Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.
Brownell WE, Bader CR, Bertrand D, de Ribaupierre Y. Evoked mechanical responses of isolated cochlear outer hair cells. Science. 1985;227(4683):194–6. pmid:3966153 doi: 10.1126/science.3966153
[3]
Cheatham MA, Zheng J, Huynh KH, Du GG, Edge RM, Anderson CT, et al. Evaluation of an independent prestin mouse model derived from the 129S1 strain. Audiol Neurootol. 2007;12(6):378–90. pmid:17664869 doi: 10.1159/000106481
[4]
Dallos P, Wu X, Cheatham MA, Gao J, Zheng J, Anderson CT, et al. Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification. Neuron. 2008;58(3):333–9. doi: 10.1016/j.neuron.2008.02.028. pmid:18466744
[5]
Fisher JA, Nin F, Reichenbach T, Uthaiah RC, Hudspeth AJ. The spatial pattern of cochlear amplification. Neuron. 2012;76(5):989–97. doi: 10.1016/j.neuron.2012.09.031. pmid:23217746
[6]
Jacob S, Pienkowski M, Fridberger A. The endocochlear potential alters cochlear micromechanics. Biophys J. 2011;100(11):2586–94. doi: 10.1016/j.bpj.2011.05.002. pmid:21641303
[7]
Liberman MC, Gao J, He DZ, Wu X, Jia S, Zuo J. Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature. 2002;419(6904):300–4. pmid:12239568 doi: 10.1038/nature01059
[8]
Liberman MC, Zuo J, Guinan JJ Jr. Otoacoustic emissions without somatic motility: can stereocilia mechanics drive the mammalian cochlea? J Acoust Soc Am. 2004;116(3):1649–55. pmid:15478431 doi: 10.1121/1.1775275
[9]
He DZ, Jia S, Sato T, Zuo J, Andrade LR, Riordan GP, et al. Changes in plasma membrane structure and electromotile properties in prestin deficient outer hair cells. Cytoskeleton (Hoboken). 2010;67(1):43–55. doi: 10.1002/cm.20423
[10]
Oghalai JS, Zhao HB, Kutz JW, Brownell WE. Voltage- and tension-dependent lipid mobility in the outer hair cell plasma membrane. Science. 2000;287(5453):658–61. pmid:10650000 doi: 10.1126/science.287.5453.658
[11]
Kamar RI, Organ-Darling LE, Raphael RM. Membrane cholesterol strongly influences confined diffusion of prestin. Biophys J. 2012;103(8):1627–36. doi: 10.1016/j.bpj.2012.07.052. pmid:23083705
[12]
Organ LE, Raphael RM. Application of fluorescence recovery after photobleaching to study prestin lateral mobility in the human embryonic kidney cell. J Biomed Opt. 2007;12(2):021003. pmid:17477710 doi: 10.1117/1.2715202
[13]
Santos-Sacchi J, Zhao HB. Excitation of fluorescent dyes inactivates the outer hair cell integral membrane motor protein prestin and betrays its lateral mobility. Pflugers Arch. 2003;446(5):617–22. pmid:12783229 doi: 10.1007/s00424-003-1053-8
[14]
Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature biotechnology. 2002;20(1):87–90. pmid:11753368 doi: 10.1038/nbt0102-87
[15]
Belyantseva IA, Adler HJ, Curi R, Frolenkov GI, Kachar B. Expression and localization of prestin and the sugar transporter GLUT-5 during development of electromotility in cochlear outer hair cells. J Neurosci. 2000;20(24):RC116. pmid:11125015
[16]
Legendre K, Safieddine S, Kussel-Andermann P, Petit C, El-Amraoui A. alphaII-betaV spectrin bridges the plasma membrane and cortical lattice in the lateral wall of the auditory outer hair cells. J Cell Sci. 2008;121(Pt 20):3347–56. doi: 10.1242/jcs.028134. pmid:18796539
[17]
Mahendrasingam S, Beurg M, Fettiplace R, Hackney CM. The ultrastructural distribution of prestin in outer hair cells: a post-embedding immunogold investigation of low-frequency and high-frequency regions of the rat cochlea. Eur J Neurosci. 2010;31(9):1595–605. doi: 10.1111/j.1460-9568.2010.07182.x. pmid:20525072
[18]
Cheatham MA, Huynh KH, Gao J, Zuo J, Dallos P. Cochlear function in Prestin knockout mice. J Physiol. 2004;560(Pt 3):821–30. pmid:15319415 doi: 10.1113/jphysiol.2004.069559
[19]
de Monvel JB, Brownell WE, Ulfendahl M. Lateral diffusion anisotropy and membrane lipid/skeleton interaction in outer hair cells. Biophys J. 2006;91(1):364–81. pmid:16603502 doi: 10.1529/biophysj.105.076331
[20]
McAnaney TB, Zeng W, Doe CF, Bhanji N, Wakelin S, Pearson DS, et al. Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism. Biochemistry. 2005;44(14):5510–24. pmid:15807545 doi: 10.1021/bi047581f
[21]
