TMHS Is an Integral Component of the Mechanotransduction Machinery of Cochlear Hair Cells

Wei Xiong,1 Nicolas Grillet,1 Heather M. Elledge,1 Thomas F.J. Wagner,1 Bo Zhao,1 Kenneth R. Johnson,2 Piotr Kazmierczak,1 and Ulrich Mu¨ller1,* 1The Dorris Neuroscience Center, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA 2The Jackson Laboratory, Bar Harbor, ME 04609, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2012.10.041

SUMMARY deflection of the stereociliary bundles, which directly control the activity of the mechanotransduction channels in stereocilia. Hair cells are mechanosensors for the perception of It is thought that tip links, fine extracellular filaments that connect sound, acceleration, and fluid motion. Mechano- the tips of neighboring stereocilia, transmit tension force onto transduction channels in hair cells are gated by tip the transduction channels (Gillespie and Mu¨ ller, 2009). links, which connect the stereocilia of a hair cell in In recent years, significant progress has been made in the direction of their mechanical sensitivity. The the identification of components of the mechanotransduction molecular constituents of the mechanotransduction machinery of hair cells (Figure 1A). These studies have shown that tip links are formed by CDH23 homodimers that interact channels of hair cells are not known. Here, we show with PCDH15 homodimers to form the upper and lower parts that mechanotransduction is impaired in mice lack- of tip links (Ahmed et al., 2006; Kazmierczak et al., 2007; ing the tetraspan TMHS. TMHS binds to the tip-link Siemens et al., 2004; So¨ llner et al., 2004). The adaptor proteins component PCDH15 and regulates tip-link assembly, harmonin and SANS and the motor protein myosin 7a (Myo7a) a process that is disrupted by deafness-causing bind in vitro to each other and to CDH23 (Adato et al., 2005; Tmhs mutations. TMHS also regulates transducer Bahloul et al., 2010; Boe¨ da et al., 2002; Siemens et al., 2002) channel conductance and is required for fast channel and colocalize at the upper insertion site of tip links (Grati and adaptation. TMHS therefore resembles other ion Kachar, 2011; Grillet et al., 2009b), suggesting that they form channel regulatory subunits such as the transmem- a protein complex important for transduction. Consistent with brane alpha-amino-3-hydroxy-5-methyl-4-isoxazole this model, Myo7a is implicated in setting resting tension in the propionic acid (AMPA) receptor regulatory proteins transduction machinery (Kros et al., 2002), while harmonin regulates channel activation and adaptation (Grillet et al., 2009b; (TARPs) of AMPA receptors that facilitate channel Michalski et al., 2009). SANS has been proposed to regulate transport and regulate the properties of pore-forming tip-link assembly (Caberlotto et al., 2011), and Myo1c, which channel subunits. We conclude that TMHS is an inte- coimmunoprecipitates with CDH23 (Siemens et al., 2004), is gral component of the hair cell’s mechanotransduc- implicated in regulating slow adaptation (Holt et al., 2002). tion machinery that functionally couples PCDH15 to Intriguingly, while null mutations in the genes encoding CDH23, the transduction channel. PCDH15, harmonin, SANS, and Myo7a disrupt stereociliary bundles and cause deaf-blindness (Usher Syndrome Type 1, USH1), subtle mutations cause less severe forms of the disease INTRODUCTION (McHugh and Friedman, 2006; Sakaguchi et al., 2009). Subtle mutations in tip-link-associated proteins might affect the proper- Our senses of hearing, balance, proprioception, and touch rely ties of the hair cells transduction machinery, a model that is on the process of mechanoelectrical transduction, the conver- supported by the analysis of mice carrying missense mutations sion of mechanical force into electrical signals. Despite the in CDH23 and harmonin (Grillet et al., 2009b; Schwander et al., importance of mechanotransduction for perception, the molec- 2009). ular mechanisms that control this process are not well under- Despite this progress, it is not known which genes encode stood. Electrophysiological recordings and imaging studies subunits of the mechanotransduction channel of hair cells. have revealed that in mechanosensory hair cells of the inner Ca2+ enters stereocilia upon mechanical stimulation near the ear mechanically gated ion channels are localized close to the lower tip-link insertion site, indicating that transduction channels tips of stereocilia, actin-rich projections that emanate from the are present in proximity to PCDH15 (Beurg et al., 2009). Mecha- apical cell surface. Sound-induced vibrations or motion lead to notransduction in mouse hair cells requires the transmembrane

Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. 1283 Figure 1. Mechanotransduction Defects in TMHS-Deficient Mice (A) Hair cell diagram showing on the right proteins that form tip links or are located in proximity to tip links. (B) Amplitude of mechanotransduction currents in mutant mouse lines. The values are expressed relative to the values in wild-type. The number of hair cells analyzed is indicated. Values are mean ± SEM. (C) In situ hybridization with TMHS antisense probe, sense control probe, and a LOXHD1 probe that reveals hair cells. The lowest panel shows vestibular hair cells, the magnified images hair cells at the apical-medial turn of the cochlea. Arrows point to hair cells. (D) SEM analysis of hair bundles from the midapical cochlea. On the right, OHCs are shown. The different rows of stereocilia have been colored. Whisker plots on the right show height differences between the first (longest) and second row of stereocilia (yellow); the second and third row (orange); the third row and surface of hair cells or lowest row (green) (number of evaluated hair bundles: control, n = 16; TmhsÀ/À, n = 21; hPDZ, n = 21; rumba, n = 12; from two animals each). Scale bars: (C) black bar: 200 mm; white bar, 50 mm; gray bar, 20 mm; (D) left panel: 5 mm; right panel 2 mm. See also Figure S1. channel-like genes Tmc1 and Tmc2 (Kawashima et al., 2011), hair cells and the mechanism by which mutations in its gene but it is unclear whether the encoded proteins are channel cause deafness are not known. subunits. To identify components of the mechanotransduction Here, we show that TMHS is an auxiliary subunit of the channel of hair cells, we have systematically screened mutant hair cells mechanotransduction channel. Ion channels most mouse lines with auditory impairment for defects in their mecha- commonly consist of pore-forming subunits and auxiliary sub- notransduction machinery. Here, we describe the phenotypic units. Examples for auxiliary subunits include the g1 subunit of consequences caused by mutations affecting TMHS (tetraspan Cav channels and the transmembrane alpha-amino-3-hydroxy- membrane protein of hair cell stereocilia), which lead to auto- 5-methyl-4-isoxazole propionic acid (AMPA) receptor regulatory somal recessive nonsyndromic hearing loss (DFNB67) in hu- protein (TARP) subunits of AMPA receptors. These auxiliary mans (Shabbir et al., 2006), and deafness in hurry-scurry mice subunits control transport of the channel complex to the plasma (Longo-Guess et al., 2005). TMHS is a member of the tetraspan membrane and regulate the gating properties of pore-forming superfamily, which encodes proteins with diverse functions such channel subunits (Dai et al., 2009; Jackson and Nicoll, 2011; as tight junction proteins, gap junction proteins, ion-channel Pongs and Schwarz, 2010; Vacher et al., 2008). Like the g1 subunits, and tetraspanins. However, the function of TMHS in subunit and TARPs, TMHS belongs to the tetraspan family. It

