Abstract: The actin-filled stereocilia of cochlear hair cells deflect in response to sound by pivoting around rootlets at their insertion points at the sensory cell surface. Kitajiri et al., 2010Kitajiri S.-i. Sakamoto T. Belyantseva I.A. Goodyear R.J. Stepanyan R. Fujiwara I. Bird J.E. Riazuddin S. Ahmed Z.M. Hinshaw J.E. et al.Cell. 2010; (this issue)PubMed Google Scholar now show that the actin-binding protein TRIOBP tightly bundles the actin filaments into rootlets, endowing these stereocilia “pivots” with unique elasticity and robustness. The actin-filled stereocilia of cochlear hair cells deflect in response to sound by pivoting around rootlets at their insertion points at the sensory cell surface. Kitajiri et al., 2010Kitajiri S.-i. Sakamoto T. Belyantseva I.A. Goodyear R.J. Stepanyan R. Fujiwara I. Bird J.E. Riazuddin S. Ahmed Z.M. Hinshaw J.E. et al.Cell. 2010; (this issue)PubMed Google Scholar now show that the actin-binding protein TRIOBP tightly bundles the actin filaments into rootlets, endowing these stereocilia “pivots” with unique elasticity and robustness. Studying inherited forms of deafness has opened up avenues for deciphering the molecular mechanisms underlying the development and function of the mammalian auditory sensory organ, the cochlea. This genetic approach bypasses a major obstacle presented by the small number of cochlear cells and offers unique experimental opportunities for improving our understanding of sound processing in the cochlea. In mouse models of human deafness, structures of unknown function can be removed or modified in a way that even the best microsurgery will not allow (Legan et al., 2000Legan P.K. Lukashkina V.A. Goodyear R. Kössl M. Russell I.J. Richardson G.P. Neuron. 2000; 28: 273-285Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, Verpy et al., 2008Verpy E. Weil D. Leibovici M. Goodyear R.J. Hamard G. Houdon C. Lefèvre G.M. Hardelin J.-P. Ridchardson G.P. Avan P. Petit C. Nature. 2008; 456: 255-259Crossref PubMed Scopus (92) Google Scholar). In this issue of Cell, Kitajiri et al., 2010Kitajiri S.-i. Sakamoto T. Belyantseva I.A. Goodyear R.J. Stepanyan R. Fujiwara I. Bird J.E. Riazuddin S. Ahmed Z.M. Hinshaw J.E. et al.Cell. 2010; (this issue)PubMed Google Scholar now use “genetic microsurgery” to elucidate the structure and function of the stereocilia rootlets of cochlear hair cells. The inner and outer hair cells of the cochlea detect sound using mechanosensitive hair bundles that protrude from their apical surfaces (Figure 1). The hair bundles contain microvilli-like structures called stereocilia that are filled with hundreds of actin filaments crosslinked into dense and ordered paracrystalline arrays (Tilney et al., 1980Tilney L.G. DeRosier D.J. Mulroy M.J. J. Cell Biol. 1980; 86: 244-259Crossref PubMed Scopus (301) Google Scholar). Each stereocilium tapers at its base immediately above the apical cell surface, where its actin core is reduced to 10–30 filaments that are tightly packed into a rootlet (Figure 1). Stereocilia rootlets form electron-dense rod-like structures that span roughly the entire tapering region and extend into the cuticular plate, a thick horizontal meshwork of actin filaments underneath the hair bundle (Furness et al., 2008Furness D.N. Mahendrasingam S. Ohashi M. Fettiplace R. Hackney C.M. J. Neurosci. 2008; 28: 6342-6353Crossref PubMed Scopus (73) Google Scholar, Tilney et al., 1980Tilney L.G. DeRosier D.J. Mulroy M.J. J. Cell Biol. 1980; 86: 244-259Crossref PubMed Scopus (301) Google Scholar). In response to mechanical stimulation, stereocilia behave like rigid rods and pivot around their tapered region (Howard and Ashmore, 1986Howard J. Ashmore J.F. Hear. Res. 1986; 23: 93-104Crossref PubMed Scopus (100) Google Scholar). This creates shearing stress between adjacent stereocilia that results in the opening of mechanosensitive transducer channels located at the tips of the stereocilia. In this way, auditory hair cells convert sound-evoked deflections of the stereocilia into graded variations of membrane potential with exquisite sensitivity. The rootlets are flexible structures responsible for the pivotal elasticity of stereocilia and are thought to anchor the stereocilia in the cuticular actin plate. Until recently, techniques were not available to allow researchers to test these ideas. In their new study, Kitajiri et al., 2010Kitajiri S.