Richards DA, De Paola V, Caroni P, Gahwiler BH, McKinney RA. AMPA-receptor activation regulates the diffusion of a membrane marker in parallel with dendritic spine motility in the mouse hippocampus. J Physiol. 2004;558(Pt 2):503–12. pmid:15169845 doi: 10.1113/jphysiol.2004.062091
[22]
Digman MA, Dalal R, Horwitz AF, Gratton E. Mapping the number of molecules and brightness in the laser scanning microscope. Biophys J. 2008;94(6):2320–32. pmid:18096627 doi: 10.1529/biophysj.107.114645
[23]
Brownell WE, Spector AA, Raphael RM, Popel AS. Micro- and nanomechanics of the cochlear outer hair cell. Annual review of biomedical engineering. 2001;3:169–94. pmid:11447061 doi: 10.1146/annurev.bioeng.3.1.169
[24]
Beurg M, Tan X, Fettiplace R. A Prestin Motor in Chicken Auditory Hair Cells: Active Force Generation in a Nonmammalian Species. Neuron. 2013. doi: 10.1016/j.neuron.2013.05.018
[25]
Brownell WE, Jacob S, Hakizimana P, Ulfendahl M, Fridberger A. Membrane cholesterol modulates cochlear electromechanics. Pflugers Arch. 2011;461(6):677–86. doi: 10.1007/s00424-011-0942-5. pmid:21373862
[26]
Rajagopalan L, Greeson JN, Xia A, Liu H, Sturm A, Raphael RM, et al. Tuning of the outer hair cell motor by membrane cholesterol. J Biol Chem. 2007;282(50):36659–70. pmid:17933870 doi: 10.1074/jbc.m705078200
[27]
Thomas PV, Cheng AL, Colby CC, Liu L, Patel CK, Josephs L, et al. Localization and proteomic characterization of cholesterol-rich membrane microdomains in the inner ear. Journal of proteomics. 2014;103C:178–93. doi: 10.1016/j.jprot.2014.03.037
[28]
Szarama KB, Gavara N, Petralia RS, Kelley MW, Chadwick RS. Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea. Development. 2012;139(12):2187–97. doi: 10.1242/dev.073734. pmid:22573615
[29]
Adachi M, Iwasa KH. Effect of diamide on force generation and axial stiffness of the cochlear outer hair cell. Biophys J. 1997;73(5):2809–18. pmid:9370475 doi: 10.1016/s0006-3495(97)78310-3
[30]
Kitani R, Park C, Kalinec F. Microdomains shift and rotate in the lateral wall of cochlear outer hair cells. Biophys J. 2013;104(1):8–18. doi: 10.1016/j.bpj.2012.11.3828. pmid:23332054
[31]
Dieler R, Shehata-Dieler WE, Brownell WE. Concomitant salicylate-induced alterations of outer hair cell subsurface cisternae and electromotility. Journal of neurocytology. 1991;20(8):637–53. pmid:1940979 doi: 10.1007/bf01187066
[32]
Pollice PA, Brownell WE. Characterization of the outer hair cell's lateral wall membranes. Hear Res. 1993;70(2):187–96. pmid:8294263 doi: 10.1016/0378-5955(93)90157-v
[33]
Yamashita T, Fang J, Gao J, Yu Y, Lagarde MM, Zuo J. Normal hearing sensitivity at low-to-middle frequencies with 34% prestin-charge density. PLoS One. 2012;7(9):e45453. doi: 10.1371/journal.pone.0045453. pmid:23029017
[34]
Wu X, Gao J, Guo Y, Zuo J. Hearing threshold elevation precedes hair-cell loss in prestin knockout mice. Brain Res Mol Brain Res. 2004;126(1):30–7. pmid:15207913 doi: 10.1016/j.molbrainres.2004.03.020
[35]
Madisen L, Mao T, Koch H, Zhuo JM, Berenyi A, Fujisawa S, et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat Neurosci. 2012;15(5):793–802. doi: 10.1038/nn.3078. pmid:22446880
[36]
Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45(9):593–605. pmid:17868096 doi: 10.1002/dvg.20335
[37]
Gorbunov D, Sturlese M, Nies F, Kluge M, Bellanda M, Battistutta R, et al. Molecular architecture and the structural basis for anion interaction in prestin and SLC26 transporters. Nature communications. 2014;5:3622. doi: 10.1038/ncomms4622. pmid:24710176
[38]
Weddell TD, Mellado-Lagarde M, Lukashkina VA, Lukashkin AN, Zuo J, Russell IJ. Prestin links extrinsic tuning to neural excitation in the mammalian cochlea. Curr Biol. 2011;21(18):R682–3. doi: 10.1016/j.cub.2011.08.001. pmid:21959151
[39]
Zhang DS, Piazza V, Perrin BJ, Rzadzinska AK, Poczatek JC, Wang M, et al. Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. Nature. 2012;481(7382):520–4. doi: 10.1038/nature10745. pmid:22246323
[40]
Abrashkin KA, Izumikawa M, Miyazawa T, Wang CH, Crumling MA, Swiderski DL, et al. The fate of outer hair cells after acoustic or ototoxic insults. Hear Res. 2006;218(1–2):20–9. pmid:16777363 doi: 10.1016/j.heares.2006.04.001
[41]
Harvey L, Arnold B, S Lawrence Z, Paul M, David B, James D. Section 5.3, Biomembranes: Structural Organization and Basic Functions. Molecular Cell Biology, 4th edition. 2000.