1284 Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. binds to PCDH15, controls the assembly of the hair cells mecha- as greater variations of the height differences between stereoci- notransduction machinery, regulates the properties of their lia in different rows compared to wild-type. The defects became mechanotransduction channels, and is required for adaptation. more pronounced along the length of the cochlear duct at P5 and increased in severity thereafter (Figure 1D; Figure S1). Morpho- RESULTS logical hair bundle defects were mild at P5 and P7 when mechanotransduction was strongly affected (Figures 1B and 1D). We Mechanotransduction Defects in TMHS-Deficient Hair also observed strong transduction defects at P0 and P4 (data Cells not shown) suggesting that the hair bundle morphological Several laboratories including ours have identified spontane- defects alone cannot explain the functional defects. ously arising and chemically induced mutant mouse lines with To further investigate the relationship between hair bundle defects in auditory function. To identify the extent to which me- morphology and function, we analyzed hair bundles in other chanotransduction is affected in these lines, we measured in mutant mouse lines with defects in transduction. We did not many of them transducer currents in postnatal day (P) seven observe a clear correlation between the two phenotypes. For outer hair cells (OHCs) using whole-cell recordings (Figure 1B). example, peak amplitude of transducer currents was reduced All experiments involving animals followed institutional guide- by 44% in rumba mice and by 60% in harmonin-PDZ2AAA lines and were under the oversight of institutional review boards. mice (Figure 1B). The former had short stereocilia (Figure 1D). We included mouse lines generated by gene targeting, such as The latter showed disruptions of the staircase that was similar harmonin-PDZ2AAA mice that carry a point mutation in harmo- in magnitude to TmhsÀ/À mice (Figure 1D). These findings sug- nin (Grillet et al., 2009b). Hair bundles were stimulated with a stiff gest that mechanotransduction defects in OHCs from TmhsÀ/À probe. In wild-type mice, excitatory stimuli elicited transducer mice are likely not explained primarily by the defects in hair currents that reached peak currents of 571.9 ± 20 pA (n = 9). bundle morphology. Peak amplitude of transducer currents was not reduced in some mutant lines such as pejvakin-deficient sirtaki mice and Tip-Link Defects in TmhsÀ/À Mice CDH23-deficient salsa mice, but more severely in others such We next determined the TMHS expression pattern by immuno- as whirlin-deficient rumba mice, MyoXVa-deficient pogo mice, histochemistry. Between P4 and P6, TMHS was expressed in and harmonin-PDZ2AAA mice (Figure 1B). The most striking stereocilia in a punctate pattern (Figure 2A). The pattern resem- defect was observed in two lines of TMHS-deficient mice, with bled the distribution of the tip-link components CDH23 and 90% decrease in peak amplitude (Figure 1B). One of these PCDH15, which are at this age localized throughout the bundle lines, hurry-scurry, carries a point mutation in TMHS (Longo- with only some protein at tip links (Lagziel et al., 2005; Michel Guess et al., 2005). The second line, Tmhstm1kjn (referred to as et al., 2005; Siemens et al., 2004). TMHS staining was also TmhsÀ/À) was generated by knocking LacZ into the Tmhs locus detectable at the tip-link region (Figure 2A, arrows). HA-tagged (Longo-Guess et al., 2007). Subsequent studies were carried out TMHS that was expressed in OHCs by electroporation was tar- with TmhsÀ/À mice, since they contain a Tmhs-null allele (Longo- geted toward the tips of stereocilia with some of the protein Guess et al., 2007). located in the tip-link region (Figure 2B). After P7, the amount of TMHS detectable by immunostaining drastically decreased. TMHS Expression in Hair Cells and Hair Bundle Since TMHS mRNA is robustly expressed in adult hair cells (Fig- Morphology in TmhsÀ/À Mice ure 1C), low levels of TMHS protein likely persist but were difficult It has been reported that TMHS is transiently expressed in devel- to detect. Similar observations have been made for other tip-link- oping hair cells, and that TmhsÀ/À mice have mild morphological associated proteins such as CDH23 and harmonin, which are hair bundle defects (Longo-Guess et al., 2007). To extend these detected in mature hair cells with few antibodies (Boe¨ da et al., findings and to determine the extent to which the mechanotrans- 2002; Grillet et al., 2009b; Kazmierczak et al., 2007; Lagziel duction defect in TmhsÀ/À hair cells is a secondary consequence et al., 2005; Lefe` vre et al., 2008; Michel et al., 2005; Siemens of morphological defects, we investigated by in situ hybridization et al., 2004). the Tmhs expression pattern, and we analyzed hair bundle The localization of TMHS toward stereociliary tips suggested morphology. TMHS mRNA was expressed in inner hair cells that defects in mechanotransduction in TmhsÀ/À mice might be (IHCs) and OHCs of the cochlear as well as in vestibular hair cells caused by abnormal tip-link function. We therefore analyzed (Figure 1C). Expression levels correlated with maturation of the by SEM the integrity of tip links in hair cells from TmhsÀ/À transduction machinery. At the cochlear apex, which functionally mice. At P7, stereocilia from control mice were connected by matures after the cochlear base (Waguespack et al., 2007), an array of extracellular filaments as reported (Figure 2C) (Good- expression levels were low at P1 and increased thereafter (Fig- year et al., 2005). Higher magnification views revealed the pres- ure 1C). Robust expression was observed in mature hair cells ence of tip links in IHCs and OHCs (Figure 2D). Stereocilia in hair throughout the cochlea at P25, the last time point analyzed bundles from TmhsÀ/À mice were also connected by extracel- (Figure 1C). lular filaments, but tip links were rarely observed (Figures 2C We next evaluate hair bundle morphology by scanning elec- and 2D). To quantify tip-link numbers, we focused in P7 hair cells tron microscopy (SEM). To obtain quantitative values, we deter- on linkages between the longest rows of stereocilia and their mined the height difference between the rows of stereocilia next shorter neighbors. Tip links were at this age more difficult (Figure S1 available online). In TmhsÀ/À mice, hair bundles at to distinguish from other linkages in the shorter rows of stereoci- the cochlear base showed mild defects at P3, which manifested lia. At P7, control IHCs (n = 17 IHCs) from the medial part of the

Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. 1285 Figure 2. Tip-Link Defects in Hair Cells from TmhsÀ/À Mice (A) Hair cells from wild-type mice at P5 were stained with antibodies to TMHS (red) and with phalloidin (green). Arrows point to TMHS in the tip-link region. (B) OHCs were electroporated to express HA- TMHS (electroporated cells labeled with an asterisk). Cells were stained for HA (red in upper panel, green in lower panel) and with phalloidin (green in upper panel, red in lower panel). (C) SEM analysis of OHCs at P7. Arrows point to tip links. (D) High-resolution images showing tip links (arrows) in OHCs and IHCs. (E) Quantiﬁcation of tip-link numbers at P7. Values are mean ± SD (**p < 0.01; ***p < 0.001). Scale bar: (A and B) 3 mm; (C) 0.5 mm; (D) 0.25 mm.

ure 3A). At P0 and P3, residual levels of PCDH15 were present in stereocilia (data not shown), indicating that loss of PCDH15 localization to hair bundles was progressive. These data suggest that TMHS is required for the efﬁcient localization of PCDH15 to hair bundles and vice versa. Perturbations in the formation of tip links in TmhsÀ/À mice are therefore likely caused, at least in part, by inefﬁcient localization of PCDH15 to stereocilia.

TMHS Binds PCDH15 To define whether TMHS affects the distribution of PCDH15 in hair cells directly or indirectly, we next asked whether TMHS and PCDH15 are part of a protein complex. We coexpressed TMHS-HA and PCDH15 in HEK293 cells, focusing on PCDH15-CD1, -CD2, and -CD3, three PCDH15 isoforms that differ in their cyto- cochlea contained 2.8 times more tip links compared to TmhsÀ/À plasmic domains (Ahmed et al., 2006). TMHS coimmunoprecipi- (n = 14 IHCs), while OHC from the apical/medial part of the tated with all three isoforms (Figures 3B and 3C; data not shown), cochlea contained 2.2 times more tip links in controls (n = 7 but not with N-cadherin (Figure 3C), an unrelated cadherin. OHCs) compared to mutants (n = 7 OHCs) (Figure 2E). The PCDH15-CD1, -CD2, and -CD3 cytoplasmic domains share a conserved membrane proximal (CR) domain followed Interdependence of PCDH15 and TMHS Localization by isoform specific N-terminal domains (Ahmed et al., 2006). To determine the mechanism by which mutations in TMHS affect TMHS coprecipitated with a PCDH15 mutation truncated imme- tip links, we analyzed the TMHS expression pattern in mice diately at the C terminus of the CR domain (Figure 3C), and with with mutations in CDH23 and PCDH15. Similarly, we evaluated a PCDH15 fragment consisting of the CR and CD3 domains, but CDH23 and PCDH15 expression in TmhsÀ/À mice. Stereocilia not with a fragment consisting of the CD3-specific domain only are splayed in PCDH15- and CDH23-deficient hair bundles (Ala- (Figure 3C). Finally, TMHS coimmunoprecipitated with a chimeric gramam et al., 2001; Di Palma et al., 2001; Wilson et al., 2001). At protein, where the N-cadherin transmembrane domain was sub- P1, TMHS expression at tips of stereocilia was maintained in the stituted with the PCDH15 transmembrane domain (Figure 3D). splayed bundles from CDH23-deficient waltzerv2J/v2J mice, but We conclude that the transmembrane and membrane proximal expression was drastically reduced in PCDH15-deficient Ames CR domains of PCDH15 mediate interaction with TMHS. waltzerav3J/av3J mice (Figure 3A). Conversely, CDH23 was ex- TMHS does not bind to CDH23, harmonin, or SANS (Figure 3E) pressed at P6 in hair bundles from TmhsÀ/À hair cells, while providing further evidence for a specific functional link between PCDH15 expression levels were reduced in the mutants (Fig- PCDH15 and TMHS.

1286 Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. Figure 3. Interactions between TMHS and PCDH15 (A) IHCs from P1 wild-type mice, PCDH15-deficient Ames waltzerav3J/av3J mice, CDH23-deficient waltzerv2J/v2J mice, and TmhsÀ/À mice were stained with antibodies to TMHS, PCDH15, and CDH23 (red) and phalloidin (green). Note the reduced staining of TMHS in PCDH15 mutants and of PCDH15 in TMHS mutants. (B) Diagram of the constructs used for biochemical experiments. (C–E) HEK293 cells were transfected with the constructs indicated on top of each panel. Immunoprecipitations were carried out with HA antibodies that recognize HA-TMHS, followed by western blotting to detect PCDH15 constructs (C and D), CDH23, SANS, and harmonin (E). GFP-tagged PCDH15 and N-cadherin proteins were detected with an antibody to GFP, the remaining proteins with antibodies specific to the individual proteins. The lower rows show input protein, the upper rows coimmunoprecipitation (Co-IP) results. Note the specific interaction between TMHS and PCDH15. Scale bars: (A) 4 mm.

