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1 TM TM tip link horizontl top connectors 1 leucine-rich (21 %) otoncorin-like 1809 ntigenic peptides B D signl peptide hydrophoic segment proline/threonine-rich (79 %) Supplementry Figure 1. () The outer hir cell s hir undle (mouse). Stereocili re rrnged in three rows of grded heights. The tllest stereocili hve their tips nchored in the tectoril memrne (TM). The tip link extends from the tip of stereocilium to the side of the djcent tller one, nd is thought to gte the mechnoelectricl trnsducer chnnel upon sound-induced deflection of the hir undle. The horizontl top connectors connect the upper prts of djcent stereocili, oth within nd etween rows. They hve zipper-like ppernce, due to the presence of centrl density (in green). () Predicted structure of murine stereocilin nd loction of the ntigenic peptides. The sequences of stereocilin nd otoncorin, defective in the DFNB16 4 nd DFNB22 35 recessive forms of defness, respectively, re 30 % identicl in 260/255 mino-cid frgment locted in their C-terminl regions, i.e (stereocilin) nd (otoncorin). 1

2 Bgl II proe F2 F1 R2 R1 Bgl II Bgl II +/ fl +/ F1 F2 HYGRO HR k 2.2 k R k 11.3 k c (+) DS AS d Strc + /+ Strc - /- (-) 1 4 M +/+ +/- -/- M 1140 p 329 p Supplementry Figure 2. Inctivtion of the Strc gene. () Structure of the Strc 5 region nd the trgeting construct used to produce floxed Strc llele with loxp sequences (tringles) flnking exons 2 nd 3. Nucleotide primers used for PCR genotyping re indicted y rrows. () Southern lot of Bgl II- digested genomic DNA shows wild-type (+) nd floxed (fl) Strc lleles. The proe hyridises to 11.3 k frgment from the wild-type llele nd to 13.5 k frgment from the floxed llele crrying the hygromycin resistnce gene. (c) RT-PCR nlysis of Strc expression in Strc +/+ (+/+), Strc +/- (+/-) nd Strc -/- (-/-) P15 inner ers. The expected 1140 p mplicon is seen in the +/+ nd +/- lnes, together with products of lower moleculr mss. Some of these products result from the use of lterntive splice donor (DS, GT consensus t cdna position 377) nd cceptor (AS, AG consensus t cdna position 827) sites locted in exon 2. Some others re likely to correspond to heteroduplex molecules formed y different mplicons. The 329 p mplicon from the deleted Strc -/- llele is present in the +/- lne nd is the only product seen in the -/- lne. If trnslted, the Strc -/- llele, crrying the 811 p frme shifting deletion of exons 2 nd 3, would produce n incomplete signl peptide (19 out of 22 mino cids) followed y 30 out-of-frme mino-cids. M, 100 p DNA ldder (Biols). (d) Compressions of z-stck confocl imges of whole mount preprtions of cochler sensory epitheli from P14 Strc +/+ nd Strc -/- mice stined for stereocilin (green) nd ctin (red). The stereocilin lelling is detected in the Strc +/+, ut not Strc -/- cochle. Scle rs : 10 µm. 2

3 frequency (khz) P P15 P P45 P60 80 hering loss (db HL) Supplementry Figure 3. Progressive elevtion of hering thresholds in Strc -/- mice. To evlute the effect of sent stereocilin on CAP thresholds in Strc -/- mice, the confounding effect of neurl immturity is removed y sutrcting the CAP thresholds of ge-mtched Strc +/+ mice t ech postntl stge. Thus t P14, the reference CAP udiogrm ws tht represented on Fig. 1 (lck line). At P15, the CAP udiogrm of Strc +/+ mice (grey line, Fig. 1) ws slightly more mture thn t P14. From P30 on, the CAP udiogrm of dult (P60) Strc +/+ mice ws used (Fig. 1, lck line). The resulting plots in db HL -for Hering Loss- (mens ± one s.e.m.) revel progressive hering loss in Strc -/- mice older thn P14. With incresing ge, the verge hering loss tends to tht expected in cse of totl filure of the cochler mplifier, tht is 60 db for sound frequencies ove 10 khz nd out 40 db t lower frequencies 16. The progressive mturtion of Strc +/+ CAP udiogrms with postntl ge mtches tht reported y Song et l. who report conspicuous mturtion of the ltencies nd mplitudes of neurl responses etween P13 nd P

