Article |
Address correspondence to Sachiko Tsukita, Dept. of Cell Biology, Kyoto University Faculty of Medicine, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: (81) 75-753-4373. Fax: (81) 75-753-4660. email: atsukita{at}mfour.med.kyoto-u.ac.jp
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Abstract |
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Key Words: ERM; radixin; stereocilia; cochlea; deafness
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Introduction |
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Stereocilia are specifically developed giant microvilli (finger-like projections of plasma membrane that are underlain by actin filaments). Microvilli show significant variations in morphology, diameter, and length depending on cell types; some are formed by densely packed rigid-looking actin filaments, whereas others are formed by loosely packed fragile-looking actin filaments (Furukawa and Fechheimer, 1997; Bartles, 2000; DeRosier and Tilney, 2000). Of these microvilli, the stereocilia of cochlear sensory hair cells are the most specialized with a characteristic morphology (DeRosier et al., 1980; Gillespie and Walker, 2001; Zuo, 2002). The stereocilia of vestibular sensory hair cells show a more fragile appearance (Sobin and Flock, 1983; Denman-Johnson and Forge, 1999). Besides the general microvilli cytoskeletal proteins such as actin, fimbrin, and myosin Ic, which were immunolocalized to stereocilia (Tilney et al., 1989; Drenckhahn et al., 1991; Skowron et al., 1998; Dumont et al., 2002), the positional cloning of deafness genes in humans and mice has enabled the identification of the novel cytoskeleton-associated constituents of stereocilia such as espin, harmonin, SANS, whirlin, unconventional myosins (VI/VIIa/XV), and cadherins (cadherin 23 and protocadherin 15); these proteins are most likely implicated during stereocilia formation and/or maintenance (Avraham et al., 1995; Gibson et al., 1995; Probst et al., 1998; Liang et al., 1999; Littlewood Evans and Müller, 2000; Zheng et al., 2000; Alagramam et al., 2001; Di Palma et al., 2001; Wada et al., 2001; Boeda et al., 2002; Karolyi et al., 2003; Mburu et al., 2003; Weil et al., 2003). Despite these advances, we are still far from having a comprehensive view of how the stereocilia of hair cells are built during development and maintained throughout life.
Ezrin/radixin/moesin (ERM) proteins are three closely related proteins in the band 4.1 superfamily that are thought to function as cross-linkers between plasma membranes and actin filaments, thus integrating the actin cortical layer, especially the microvilli (Sato et al., 1992; Berryman et al., 1993; Mangeat et al., 1999; Tsukita and Yonemura, 1999; Bretscher et al., 2002). The COOH-terminal domains of these ERM proteins bind to the actin filaments (Algrain et al., 1993; Turunen et al., 1994; Martin et al., 1995), and the NH2-terminal halves, the FERM domains (band 4.1/ezrin/radixin/moesin homology domains; Algrain et al., 1993; Henry et al., 1995; Bretscheret al., 2002), associate directly with the cytoplasmic domains of several integral membrane proteins such as CD43, CD44, and ICAM-1/2/3 (Tsukita et al., 1994; Helander et al., 1996; Serrador et al., 1997; Yonemura et al., 1998, 1999; Shaw, 2001). These NH2-terminal halves are also indirectly associated with membrane transporters/channels such as the Na+/H+ exchanger 3, CFTR (cystic fibrosis transmembrane conductance regulator), ß2-adrenergic receptor, and PDGF-receptor, via a cytoplasmic phosphoprotein, EBP50/NHE-RF (Reczek et al., 1997; Yun et al., 1997; Murthy et al., 1998; Short et al., 1998; Maudsley et al., 2000; Bretscher et al., 2002). The cross-linking activity of ERM proteins between actin filaments and plasma membranes is thought to be regulated by PIP2 in the downstream of Rho (Hirao et al., 1996; Mackay et al., 1997; Heiska et al., 1998; Matsui et al., 1999; Yonemura et al., 2002).
The suppression of ERM protein expression in lymphocytes using antisense oligonucleotides results in the complete disappearance of microvilli, suggesting that they play an essential role in cortical microvilli formation (Takeuchi et al., 1994). To examine the physiological functions of ERM proteins at the entire body level (Ingraffea et al., 2002), moesin- and radixin-deficient mice have been generated (Doi et al., 1999; Kikuchi et al., 2002). Moesin-deficient mice showed no abnormalities when examined under laboratory conditions (Doi et al., 1999). By contrast, radixin deficiency caused congenital conjugated hyperbilirubinemia with losses of multidrug resistance protein 2, a bilirubin transporter, from the bile canalicular membranes of the hepatocytes with concomitant decrease of microvilli in number as well as in length (Kikuchi et al., 2002). In wild-type mice, radixin was the dominant ERM protein in the hepatocytes, and was not substituted by either ezrin or moesin in Rdx/ mice.