TMHS Regulates PCDH15 Cell-Surface Expression cell-surface transport. We therefore analyzed the subcellular Based on the interdependence of PCDH15 and TMHS for distribution of PCDH15 and TMHS in heterologous cells. We their efﬁcient localization to stereocilia, we reasoned that inter- chose RPMI-2650 cells for this purpose because they have action between the two proteins might regulate their effective a ﬂat morphology with an extensive cytoplasm. In the absence

Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. 1287 Figure 4. Wild-Type, but Not Mutant TMHS, Facilitates PCDH15 Transport to the Plasma Membrane (A) RPMI-2650 cells were transfected with expression vectors for TMHS and/or PCDH15 and analyzed for protein expression and localization 1 day after transfection (a–f) or 4 days after transfection (g–i). TMHS is shown in green, PCDH15 in red. Nuclei (a–c, blue) and actin (b and c, blue) were stained with DAPI and phalloidin, respectively. Note the low levels of PCDH15 at the cell surface in the absence of TMHS (c, arrows), and extensive colocalization of PCDH15 and TMHS at the surface of double-transfected cells (d–f, arrows). The insert in (e) show staining of nonpermeabilized cells with an antibody to the PCDH15 extracellular domain. (g–i) show colocalization of TMHS and PCDH15 in small aggregates in cell protrusions (arrows) after 4 days in culture. (B) Quantiﬁcation of expression of proteins in the cytosol/at the plasma membrane in RPMI- 2650 cells transfected to express TMHS and/or PCDH15 (at least 100 cells were quantiﬁed in three independent experiments). Values are mean ± SEM. (C) Diagram of TMHS and location of mutations within the protein. (D) Coimmunoprecipitation experiments were carried out with extracts from transfected HEK293 cells. Transfected constructs are indicated on top. The lower rows show input protein, the upper rows coimmunoprecipitation (Co-IP) results. (E) RPMI-2650 cells were cotransfected with expression vectors for PCDH15 and TMHS carrying the hurry-scurry mutation. TMHS is in green, PCDH15 in red, and nuclei and actin in blue. Scale bars: (A) 7 mm; (B) 5 mm.

TMHS prominently colocalized with PCDH15 at the plasma membrane (Fig- ures 4Ad–4Af, 4B, and 4C). Staining of nonpermeabilized cells with an antibody to the extracellular PCDH15 domain conﬁrmed that PCDH15 was localized at the cell surface upon coexpression with TMHS (Figure 4Ae, insert). After 4 days in culture, PCDH15 and TMHS accumulated in the plasma membrane in a punctate pattern (Figure 4Ag–4Ai), indicating that the two proteins clustered into membrane microdomains. We con- of PCDH15, TMHS accumulated in clusters in the cyto- clude that PCDH15 and TMHS facilitate each other’s cell-surface plasm (Figures 4Aa and 4B). Similarly, without TMHS, PCDH15 transport. accumulated around the nucleus of transfected cells in rod- like aggregates (Figures 4Ab and 4B). Few cells showed low Tmhs Mutations that Cause Deafness Affect PCDH15 levels of PCDH15 in cell protrusions (Figure 4Ac, arrows; Cell-Surface Expression Figure 4B) indicative of inefﬁcient protein transport. The distribu- Several Tmhs mutations have been reported that cause hearing tion of TMHS and PCDH15 was altered upon coexpression of loss (Figure 4C) (Longo-Guess et al., 2005; Longo-Guess et al., the two proteins (Figure 4Ad–4Af). One day after transfection, 2007; Shabbir et al., 2006). None of the mutations affected