-i. Sakamoto T. Belyantseva I.A. Goodyear R.J. Stepanyan R. Fujiwara I. Bird J.E. Riazuddin S. Ahmed Z.M. Hinshaw J.E. et al.Cell. 2010; (this issue)PubMed Google Scholar, using a genetic approach, identify the actin-binding protein TRIOBP as a critical component of stereocilia rootlet formation and auditory function. They also demonstrate how TRIOBP may organize the actin filaments of stereocilia rootlets and how these pivot-like structures affect the mechanics of hair bundles. In humans, mutations in the TRIOBP gene cause a type of recessively inherited profound deafness called DFNB28 (Riazuddin et al., 2006Riazuddin S. Khan S.N. Ahmed Z.M. Ghosh M. Caution K. Nazli S. Kabra M. Zafar A.U. Chen K. Naz S. et al.Am. J. Hum. Genet. 2006; 78: 137-143Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, Shahin et al., 2006Shahin H. Walsh T. Sobe T. Abu Sa'ed J. Abu Rayan A. Lynch E.D. Lee M.K. Avraham K.B. King M.C. Kanaan M. Am. J. Hum. Genet. 2006; 78: 144-152Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To uncover the function of TRIOBP in the auditory system, Kitajiri et al. engineered mice that lacked two isoforms, TRIOBP-4 and TRIOBP-5, which are both defective in DFNB28 patients. They discovered that the cochlear hair bundles of the deaf mutant mice lack stereocilia rootlets. In the mouse cochlea, TRIOBP-4 is present in stereocilia from the apical tip to the tapered region, whereas TRIOBP-5 is mainly associated with stereocilia rootlets. The latter form shortly after birth and grow to >2 μm in length (Figure 1). An actin treadmilling process drives the renewal of the actin paracrystalline core of stereocilia and also may drive the final phase of stereocilia elongation that occurs concomitantly with rootlet formation. The length of stereocilia correlates with that of their rootlets (Furness et al., 2008Furness D.N. Mahendrasingam S. Ohashi M. Fettiplace R. Hackney C.M. J. Neurosci. 2008; 28: 6342-6353Crossref PubMed Scopus (73) Google Scholar). However, the genetic evidence in the Kitajiri et al. study now indicates that rootlet formation is driven by TRIOBP-dependent actin polymerization that is distinct from the actin treadmilling that drives formation of the stereocilia actin core. Taking advantage of their mutant mice, Kitajiri and colleagues determined how the absence of rootlets affects the mechanotransduction process and the mechanics of hair bundles. At equal deflections of the hair bundle, they found that the amplitude and kinetics of the current flowing through transducer channels in hair cells lacking TRIOBP-4 and TRIOBP-5 differed little from the currents observed in the hair cells of control mice. Therefore, the authors hypothesized that the absence of rootlets in hair bundles might instead modify the sound-evoked mechanical input to the transducer channels. The hair bundles of wild-type cells display a linear elastic behavior over a wide range of deflections, with stiffness in the mN/m range (Howard and Ashmore, 1986Howard J. Ashmore J.F. Hear. Res. 1986; 23: 93-104Crossref PubMed Scopus (100) Google Scholar). Using a fluid-jet to stimulate the stereocilia, Kitajiri et al. found that the hair bundles from mutant mice lacking TRIOBP-4 and TRIOBP-5 are abnormally compliant. When probed with a given force, they deflected with amplitudes 2–4 times larger than those of hair bundles from control mice. Moreover, the hair bundles of mutant cells displayed conspicuous nonlinear and nonelastic mechanical behavior (Figure 1); their pivotal stiffness decreased irreversibly after applying stimulation that did not affect the stiffness of normal hair bundles. Yet, for deflection amplitudes in the ranges expected to occur in vivo, the static pivotal stiffness of the hair bundle did not appear greatly reduced in the absence of rootlets but rather remained elastic and linear. However, during and after hearing onset, the stereocilia must withstand the oscillating mechanical stress imposed by acoustic stimuli. Thus, it is the dynamic stiffness of the hair bundle at acoustic frequencies (i.e., the amplitude ratio of an applied oscillating force to the resulting oscillating deflection) that sets its amplitude of