Taking all of the above into consideration, it would seem important that the possible involvement of ERM proteins in the formation, maintenance, and functions of stereocilia on hair cells be examined. In this work, we found that radixin was the only detectable species of ERM protein in the stereocilia of the cochlea of adult wild-type mice, whereas vestibular stereocilia appeared to contain a detectable amount of ezrin in addition to radixin. Adult Rdx/ mice exhibited profound deafness, but not imbalance; consistent with this, stereocilia were significantly defective in the outer and inner hair cells of the cochlea, but not in the hair cells of the vestibule. Furthermore, we found that in the cochlea of Rdx/ mice, ezrin-based stereocilia appeared to develop normally from birth to postnatal day 14 (P14), the onset of hearing, after which they progressively degenerated. Thus, the radixin knockout provides a type of mouse deafness model caused by mutation in cytoskeletal components in stereocilia, which presents with progressive degeneration of cochlear (but not vestibular) stereocilia. The present paper provides a new insight into the ERM-based molecular mechanisms for the formation and maintenance of stereocilia, and the functional specificity/redundancy among ERM proteins in situ.
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Results |
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Radixin deficiency causes progressive degeneration of stereocilia of cochlear hair cells after the onset of hearing
The question then naturally arose as to what is the consequence of radixin deficiency in the cochlear stereocilia in Rdx/ mice. Isolated cochleae of P40 Rdx+/+ and Rdx/ littermates were examined by scanning EM (Fig. 3 A, af). When removing the lateral wall and tectorial membrane of the cochlea in P40 Rdx/ mice to expose the organ of Corti, the cellular arrangement of the outer and inner hair cells as well as the supporting cells appeared normal, but very interestingly, the stereocilia in both outer and inner hair cells were significantly defective compared with those in Rdx+/+ organ of Corti. Instead of being regularly arranged in a "W" shape, stereocilia of the outer hair cells of Rdx/ mice were deformed to 13 residual knoblike protrusions on the apical surfaces (Fig. 3 A, d). Inner hair cells had several shorter fused protrusions instead of stereocilia (Fig. 3 A, f). When the organ of Corti isolated from these Rdx/ cochleae was whole-mount stained with ezrin-, radixin-, and moesin-specific mAbs, ezrin, radixin, or moesin was not detected in the residual structures of outer and inner stereocilia, i.e., knoblike protrusions and shorter fused protrusions, respectively (Fig. 3 A, gj). Diffuse ezrin staining in the organ of Corti and intense moesin staining in blood vessels did not appear to be affected. Considering that stereocilia play a central role in transducing acoustic stimuli into electrical signals in the cochlea, it is likely that these defects in cochlear stereocilia are responsible for the hearing impairments observed in adult Rdx/ mice.
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We then pursued the possible postnatal degeneration process of stereocilia in Rdx/ mice (Fig. 4). At P6, scanning EM did not detect any significant differences between Rdx+/+ and Rdx/ cochlea, indicating that there was still no sign for stereocilia degeneration (Fig. 4 A, af). However, in Rdx+/+ cochlea, ezrin became weaker during P1 to P14, almost undetectable from stereocilia at P14, and radixin was dominantly detected among ERM proteins in stereocilia (Fig. 4 A, g and i). Supporting cells were intensely stained for ezrin and radixin. In P6 Rdx/ cochlea, ezrin was highly concentrated in stereocilia on both inner and outer hair cells (Fig. 4 A, h) with no radixin staining (Fig. 4 A, j). Around age P14 (onset of hearing in mice), in Rdx/ cochlea, the sign for stereocilia degeneration began to be detected on outer hair cells by scanning EM (Fig. 4 B, af): The central part of the W-shaped row of stereocilia was lost, leaving discontinuous and disorganized arrays of stereocilia. Ezrin was still concentrated in these degenerating stereocilia in large amounts, though in Rdx+/+ hair cells, radixin, but not ezrin, was enriched at stereocilia (Fig. 4 B, gj). We concluded that in Rdx/ cochleae, ezrin can counterbalance radixin deficiency to normally develop stereocilia up to the onset of hearing, but not to maintain these once-developed stereocilia to transmit acoustic stimuli into electrical signals.
