). Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure. J. Microsc. 212, 71–80. 10.1046/j.1365-2818.2003.01250.x10.1046/j.1365-2818.2003.01250.x14516364Nygren A., Halter J. A. (1999). A general approach to modeling conduction and concentration dynamics in excitable cells of concentric cylindrical geometry. J. Theor. Biol. 199, 329–358. 10.1006/jtbi.1999.096210.1006/jtbi.1999.096210433897Oghalai J. S., Patel A. A., Nakagawa T., Brownell W. E. (1998). Fluorescence-imaged microdeformation of the outer hair cell lateral wall. J. Neurosci. 18, 48–58. 10.1523/jneurosci.18-01-00048.199810.1523/jneurosci.18-01-00048.1998PMC67934069412485Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., et al. . (2004). UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612. 10.1002/jcc.2008410.1002/jcc.2008415264254Raphael R. M., Popel A. S., Brownell W. E. (2000). A membrane bending model of outer hair cell electromotility. Biophys. J. 78, 2844–2862. 10.1016/s0006-3495(00)76827-510.1016/s0006-3495(00)76827-5PMC130087210827967Santos-Sacchi J., Dilger J. P. (1988). Whole cell currents and mechanical responses of isolated outer hair cells. Hear. Res. 35, 143–150. 10.1016/0378-5955(88)90113-x10.1016/0378-5955(88)90113-x2461927Santos-Sacchi J., Kakehata S., Kikuchi T., Katori Y., Takasaka T. (1998). Density of motility-related charge in the outer hair cell of the guinea pig is inversely related to best frequency. Neurosci. Lett. 256, 155–158. 10.1016/s0304-3940(98)00788-510.1016/s0304-3940(98)00788-59855363Santos-Sacchi J., Navaratnam D., Raphael R. M., Oliver D. M. (2017). “Prestin: molecular mechanisms underlying outer hair cell electromotility,” in Understanding the Cochlea, eds Manley G., Gummer A., Popper A., Fay R. (Cham: Springer; ), 113–145.Sato T. (1968). A modified method for lead staining of thin sections. J. Electron Microsc. 17, 158–159.4177281Serysheva I. I., Hamilton S. L., Chiu W., Ludtke S. J. (2005). Structure of Ca2+ release channel at 14 A resolution. J. Mol. Biol. 345, 427–431. 10.1016/j.jmb.2004.10.07310.1016/j.jmb.2004.10.073PMC297851215581887Slepecky N. B. (1996). “Structure of the mammalian cochlea,” in The Cochlea, eds Dallos P., Richard R. F. (New York, NY: Springer-Verlag; ), 44–129.Slepecky N. B., Ligotti P. J. (1992). Characterization of inner ear sensory hair cells after rapid-freezing and freeze-substitution. J. Neurocytol. 21, 374–381. 10.1007/bf0119170510.1007/bf011917051376772Song L., Santos-Sacchi J. (2015). An electrical inspection of the subsurface cisternae of the outer hair cell. Biophys. J. 108, 568–577. 10.1016/j.bpj.2014.12.01010.1016/j.bpj.2014.12.010PMC431753925650924Spector A. A., Deo N., Grosh K., Ratnanather J. T., Raphael R. M. (2006). Electromechanical models of the outer hair cell composite membrane. J. Membr. Biol. 209, 135–152. 10.1007/s00232-005-0843-710.1007/s00232-005-0843-716773498Tolomeo J. A., Steele C. R., Holley M. C. (1996). Mechanical properties of the lateral cortex of mammalian auditory outer hair cells. Biophys. J. 71, 421–429. 10.1016/s0006-3495(96)79244-510.1016/s0006-3495(96)79244-5PMC12334938804625Triffo W. J., Palsdottir H. K., McDonald K. L., Lee J. K., Inman J., Bissell M. J., et al. . (2008). Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography. J. Microsc. 230, 278–287. 10.1111/j.1365-2818.2008.01986.x10.1111/j.1365-2818.2008.01986.xPMC273414018445158Ulfendahl M., Slepecky N. (1988). Ultrastructural correlates of inner ear sensory cell shortening. J. Submicrosc. Cytol. Pathol. 20, 47–51.3370621Wada H., Kimura K., Gomi T., Sugawara M., Katori Y., Kakehata S., et al. . (2004). Imaging of the cortical cytoskeleton of guinea pig outer hair cells using atomic force microscopy. Hear. Res. 187, 51–62. 10.1016/s0378-5955(03)00334-410.1016/s0378-5955(03)00334-414698087Walther P., Ziegler A. (2002). Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water. J. Microsc. 208, 3–10. 10.1046/j.1365-2818.2002.01064.x10.1046/j.1365-2818.2002.01064.x12366592Zelenskaya A., de Monvel J. B., Pesen D., Radmacher M., Hoh J. H., Ulfendahl M. (2005). Evidence for a highly elastic shell-core organization of cochlear outer hair cells by local membrane indentation. Biophys. J. 88, 2982–2993. 10.1529/biophysj.104.05222510.1529/biophysj.104.052225PMC130539215653728Zheng J., Shen W., He D. Z., Long K. B., Madison L. D., Dallos P. (2000). Prestin is the motor protein of cochlear outer hair cells. Nature 405, 149–155. 10.1038/3501200910.1038/3501200910821263