[42]
Santi PA, Mancini P, Barnes C. Identification and localization of the GM1 ganglioside in the cochlea using thin-layer chromatography and cholera toxin. J Histochem Cytochem. 1994;42(6):705–16. pmid:8189033 doi: 10.1177/42.6.8189033
[43]
Sturm AK, Rajagopalan L, Yoo D, Brownell WE, Pereira FA. Functional expression and microdomain localization of prestin in cultured cells. Otolaryngology—head and neck surgery: official journal of American Academy of Otolaryngology-Head and Neck Surgery. 2007;136(3):434–9. doi: 10.1016/j.otohns.2006.10.030
Santos-Sacchi J. Reversible inhibition of voltage-dependent outer hair cell motility and capacitance. J Neurosci. 1991;11(10):3096–110. pmid:1941076
[46]
Shehata WE, Brownell WE, Dieler R. Effects of salicylate on shape, electromotility and membrane characteristics of isolated outer hair cells from guinea pig cochlea. Acta oto-laryngologica. 1991;111(4):707–18. pmid:1950533 doi: 10.3109/00016489109138403
[47]
Burchard RP. Gliding motility of prokaryotes: ultrastructure, physiology, and genetics. Annual review of microbiology. 1981;35:497–529. pmid:6117246 doi: 10.1146/annurev.mi.35.100181.002433
[48]
Hoiczyk E, Baumeister W. Envelope structure of four gliding filamentous cyanobacteria. J Bacteriol. 1995;177(9):2387–95. pmid:7730269
[49]
Martiniere A, Lavagi I, Nageswaran G, Rolfe DJ, Maneta-Peyret L, Luu DT, et al. Cell wall constrains lateral diffusion of plant plasma-membrane proteins. Proc Natl Acad Sci U S A. 2012;109(31):12805–10. doi: 10.1073/pnas.1202040109. pmid:22689944
[50]
Peters A, Proskauer CC, Kaiserman-Abramof IR. The small pyramidal neuron of the rat cerebral cortex. The axon hillock and initial segment. J Cell Biol. 1968;39(3):604–19. pmid:5699934 doi: 10.1083/jcb.39.3.604
[51]
Sloper JJ, Powell TP. A study of the axon initial segment and proximal axon of neurons in the primate motor and somatic sensory cortices. Philosophical transactions of the Royal Society of London Series B, Biological sciences. 1979;285(1006):173–97. pmid:88058 doi: 10.1098/rstb.1979.0004
[52]
Xu K, Zhong G, Zhuang X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science. 2013;339(6118):452–6. doi: 10.1126/science.1232251. pmid:23239625
[53]
Somers KL, Royals MA, Carstea ED, Rafi MA, Wenger DA, Thrall MA. Mutation analysis of feline Niemann-Pick C1 disease. Molecular genetics and metabolism. 2003;79(2):99–103. pmid:12809639 doi: 10.1016/s1096-7192(03)00074-x
[54]
Vite CH, Ding W, Bryan C, O'Donnell P, Cullen K, Aleman D, et al. Clinical, electrophysiological, and serum biochemical measures of progressive neurological and hepatic dysfunction in feline Niemann-Pick type C disease. Pediatric research. 2008;64(5):544–9. doi: 10.1203/PDR.0b013e318184d2ce. pmid:18614965
[55]
Crumling MA, Liu L, Thomas PV, Benson J, Kanicki A, Kabara L, et al. Hearing loss and hair cell death in mice given the cholesterol-chelating agent hydroxypropyl-beta-cyclodextrin. PLoS One. 2012;7(12):e53280. doi: 10.1371/journal.pone.0053280. pmid:23285273
[56]
Ward S, O'Donnell P, Fernandez S, Vite CH. 2-hydroxypropyl-beta-cyclodextrin raises hearing threshold in normal cats and in cats with Niemann-Pick type C disease. Pediatric research. 2010;68(1):52–6. pmid:20357695 doi: 10.1203/pdr.0b013e3181df4623
[57]
Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976;16(9):1055–69. pmid:786399 doi: 10.1016/s0006-3495(76)85755-4