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Ezrin compensates radixin deficiency in the development/maintenance of stereocilia in the vestibular hair cells
As shown in Fig. 1, in the vestibule of wild-type mice aged 510 wk, radixin was predominantly concentrated in stereocilia among ERM proteins, but distinct from cochlear stereocilia, these stereocilia contained detectable amounts of ezrin. The question then arose as to the structure and function of vestibular stereocilia in adult Rdx/ mice. Structurally, scanning EM identified no abnormality in the number/density and morphology of stereocilia at the crista ampullaris of the vestibule of adult Rdx/ mice (Fig. 6 A). Also, scanning EM identified no abnormality in the otolith organs (the utricle and saccule) of adult Rdx/ mice (unpublished data). Functionally, Rdx/ vestibular stereocilia appeared to be normal because adult Rdx/ mice showed no signs of imbalance in their behavior as far as we examined up to 100 d after birth. Then, their vestibulo-ocular reflex (VOR) was measured, in which the function of horizontal semicircular canals, but not otolith organs (the utricle and saccule), can be evaluated. Heads of Rdx+/+, Rdx+/, or Rdx/ mice aged 510 wk were rotated sinusoidally and eye positions were recorded using a CCD camera at various head rotation frequencies. As shown in Fig. 6 B, the VOR gains of Rdx/ mice were normal at all head rotation frequencies.
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Discussion |
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To discuss the functions of ERM proteins and their possible redundancy in situ, the inner ear provides an advantageous system, as in it there are two distinct types of stereocilia, cochlear and vestibular stereocilia, which play central roles in transmitting acoustic and acceleration stimuli, respectively, into electrical signals. (Roberts et al., 1988; Hudspeth, 1989; Pickles and Corey, 1992; Eatock et al., 1998; Corey, 2003). In wild-type cochlea, before P14, ezrin was detectable in addition to large amounts of radixin in stereocilia, and then progressively disappeared, leaving purely radixin-based stereocilia. In the Rdx/ cochlea, ezrin was significantly up-regulated, and purely ezrin-based stereocilia developed normally up to
P14 and then progressively degenerated. By contrast, in the Rdx+/+ vestibule, stereocilia contained a small amount of ezrin in addition to large amounts of radixin throughout life, whereas in the Rdx/ vestibule, stereocilia containing up-regulated ezrin developed and was maintained normally throughout the life. These findings would provide the first clear example that these members of the ERM family, ezrin and radixin, function redundantly in situ at the whole-body level: ezrin compensates for radixin deficiency functionally in stereocilia. In Rdx+/+ mice, the cochlear hair cells up to
P14 and the vestibular hair cells throughout the life expressed both radixin and ezrin in large and small amounts, respectively, and probably due to the difference in their expression levels or affinity to plasma membranes, radixin was the main contributor to the formation of stereocilia. In these hair cells, in the absence of radixin (Rdx/ mice), a larger amount of ezrin was recruited, which appeared to compensate for radixin deficiency in the formation of stereocilia.
The molecular mechanism behind the progressive degeneration of the cochlear stereocilia after the onset of hearing (P14) remained elusive. At present, two explanations appear possible. First, if the expression of ezrin is genetically programmed to be suppressed in cochlear hair cells after the onset of hearing in the wild-type mice, and if this program is not changed by radixin deficiency, in Rdx/ mice, cochlear hair cells lack both ezrin and radixin after the onset of hearing, resulting in progressive degeneration of stereocilia. As the total lack of ERM proteins was reported to cause cell death (Kondo et al., 1997), this mechanism can explain why cochlear hair cells were eliminated in older Rdx/ mice. The second possibility is that sound stimulation induces the progressive degeneration of ezrin-based stereocilia in the Rdx/ cochlea. It would be possible that these ezrin-based stereocilia are more labile against the noise stimulation (i.e., frequent deflection) than the wild-type radixin-based stereocilia due to the qualitative or quantitative difference between radixin in wild-type stereocilia and ezrin in Rdx/ stereocilia. Consistently, the noise-induced loss of stereocilia was reported in the wild-type mice as well as humans (Ou et al., 2000). As deflection frequency for vestibular stereocilia is expected to be much lower than for cochlear stereocilia, Rdx/ vestibule ezrin-based stereocilia could be maintained without degeneration. According to Western blotting of the whole cochlea and vestibule, there were no signs for up-regulation of the expression of ezrin (or moesin), but as it was technically difficult to examine the expression of ERM proteins in individual sensory hair cells, the two above explanations cannot be further evaluated for now. Furthermore, to understand the molecular mechanism behind the progressive degeneration of the cochlear stereocilia, identification of the specific molecular partners of radixin and ezrin, including integral membrane proteins, within stereocilia would be important, although it is again technically difficult due to the small number of stereocilia in the cochlea. Indeed, as described in the results, we performed immunoprecipitation and gel overlay experiments, but were unable to identify the radixin- or ezrin-binding partners in stereocilia. To be successful, any future work will need to overcome several technical difficulties.
