Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2 Reo Maedaa,b,1, Katie S. Kindta,b,1,2, Weike Moa,b,1, Clive P. Morgana,b, Timothy Ericksona,b, Hongyu Zhaoa,b, Rachel Clemens-Grishama,b, Peter G. Barr-Gillespiea,b, and Teresa Nicolsona,b,3 aOregon Hearing Research Center and bVollum Institute, Oregon Health and Science University, Portland, OR 97239 Edited by A. J. Hudspeth, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, and approved July 23, 2014 (received for review February 3, 2014) The tip link protein protocadherin 15 (PCDH15) is a central compo- vestibular deficits, along with the complete absence of normal nent of the mechanotransduction complex in auditory and vestib- mechanotransduction currents in auditory and vestibular hair cells ular hair cells. PCDH15 is hypothesized to relay external forces to the (17). Changes in calcium permeability through the transduction mechanically gated channel located near its cytoplasmic C terminus. channel of cochlear hair cells were observed for Tmc1 Tmc2 How PCDH15 is coupled to the transduction machinery is not clear. double-mutant mice, as well as in single mutants of either gene Using a membrane-based two-hybrid screen to identify proteins (10, 18, 19). In further support of the idea that TMCs are pore- that bind to PCDH15, we detected an interaction between zebrafish forming subunits of the transduction channel, mouse vestibular Pcdh15a and an N-terminal fragment of transmembrane channel- hair cells that express only the dominant Beethoven (M412K) al- like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which lele of Tmc1, in the absence of any wild-type TMC1 or TMC2, along with TMC1 has been implicated in mechanotransduction in display altered single-channel transduction currents (10). Curi- mammalian hair cells. Using the above-mentioned two-hybrid assay, ously, an 180° phase-shifted transduction current can be evoked in we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 cochlear hair cells expressing only the deafness allele of Tmc1 − − or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction (Tmc1dn/dn Tmc2 / ) (19). The source of this anomalous current is depends on the common region shared between the two Pcdh15 not clear, but a similar current is also observed in hair cells where isoforms. Moreover, an interaction between mouse PCDH15-CD3 tip links have been disrupted by either genetic lesions (Pcdh15av3J, and TMC1 or TMC2 was observed in both yeast two-hybrid assays Cdh23v2J) or by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tet- and coimmunoprecipitation experiments. To determine whether the raacetic acid (BAPTA) treatment (7, 19, 20). Although the − − Pcdh15–Tmc interaction is relevant to mechanotransduction in vivo, anomalous current seen in Tmc1dn/dn Tmc2 / hair cells calls into we overexpressed N-terminal fragments of Tmc2a in zebrafish hair question whether TMC1 and TMC2 serve as pore-forming sub- cells. Overexpression of the Tmc2a N terminus results in mislocaliza- units of the mechanotransduction complex, it is nonetheless clear tion of Pcdh15a within hair bundles, together with a significant de- that these proteins are essential for normal transduction and crease in mechanosensitive responses, suggesting that a Pcdh15a– modulate the channel’s properties. However, how the TMCs in- Tmc complex is critical for mechanotransduction. Together, these teract with other components of the mechanotransduction com- results identify an evolutionarily conserved association between plex has not yet been investigated. the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, sup- Given that PCDH15 may be located at the site of mechano- porting the notion that TMCs are key components of the transduc- transduction, we performed a split-ubiquitin yeast two-hybrid tion complex in hair cells. screen with zebrafish Pcdh15a to identify proteins that could serve as subunits of the mechanotransduction complex. Our ensory hair cells are specialized mechanosensory cells that unbiased screen identified an interaction with the N terminus of Srespond to auditory and vestibular stimuli with their apical zebrafish Tmc2a, an ortholog of mammalian TMC2. Both Tmc1 hair bundles. Each bundle consists of rows of stereocilia that are and Tmc2a from zebrafish interact with the CD1 and/or CD3 interconnected by extracellular tip links. Collective evidence suggests that tip link filaments are composed of protocadherin Significance 15 (PCDH15) and cadherin 23 (CDH23) (1), which are each encoded by deafness genes identified in humans, mice, and fish Our understanding of the molecular basis of our sense of hearing (1–5). Tip links are required for proper gating of mechano- and balance has improved significantly, although some of the transduction channels (1, 6, 7), and calcium imaging of trans- key players in sensory hair cells have yet to be identified. Sensory duction in rodent auditory hair cells indicates that transduction