1288 Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. interactions between TMHS and PCDH15 as analyzed by coim- Instead, our histochemical and biochemical data suggest that munoprecipitation experiments (Figure 4D). When we coex- effects of TMHS mutations on mechanotransduction are caused pressed PCDH15 and TMHS carrying the hurry-scurry mutation by inefficient PCDH15 transport. If this is true, overexpression in RPMI-2650 cells, PCDH15 and TMHS still colocalized (Fig- of PCDH15 in hair cells from TmhsÀ/À mice might rescue trans- ure 4E), but the protein complex remained in aggregates within duction. To test this model, we generated a cDNA expression the cytoplasm, suggesting that some TMHS mutations cause vector for PCDH15-CD3. As a control, we expressed PCDH15- deafness by affecting PCDH15 transport. Since we did not CD3 in PCDH15-deficient OHCs from Ames waltzerav3J/av3J observe accumulation of PCDH15/TMHS in the cell bodies of mice. Acute expression of PCDH15-CD3 rescued their mecha- hair cells in TMHS-/PCDH15-deficient mice (data not shown), notransduction defect, demonstrating the effectiveness of our defects in protein transport likely affect protein stability in vivo. expression vector to restore PCDH15 function (Figure 5G). Sig- nificantly, expression of PCDH15 in OHCs from TmhsÀ/À mice Expression of TMHS and PCDH15 in TMHS-Deficient also restored transduction (Figure 5H). The data provide compel- Hair Cells Restores Mechanotransduction ling evidence that transduction defects in TmhsÀ/À mice are To exclude that general defects in hair cell development account caused by defects in PCDH15 function and support the model for the mechanotransduction defect in OHCs from TmhsÀ/À that PCDH15 can be transported to stereocilia in the absence mice, we determined whether acute expression of TMHS in of TMHS but only when expressed at high levels. mutant hair cells rescues transduction. To facilitate the experi- TMHS expression in OHCs from PCDH15-deficient Ames ments, we developed an electroporation method for the expres- waltzerav3J/av3J mice did not restore transduction (Figure 5I). sion of cDNAs in hair cells, combined with the analysis of mecha- This is not surprising since TMHS is not expected to restore tip notransduction currents using the genetically encoded Ca2+ links in the absence of the essential tip-link component PCDH15. sensor G-CaMP3 (Tian et al., 2009). We confirmed by patch clamp physiology that the electroporation method was compat- Macroscopic Mechanotransduction Currents ible with measuring transduction currents (Figure S2). Next, we The g subunits of Cav channels and the TARPs of AMPA recep- evaluated the feasibility to use G-CaMP3 as a readout for me- tors are required for efficient plasma membrane localization of chanotransduction currents. We electroporated hair cells from their respective ion channels, but they also regulate ion channel wild-type mice at P4 with G-CaMP3, cultured them for 1 day (Fig- pore properties (Dai et al., 2009; Jackson and Nicoll, 2011; ure 5A) and activated transduction channels in OHCs with a fluid Pongs and Schwarz, 2010; Vacher et al., 2008). We wondered jet, by applying three sequential stimuli of increasing duration. whether TMHS might similarly affect the assembly of the hair Following stimulation, fluorescence intensity increased robustly cells mechanotransduction machinery and channel function. in OHCs expressing G-CaMP3 (Figure 5B). Little increase was To test this model, we analyzed the remaining mechanotrans- observed in OHCs from TmhsÀ/À mice (Figure 5B), or in the pres- duction currents in OHCs from TmhsÀ/À mice. ence of the transduction channel blocker dihydrostreptomycin Hair bundles were stimulated with a stiff glass probe. As re- (Figure 5C). We conclude that the fluorescence signal is a useful ported (Grillet et al., 2009b; Kennedy et al., 2003; Kros et al., readout for the activation of transduction channels, although 2002; Stauffer et al., 2005; Waguespack et al., 2007), control changes in fluorescence intensity likely also depend on the acti- OHCs had rapidly activating transducer currents, which subse- vation of Ca2+ channels downstream of the transducer channel. quently adapted with a fast and slow time constant (Figure 6A). Previous studies have shown that the mechanotransduction The amplitude of saturated mechanotransduction currents was machinery of cochlear hair cells matures in the first few postnatal at 540.9 ± 27.8 pA. In hair cells from mutant mice, peak ampli- days, during which the amplitude of the transducer currents tudes reached 58.7 ± 5.9 pA (Figures 6A and 6B), suggesting increases over time (Waguespack et al., 2007). Consistent with that the number of active mechanotransduction channels was this finding, we observed that fluid-jet-induced changes in reduced, which is consistent with the reduced number of tip links Ca2+ fluorescence intensity were significantly higher in wild- in TmhsÀ/À OHCs. The shape of the current traces obtained from type OHCs electroporated at P4 when compared to P1 (Fig- mutant OHCs were also altered and revealed an apparent lack of ure 5D). We could evoke minimal changes in Ca2+ fluorescence adaptation (Figure 6A). To normalize for variations in amplitude, intensity in OHCs from TmhsÀ/À mice transfected at either age we plotted the open probability of the transducer channel (Po) (Figure 5D). against displacement (Figure 6C). The resulting curve from Next, we determined the extent to which mechanotransduc- mutants was significantly shifted to the right and broadened tion in OHCs from TmhsÀ/À mice is rescued by expression of when compared to wild-type, indicative of less coordinated TMHS. Expression of wild-type TMHS or TMHS carrying the channel gating throughout the hair bundle and reduced sensi- hurry-scurry mutation did not affect mechanically evoked trans- tivity to deflection. ducer currents in wild-type OHCs (Figure 5E). However, wild- To analyze adaptation further, we applied a set of deflections type but not mutant TMHS restored mechanical sensitivity in ranging from À100 nm to 1,000 nm to hair bundles. Each deflec- OHCs from TmhsÀ/À mice transfected at P2 (Figure 5E) or P5 (Fig- tion lasted for 30 ms, after which adaptation is largely complete. ure 5F). These data were confirmed by analyzing mechanically To compare the kinetic properties of the mechanotransducer evoked transducer currents using whole-cell recordings (Fig- currents, we superimposed current traces from controls and ure S2). We conclude that the transduction defect in TmhsÀ/À mutants based on similar steady-state currents (Figure 6D). mice is likely not a secondary consequence of defects in overall The fast adaptation phase was no longer observable in OHCs hair cell development. from TmhsÀ/À mice. To quantify the changes, we plotted the

Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. 1289 Figure 5. Rescue of Mechanotransduction in TmhsÀ/À Mice by Acute Expression of TMHS and PCDH15 (A) Example of G-CaMP3 expression (green) in transfected OHC. Hair bundles of transfected cells are morphologically intact (phalloidin staining, red), which can be seen in detail (asterisk) in the bottom-right panel. (B) Representative example demonstrating fluid-jet-induced Ca2+ response in control and TmhsÀ/À OHCs. OHCs were transfected at P4 and cultured for 1 day in vitro (DIV). Fluid-jet pulse duration was increased from 0.1 s, to 0.3 s, to 0.5 s. For quantitative analysis (C–I), the amplitude of the second Ca2+ response peak was measured. (C) Ca2+ response of wild-type OHCs with/without dihydrostreptomycin (DHS). (D) Quantification of peak amplitude in OHCs from control and TmhsÀ/À mice transfected at P0 or P4 and cultured for 1 DIV. (E) Response of OHCs from control and TmhsÀ/À mice transfected to express G-CaMP3 only (À), wild-type TMHS (TMHS), or TMHS carrying the hurry-scurry mutation (mtTMHS). (F) OHCs from TmhsÀ/À mice at P5 were transfected with wild-type and mutant TMHS and analyzed after 2 DIV for mechanically evoked Ca2+ currents. (G) Ca2+ responses in OHCs from PCDH15-deficient Ames-waltzerav3J/av3J mice electroporated to express PCDH15-CD3. (H) Ca2+ responses in OHCs from TmhsÀ/À mice electroporated to express PCDH15-CD3. (I) Ca2+ responses in OHCs from PCDH15-deficient Ames-waltzerav3J/av3J mice electroporated to express TMHS. Please note that the changes in fluorescence intensity vary in magnitude between panels since we used for the experiments hair cells of different ages. All values are mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001). Scale bars: (A) 6 mm. See also Figure S2.