deflection in response to sound. Dynamic stiffness might be much more affected than static stiffness, which could explain why mice lacking TRIOBP-4 and TRIOBP-5 become profoundly deaf. At any rate, the nonelastic behavior of mutant hair bundles when deflected by physiologically large amplitudes suggests that in the absence of rootlets, these deflections cause irreversible damage to the stereocilia. In the cochlear outer hair cells, which amplify the movement of auditory sensory epithelia in response to sound stimuli, some peripheral rootlets make contacts with the junctions between these cells and their supporting cells (Furness et al., 2008Furness D.N. Mahendrasingam S. Ohashi M. Fettiplace R. Hackney C.M. J. Neurosci. 2008; 28: 6342-6353Crossref PubMed Scopus (73) Google Scholar, Etournay et al., 2010Etournay R. Lepelletier L. Boutet de Monvel J. Michel V. Cayet N. Leibovici M. Weil D. Foucher I. Hardelin J.-P. Petit C. Development. 2010; 137: 1373-1383Crossref PubMed Scopus (28) Google Scholar). The absence of rootlets in mice lacking TRIOBP-4 and TRIOBP-5 also may alter the mechanical coupling between hair bundles of neighboring outer hair cells. This could in turn reduce hearing sensitivity by affecting the collective amplifying behavior of these cells. Finally, by examining transmission electron microscopy images of actin filaments formed on artificial lipid monolayers, Kitajiri et al. demonstrate that TRIOBP-4 organizes F-actin into bundles of 10 to 40 filaments. In addition, direct imaging of actin polymerization reveals that TRIOBP-4 promotes both increased stability of these bundles and prompt fusion of nearby growing bundles. Notably, the distance separating two actin filaments within these bundles is comparable to a filament's diameter. This lack of space between actin filaments is unusual and suggests that, instead of crosslinking them by intercalation, TRIOBP might wrap around them like a rope around a bundle of hay. Such wrapping would allow actin filaments to slide along each other, which would increase the range of shearing that the bundle can accommodate despite its dense packing. This sliding model could explain how rootlets can sustain the repeated mechanical stress concentrated at the stereocilia “pivots” while simultaneously allowing stereocilia to respond to a wide range of deflective forces. The force required to buckle an elastic rod composed of N = 20 rigidly crosslinked actin filaments of radius r ≈ 4 nm and length l = 2 μm is about 60 pN (F ≈ EN2r4/l2 with a Young's modulus of E ≈ 2.3 GPa). However, if the filaments are allowed to slide independently along each other, this force decreases by a factor of N (the number of filaments). This gives a buckling force of 3 pN, which is a more realistic estimate for a structure responding to forces in the pN range. Prior structural and functional studies suggest that rootlets are in fact dynamic structures that adapt to mechanical stress (Liberman and Dodds, 1987Liberman M.C. Dodds L.W. Hear. Res. 1987; 26: 45-64Crossref PubMed Scopus (109) Google Scholar). Thus, during adulthood, TRIOBP might be important for allowing rootlets to adapt to changes in the levels of ambient noise or to repair rootlets damaged by acoustic trauma. The study by Kitajiri et al. not only reveals how TRIOBP densely packs actin filaments into the rootlets of stereocilia but also paves the way to a better understanding of the role of the rootlets in hair bundle micromechanics. The widespread expression of TRIOBP protein isoforms in mice (Riazuddin et al., 2006Riazuddin S. Khan S.N. Ahmed Z.M. Ghosh M. Caution K. Nazli S. Kabra M. Zafar A.U. Chen K. Naz S. et al.Am. J. Hum. Genet. 2006; 78: 137-143Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) suggests that TRIOBP-mediated F-actin bundling might be an important new mechanism for building highly elastic and robust F-actin structures in cells. We thank Cecile Sykes, Paul Avan, and Jean-Pierre Hardelin for helpful comments. Actin-Bundling Protein TRIOBP Forms Resilient Rootlets of Hair Cell Stereocilia Essential for HearingKitajiri et al.CellMay 28, 2010In BriefInner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets but distinct from bundles formed by espin, an actin crosslinker in stereocilia. Full-Text PDF Open Archive