hair cells depend on extracellular filaments known as tip links to channels are associated with the lower end of tip links (8), where transduce mechanical stimuli into electrical signals. We demon- PCDH15 localizes (6, 9). Thus, integral membrane proteins that strate that the tip link protein PCDH15 interacts with two integral interact with PCDH15 are likely to be critical for mechano- member proteins, TMC1 and TMC2, which have recently been put transduction and may constitute subunits or accessory proteins forth as candidates for the mechanotransduction channel. of the transduction channel. To date, neither the identity of the transduction channel nor the mechanism of interaction between Author contributions: R.M., K.S.K., W.M., C.P.M., T.E., P.G.B.-G., and T.N. designed re- search; R.M., K.S.K., W.M., C.P.M., T.E., and R.C.-G. performed research; H.Z. contributed the tip link and the channel are known. new reagents/analytic tools; R.M., K.S.K., W.M., C.P.M., T.E., P.G.B.-G., and T.N. analyzed Recently, TMC1 and TMC2 were put forth as candidates for data; and K.S.K., T.E., P.G.B.-G., and T.N. wrote the paper. NEUROSCIENCE the pore-forming subunits of the mechanotransduction channel The authors declare no conflict of interest. in sensory hair cells (10). Each protein is predicted to have mul- This article is a PNAS Direct Submission. tiple membrane-spanning domains reminiscent of an ion channel 1R.M., K.S.K., and W.M. contributed equally to this work. structure (11, 12); in addition, the Caenorhabditis elegans family 2Present address: Section on Sensory Cell Development and Function, National Institute member TMC-1 has been shown to be a putative channel suffi- on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, cient for detection of high salt (13). Mammalian TMC1 has been MD 20892. implicated in both recessive and dominant forms of nonsyndromic 3To whom correspondence should be addressed. Email: [email protected]. – hearing loss in humans and mice (14 16). Moreover, double This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. knockout of mouse Tmc1 and Tmc2 results in severe auditory and 1073/pnas.1402152111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1402152111 PNAS | September 2, 2014 | vol. 111 | no. 35 | 12907–12912 Downloaded by guest on September 26, 2021 isoforms of Pcdh15a, and these interactions require the common region present in both cytoplasmic domains. Likewise, using two different methods of yeast two-hybrid assays and immunopre- cipitation experiments in a heterologous expression system, we show that mouse PCDH15-CD3 can interact with either TMC1 or TMC2. Finally, we overexpressed an N-terminal fragment of Tmc2a to disrupt the Tmc–Pcdh15 interaction in zebrafish hair cells and found a reduction in mechanically evoked calcium tran- sients. Thus, our work shows that a Tmc–Pcdh15 interaction is critical for hair cell mechanosensitivity. Results Membrane-Based Screen for Components of the Mechanotransduction Complex. To identify members of the mechanotransduction com- plex in hair cells, we performed a molecular screen for proteins that bind to the cytoplasmic tail of Pcdh15a. We took advantage of the split-ubiquitin two-hybrid screening method, which is spe- cifically designed to detect membrane protein interactions at the cytoplasmic face of the endoplasmic reticulum of yeast cells (21). This method is ideal for bait proteins that contain single or multiple transmembrane domains, such as cadherins. To generate the bait vectors, we first identified splice variants of pcdh15a in 5-d-old wild-type larvae by using RACE PCR to amplify the 3′ end of pcdh15a. In mice and humans, three major isoforms of PCDH15 have been reported: CD1, CD2, and CD3 (22, 23). As reported in previously (5), we detected the CD1 isoform in zebrafish, along with amplicons corresponding to a CD3 isoform (Fig. S1). We were unable to detect any products corresponding to a CD2 isoform. As in other vertebrates, a common region Fig. 1. Interaction of zebrafish Pcdh15a with Tmc2a and Tmc1 using a split- ubiquitin yeast two-hybrid assay. (A) The N terminus of zebrafish Tmc2a adjacent to the transmembrane domain is shared by both CD1 – and CD3 isoforms of Pcdh15a (Fig. S1). (1 117 aa) can interact with either CD1 or CD3 isoforms of Pcdh15a, and binding is dependent upon the common region (CR). Transfection efficiency For the bait vectors, we used truncated versions of Pcdh15a- is shown on SD-LeuTrp medium (Left). Protein–protein interactions were CD1 or Pcdh15a-CD3 containing the transmembrane domain detected on SD-LeuTrpHisAde selective media (Right). Shown from left to and the full cytoplasmic domain (Fig. S1). Prey vectors contained right on each plate is a fivefold serial dilution series of yeast cells. The Ost1- cDNA generated from the total RNA of ∼1,500 zebrafish larval NubI ubiquitin moiety was used as a positive control, and empty prey vector ears at 5 d postfertilization (dpf).