ratio of steady-state current to peak current (Rsteady-state/peak) OHCs, the activation time constant ranged from 85 ± 3to97± versus the channels open probability (Po)(Figure 6E). The 7 ms, which was within the rise time of our stimulus probe (Fig- Rsteady-state/peak of the control increased from 0.36 ± 0.02 to ure 6F). In mutant OHCs, activation kinetics was significantly 0.73 ± 0.02, while the Rsteady-state/peak of mutants ranged slower ranging from 252 ± 26 to 426 ± 150 ms(Figure 6F). In from 0.64 ± 0.06 to 0.80 ± 0.18, consistent with perturbed fast wild-type a relative stable t with increasing deflection was adaptation. Since current amplitudes in OHCs from mutant observed, which is in agreement with published findings for rat mice were small and peak currents were sometimes difficult and mouse hair cells (Kennedy et al., 2003; Ricci et al., 2005). to determine, we did not calculate the time constant for fast In mutant hair cells, we did not observe a change in t with adaptation. increasing deflections either and activation was significantly The activation of transduction channels in OHCs from slowed down compared to controls (Figure 6D). These changes TmhsÀ/À mice was also affected (Figure 6D, inset). In control in channel activation could be caused by alterations in the

1290 Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. Figure 6. Macroscopic Mechanotransduc- tion Currents in OHCs from TmhsÀ/À Mice (A) Examples of transduction currents in OHCs from wild-type and TmhsÀ/À mice in response to a set of 10 ms hair bundle deﬂections ranging from À400 to 1000 nm (100 nm steps). (B) Current displacement plots obtained from similar data as shown in (A). The number of analyzed hair cells is indicated.

(C) Open probability (Po) displacement plots calculated from similar data as shown in (A). The number of analyzed hair cells is indicated. (D) Representative current traces to show adaptation in OHCs from control and TmhsÀ/À mice. Traces were chosen from controls and mutants based on similar steady state currents and similar rate of slow adaptation. Deﬂection was for 30 ms. Inset showed magniﬁed traces for the activation phase of mechanically induced currents. (E) Ratio of steady-state current versus peak current plotted against the channel open proba-

bility (Po).

(F) Activation time constant (tactivation) plotted

against Po. All values are mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001). See also Figure S3. channel protein and/or in its environment such as in the stiffness a Piezo stimulator and channel activity was recorded in the of the membrane. whole-cell configuration. Figures 7A and 7B show typical Analysis of the voltage dependence of transducer currents single-channel events obtained with a 300 nm deflection in and its sensitivity to external Ca2+ did not reveal differences controls (left panel) and mutants (right panel). Single-channel between hair cells from wild-type and mutant mice (Figure S3), events of a similar amplitude and duration as reported earlier indicating that the observed changes in the properties of the (Beurg et al., 2006; Ricci et al., 2003) were observed in control transducer currents in the absence of TMHS are not a con- OHCs. Transducer current ensemble averages from wild-type sequence of alterations in the way external Ca2+ blocks the OHCs (Figure 7A; left panel, middle) resembled macroscopic channel. currents remarkably well, where transducer currents rapidly acti- We conclude that fewer transduction channels are activated in vated, followed by adaptation. In contrast, ensemble averages in mutant TmhsÀ/À OHCs compared to wild-type, likely because TmhsÀ/À OHCs (Figure 7A; right panel, middle) lacked the adap- tip-link numbers are reduced in the mutants. We also observed tation phase. Amplitude histograms are shown to indicate open variable onset of transducer currents, indicating that the function and close state of single-channel events (Figure 7A; bottom). of the remaining tip-link complexes was altered directly or as Analysis of single-channel events from current traces of a a consequence of less coherent force propagation through the number of cells (see figure legend for numbers) established hair bundle. Moreover, adaptation appeared to be affected, several differences in the behavior of single channels from which could be a consequence of a reduction in the number of TmhsÀ/À OHCs: (1) the average conductance in controls was active channels, delay in channel activation, and less coordi- at 86.8 ± 1.6 pS and in the mutants at 68.1 ± 1.7 pS (Figure 7C); nated activation by remaining tip links. (2) the latency between the onset of deflection and the first channel event was longer in mutant OHCs (2.3 ms) compared Single-Channel Properties to wild-type (1.3 ms) (Figure 7D); (3) the t of the average open The effects of TMHS loss on macroscopic mechanotransduction time of single event in mutant OHCs was 1.7-fold increased currents could be caused by a reduction in the number of active compared to controls (Figure 7B). channel complexes within a hair bundle. However, since TMHS binds to PCDH15, which is in close proximity to the transduction DISCUSSION channel, we wondered whether TMHS might affect the channel directly. We carried out single-channel recordings, taking advan- We provide here evidence that TMHS is intimately linked to the tage of the observation that tip links can be broken by Ca2+ function of the hair cells mechanotransduction machinery. First, removal, reducing the number of active transduction channels TMHS is localized at least in part to the tips of hair cell stereocilia until single-channel events can be recorded (Beurg et al., in close proximity to their tip links. While TMHS mRNA is present 2006; Ricci et al., 2003). We delivered with a fluid jet an EGTA in developing and adult hair cells, it is difficult to detect TMHS solution to wild-type and mutant hair bundles at a similar position protein after P7. This is likely due to the limited sensitivity of (40% of the distance along the cochlea from the low-frequency our antibody. Second, TMHS binds to the transmembrane and end). Mechanotransduction channels were then activated with CR domains of PCDH15, which is located in close proximity to

Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. 1291 Figure 7. Single-Channel Responses in OHCs from TmhsÀ/À Mice (A) Upper panels: representative single-channel recordings from a control (black) and TmhsÀ/À (gray) OHC. Close (C) and open state (O) induced by a 300 nm deflection are shown. Middle panels: ensemble averages from 25 responses in controls and from 52 responses in mutants. The adaptation phase was only evident in ensemble averages from control OHC. Bottom panels: amplitude histograms generated with the data from the second traces in upper panels. Gaussian fits of the two peaks in the histograms determine a single- channel current of 6.8 pA in control OHC and 5.2 pA in TmhsÀ/À OHC. (B) Open-time histograms of single-channel events from all recorded cells (control: 1691 events from 24 cells; mutant: 1663 events from 18 cells). The histograms were fitted with exponential curves with time constants of 0.94 ms in control (black) OHCs and 1.57 ms in TmhsÀ/À (gray) OHCs. The first bin was not included for fitting as described (Ricci et al., 2003). (C) Averaged single-channel conductance from all recorded OHCs. Single-channel conductance in OHCs from controls were at 86.8 ± 1.6 pS and at 68.1 ± 1.7 pS in OHCs from TmhsÀ/À mice (controls: 150 traces from 19 cells; mutants: 96 traces from eight cells). (D) Latency between the onset of deflection and first event from all recorded OHCs. Latency was at 1.3 ± 0.1 ms in control OHCs and at 2.2 ± 0.1 ms in TmhsÀ/À OHCs (controls: 171 traces from five cells; mutants: 263 traces from ten cells). All values are mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001). (E) Model of the function of TMHS in hair cells. According to the model, TMHS is a regulatory subunit of the mechanotransduction channel that connects the pore-forming subunits of the channel to PCDH15 and might also affect membrane properties.

the transduction channel. Third, TMHS is required for tip-link required for fast adaptation, and for efﬁcient PCDH15 locali- integrity. Finally, transducer channel properties are altered in zation to stereocilia. In the absence of TMHS, some tip links TmhsÀ/À OHCs. We conclude that TMHS is associated with form, suggesting that TMHS is not absolutely essential for PCDH15 and the mechanotransduction channel and regulates PCDH15 transport. Consistent with this model, small amounts the response of the transduction channel to mechanical stimula- of PCDH15 are transported to the surface of transfected cells in tion (Figure 7E). the absence of TMHS. In hair cells, PCDH15 is expressed at Ion channels usually consist of pore-forming and accessory high levels during development; its expression level decreases proteins. Some ion channel subunits share with TMHS a four subsequently (Ahmed et al., 2006; Webb et al., 2011). Developing transmembrane topology. Examples include the pore-forming hair bundles may depend less on TMHS, which is consistent subunits of the CRAC channel (Feske et al., 2006; Vig et al., with the observation that TMHS-deﬁcient hair bundles show

2006), the accessory g-1 subunit of Cav channels (Jay et al., less severe morphological defects than PCDH15-deficient hair 1990), and the TARP subunits of AMPA receptors such as starga- bundles. While mechanotransduction currents can be elicited in zin (Letts et al., 1998). Our findings suggest that TMHS is func- TmhsÀ/À stereocilia that contain tip links, channel activation is tionally similar to accessory ion channel subunits such as g-1 or delayed, suggesting that coupling between the transduction stargazin, which regulate ion channel transport and conductance channel and PCDH15 is less efficient. TMHS might stabilize inter- (Dai et al., 2009; Jackson and Nicoll, 2011; Pongs and Schwarz, actions between PCDH15 and the channel, and/or it might affect 2010; Vacher et al., 2008). In analogy, TMHS regulates conduc- membrane properties locally, which is a function attributed to tance through the hair cell’s mechanotransduction channel, is some tetraspans (Ya´ n˜ ez-Mo´ et al., 2009).