4 input system output (= A o X input) input + A = A o /(1 - βa o ) system output (= A X input) feedck β % extnt functionl MET chnnels in OHCs predicted hering loss (db) Supplementry Figure 4. Models of cochler mplifier gin in reltion to OHC-sed electromechnicl feedck. () Principle of regenertive feedck mplifier. The gin A of simple feedck-sed mplifier (tht is, the rtio output / input) is A = A 0 / (1 - βa 0 ); A 0 is the gin in the sence of feedck (upper digrm), nd β is the portion of the output tht is fed ck to the input of the system (lower digrm). If βa 0 is just slightly < 1, A is much lrger thn A 0 nd the system possesses lrge gin, ensuring high sensitivity. Tuning occurs s result of feedck eing effective only t the resonnce frequency. () Effect of decresed feedck strength. Improved models ccount for the specifics of cochler micromechnics 17,37. They posit tht β is proportionl to the percentge n of functionl MET chnnels in OHCs, nd thus here, to the mesured cochler microphonic potentil. The predicted hering losses HL (in db) due to decresed feedck strength exhiit similr shpes (red line, Ptuzzi et l. 17, green dshed line, Neely & Kim 37, with the ltter uthors hving solved the model exctly only t n = 0.8 nd n = 0). The prediction used here (lue line) ssumed 60 db gin t high sound frequencies, with HL given y 17 : HL = 60 (1 n) / ( n). To uild Fig. 1c, experimentlly estimted n s (from the % of extnt low-frequency cochler microphonic potentil, lck line in Fig. 1c) were fed into the formul giving HL (pink shded re, Fig. 1c). For comprison, the oserved hering loss of Strc -/- mice (Fig. 1c, red line) ws derived from verging individul CAP thresholds cross the khz frequency rnge, where CAP udiogrms in db HL were rther flt (supplementry Fig. 3), nd where the norml gin is thought to e ~60 db. Of note, the model predicts tht the initil 20% drop in cochler microphonic potentil results in ~20 db-loss of gin. In contrst, the finl 20% drop dds only ~7 db to the lredy existing hering loss (Fig. 1c). In etween, i.e. from P15 to P45, the model lso predicts the existence of residul gin nd tuning. All these predictions were experimentlly confirmed (Fig. 1c, 1e). 4

5 CM (µv) Strc + /+ Strc - /- 5 µv time (ms) Supplementry Figure 5. Phse of cochler microphonic response in Strc +/+ nd Strc -/- mice. Cochler microphonic (CM) responses to 10 khz tone urst t 60 db SPL in one Strc +/+ nd one Strc -/- mouse t P14 show similr phses nd onset times reltive to the sound stimulus. 5

6 c d Supplementry Figure 6. Expnded distriution of stereocilin-ssocited lterl links nd ending of djcent stereocili in hrmonin-deficient (Ush1c -/- ) mice 38. () Scnning electron microgrph of mture Ush1c -/- OHC hir undle. The lterl links extend further towrds the stereocili se, s est illustrted in one pir of stereocili (rrowheds) on the corresponding two-fold enlrgement (right pnel). This results in ending of djcent stereocili towrd ech other. (, c) Single stck confocl imges of mture Ush1c -/- OHC hir undles stined for stereocilin (green, in nd c) nd ctin (red, in c). The stereocilin lelling etween djcent stereocili distriutes cross the upper two thirds of the stereocili length, insted of eing restricted to smll distl region s in wild-type mice. Scle rs: 1 µm (), 5 µm (, c). 6

7 Supplementry Notes 35. Zwenepoel, I. et l. Otoncorin, n inner er protein restricted to the interfce etween the picl surfce of sensory epitheli nd their overlying cellulr gels, is defective in utosoml recessive defness DFNB22. Proc. Ntl. Acd. Sci. USA 99, (2002). 36. Song, L., McGee, J. & Wlsh, E. J. Frequency- nd level-dependent chnges in uditory rinstem responses (ABRS) in developing mice. J. Acoust. Soc. Am. 119, (2006). 37. Neely, S. T. & Kim, D. O. A model for ctive elements in cochler iomechnics. J. Acoust. Soc. Am. 79, (1986). 38. Lefèvre, G. et l. A core cochler phenotype in USH1 mouse mutnts implictes firous links of the hir undle in its cohesion, orienttion nd differentil growth. Development 135, (2008). 7

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