1292 Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. The current amplitude of the macroscopic transducer currents In Situ Hybridization, Antibodies, Whole-Mount Staining, and in TMHSÀ/À OHCs is reduced, which can be explained at least in Immunocytochemistry part by the reduced numbers of tip links. Channel activation is In situ hybridizations and cochlear whole-mount stainings were carried out as described (Schwander et al. 2007). Primary antibodies were as follows: slowed and adaptation is nearly absent. While some of these a-TMHS (Longo-Guess et al., 2005); a-CDH23 (Kazmierczak et al., 2007); changes might be explained by less coordinated and delayed a-PCDH15; (Kazmierczak et al., 2007); a-harmonin (Grillet et al., 2009b); a- activation of fewer mechanotransduction channels, our single- HA (clone 6E2, Cell Signaling). Additional reagents were Alexa Fluor 488-phal- À À channel analysis demonstrates that in Tmhs / mice the proper- loidin, Alexa Fluor 594 goat anti-rabbit, TOP-RO3, and Alexa Fluor 647-phalloi- ties of the transduction channel are altered. In the absence of din (Invitrogen, Carlsbad, CA). TMHS, channel conductance is reduced by 22%, indicating that TMHS affects the channel pore. Latency and open time of Scanning Electron Microscopy À À Inner ears were dissected and fixed. The stria vascularis, Reissner’s mem- single channels in TMHS / OHCs are increased by 70%, brane, and tectorial membrane were removed. Samples were dehydrated, which is consistent with the increased activation time constant processed to critical drying point, mounted, coated with iridium, and imaged. and lack of fast adaptation observed in macroscopic currents. Quantification of hair bundle morphology and tip links was carried out as The decreased amplitude of single-channel currents, together described in the Extended Experimental Procedures. with increased latency and channel open time, suggests that TMHS functionally couples the mechanotransduction channel DNA Constructs, Transfections, Immunoprecipitations, and to the tip link. Western Blots In analogy to g-1 and TARP subunits, changes in the proper- DNA constructs are described in Supplemental Information. Expression of the À/À constructs, immunoprecipitations, and western blots were carried out as ties of the mechanotransduction channel in Tmhs mice can described (Senften et al., 2006). Immunoprecipitation experiments were perhaps be explained best by an allosteric mechanism, where carried out at least three times. TMHS binds to pore-forming subunits of the mechanotransduction channel to affect the conformation of the channel complex. Cochlea Culture and Electroporation Changes in gating kinetics such as increased latency as well as The organ of Corti was dissected from P0-8 mice as described (Grillet et al., adaptation defects can be explained by direct effects of TMHS 2009b) and cut into three pieces, which were placed in DMEM/F12 medium on the channel, or by changes in the channels environment with 10% fetal bovine serum and 1.5 mg/ml ampicillin. For electroporation, glass electrodes (2 mm diameter) were used to deliver plasmid (1 mg/mlin13 such as the stiffness of the membrane. HBSS) to the sensory epithelium. For Ca2+ imaging, we used G-CaMP3 (Addg- The conductance properties of the transduction channel in ene 22692). A series of three pulses was applied at 1 s intervals with a magni- OHCs vary from the cochlear base to the apex, suggesting tude of 60V and duration of 15 ms (ECM 830 square wave electroporator; BTX). that the molecular composition of the channel varies (Ricci Hair cells were imaged on an upright Olympus BX51WI microscope. Hair et al., 2003). Since TMHS regulates channel conductance, vari- bundles were stimulated with a fluid jet applied through a glass electrode filled ations in TMHS expression could explain tonotopic differences with bath solution. Stimuli were applied using Patchmaster 2.35 software (HEKA) and 20 psi air pressure. Images were collected with a 2 s sampling in currents. Notably, TMHS is a member of a small tetraspan rate. A series of fluid-jet stimulations (0.1, 0.3, 0.5 s) was applied (80 s intervals). subfamily (Longo-Guess et al., 2005). While the expression Responses induced by 0.3 s fluid-jet stimulation were used for quantitative pattern of TMHS homologous needs to be determined, this rai- analysis. ses the possibility that different TMHS-like proteins might generate mechanotransduction channels with different proper- Electrophysiology ties. The pore-forming subunits of the hair cells mechanotrans- For recording, tissue was transferred to the recording chamber and a duction channel still need to be identified, but some candidates peristaltic pump was used to perfuse artificial perilymph. Hair bundles have emerged including TMC-1 and TMC-2 (Kawashima et al., were deflected with a glass pipette mounted on a P-885 piezoelectric stack actuator (Physik Instrument). The actuator was driven with voltage 2011) and Piezo1 and Piezo2 (Coste et al., 2010, 2012). It will steps that were low-pass filtered at 10 KHz. Whole-cell recording were be important to analyze functional interactions between TMHS carried out and currents were sampled at 100 KHz. Macroscopic currents and these proteins. were recorded essentially as we have described (Grillet et al., 2009b). For Finally, our findings are significant for understanding disease single-channel recordings, we followed published procedures (Ricci et al., mechanisms. We establish an unanticipated link in the mecha- 2003). nism by which mutations in TMHS and PCDH15 cause hearing loss. Our findings suggest that at least in some patients with muta- Data Analysis Data analysis was performed using Excel (Microsoft) and Igor pro 6 tions in TMHS and PCDH15 deafness is caused by functional (WaveMetrics, Lake Oswego, OR). Calcium signal (DF/F) was calculated with impairment of the hair cells mechanotransduction machinery. the equation: (F-F0)/F0, where F0 is the averaged fluorescence baseline at the beginning. Transduction current-displacement curves (I[X]) were fitted EXPERIMENTAL PROCEDURES with a three state Boltzmann model to calculate channel open probability (Po) as described (Grillet et al., 2009b). All data are mean ± SEM. Student’s Detailed experimental procedures are described in the Extended Experimental two-tailed unpaired t test was used to determine statistical significance (*p < Procedures. 0.05, **p < 0.01, ***p < 0.001).

Mouse Strains SUPPLEMENTAL INFORMATION Rumba, samba, sirtaki, pogo, salsa, hurry-scurry, TMHS knockout, harmonin PDZ2, Ames waltzerav3j, and waltzerv2j mice have been described (Alagramam Supplemental Information includes Extended Experimental Procedures and et al., 2001; Di Palma et al., 2001; Grillet et al., 2009a, 2009b; Longo-Guess three ﬁgures and can be found with this article online at http://dx.doi.org/10. et al., 2005, 2007; Schwander et al., 2007, 2009). 1016/j.cell.2012.10.041.

Cell 151, 1283–1295, December 7, 2012 ª2012 Elsevier Inc. 1293 ACKNOWLEDGMENTS Di Palma, F., Holme, R.H., Bryda, E.C., Belyantseva, I.A., Pellegrino, R., Ka- char, B., Steel, K.P., and Noben-Trauth, K. (2001). Mutations in Cdh23, encod- W.X. carried out the electrophysiological recordings, calcium imaging experi- ing a new type of cadherin, cause stereocilia disorganization in waltzer, the ments, and the analysis of protein localization in transfected cells. W.X. and mouse model for Usher syndrome type 1D. Nat. Genet. 27, 103–107. T.F.J.W. developed the electroporation technique. N.G. carried out in situ Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S.H., Tanasa, B., hybridizations and the analysis of hair cell morphology and tip links by SEM. Hogan, P.G., Lewis, R.S., Daly, M., and Rao, A. (2006). A mutation in Orai1 W.X., N.G., T.F.J.W., and P.K. carried out immunolocalization experiments in causes immune deficiency by abrogating CRAC channel function. Nature hair cells. H.M.E. and B.Z. generated plasmid constructs and carried out coim- 441, 179–185. munoprecipitation experiments. K.R.J. provided TMHS mutant mice and Gillespie, P.G., and Mu¨ ller, U. (2009). Mechanotransduction by hair cells: generated the TMHS antibody. W.X., N.G., T.F.J.W., P.K., and U.M. contrib- models, molecules, and mechanisms. Cell 139, 33–44. uted to the planning of the work. U.M. wrote the manuscript with help from W.X. and N.G. We thank S.W. Webb and S.R. Harkins-Perry for cloning Goodyear, R.J., Marcotti, W., Kros, C.J., and Richardson, G.P. (2005). Devel- constructs. This work was supported by the NIH (DC005965 and DC007704 opment and properties of stereociliary link types in hair cells of the mouse to U.M.), the Dorris Neuroscience Center, the Skaggs Institute for Chemical cochlea. J. Comp. Neurol. 485, 75–85. Biology, and the Bundy Foundation (to W.X. and U.M.). Grati, M., and Kachar, B. (2011). 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