Mutations in the actin-bundling protein espin have been reported in jerker mice and DFNB36, a form of nonsyndromic deafness in humans (Grüneberg et al., 1941; Zheng et al., 2000; Belyantseva et al., 2003). Adult jerker mice exhibit impaired hearing and balance dysfunctions, as well as short cochlear and vestibular stereocilia. Analyses of various aged jerker mice revealed apparently normal cochlear stereocilia development until the onset of hearing, after which the stereocilia progressively degenerate (Sjöström and Anniko, 1992). This aspect of jerker mice is very similar to Rdx/ mice, but jerker mice are completely different from Rdx/ mice in terms of the balance dysfunction. Because espin is likely to play a role in bundling actin filaments in stereocilia (Loomis et al., 2003), it was suggested that the organization of core actin filaments is critical for the maintenance of stereocilia after the onset of hearing. Furthermore, genetic studies of deafness in mice and humans have identified several genes for other cytoskeleton-related proteins, harmonin, myosin VI/VIIa, whirlin, and SANS, and for cadherin-related proteins, cadherin 23 and protocadherin 15, as causal genes (Avraham et al., 1995; Gibson et al., 1995; Probst et al., 1998; Self et al., 1998; Littlewood Evans and Müller, 2000; Zheng et al., 2000; Alagramam et al., 2001; Di Palma et al., 2001: Petit et al., 2001; Boeda et al., 2002; Mustapha et al., 2002; Karolyi et al., 2003; Kikkawa et al., 2003; Mburu et al., 2003; Weil et al., 2003). In these mice, the formation of not only cochlear, but also vestibular stereocilia was affected, resulting in the deafness associated with imbalance. All these cytoskeleton-related proteins, including ERM proteins and epsin, may act in cooperative ways to form and maintain dynamic stereocilia in cochlear and vestibular sensory hair cells.
To date, three mouse models in which there is profound hearing loss and no vestibular dysfunction have been reported: the deafness (dn) gene mutant mice (Bock and Steel, 1983), Beethoven mice (Vreugde et al., 2002), and the claudin 14-knockout mice (Ben-Yosef et al., 2003). Rdx/ mice can now be added as a new member of this list. Rdx/ mice should provide a valuable resource for further analysis of the functional redundancy of ERM proteins at the whole-body level, and of the molecular mechanisms behind the formation and maintenance of highly specialized microvilli, stereocilia; the elucidation of which will shed light on the pathogenesis of human deafness and imbalance.
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Materials and methods |
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Generation of Rdx/ mice
Rdx/ mice were generated as previously reported (Kikuchi et al., 2002). Two independent mouse J1 ES clones (129/Sv) in which the radixin gene was correctly disrupted were injected into C57BL/6 blastocysts, and the resulting chimeras were mated with C57BL/6 mice (Doi et al., 1999).
ABR measurements
ABR measurements were performed in a soundproof room (Zheng et al., 1999). In general, the ABR waveforms were recorded for 12.8 ms at a sampling rate of 40,000 Hz using 505,000-Hz filter settings; waveforms recorded from 1,024 stimuli at a frequency of 9 Hz were averaged. ABR waveforms were recorded in decreasing 5-dB SPL intervals from a maximum amplitude until no waveforms could be visualized.
VOR measurements
VOR was measured as described by Iwashita et al. (2001). Head movements were transduced to DC signals using a small angular velocity sensor (Gyrostar; Murata Corporation) that was fixed on the turntable. Eye movements were detected by LED and a CCD camera (C53500; Tokyo Electronic Industry), and eye velocities were calculated online by downloading them onto a computer through a video capture board. Both the head and eye velocity curves were fitted with sinusoidal curves using the least squares criterion, and the gain of eye velocity relative to the head velocity was obtained.
Immunofluorescence microscopy
Temporal bones were removed from 1-, 6-, 14-, or 40-d-old mice, and together with the small holes in the cochlear apical turn and superior semicircular canal, the round and oval windows were opened. The lymphatic space was gently perfused with 10% TCA through a round window (Hayashi et al., 1999; Kitajiri et al., 2004). Samples were then immersed in 10% TCA for 1 h at 4°C, washed three times with PBS, and decalcified with 5% EDTA in PBS for 3 d at 4°C. The cochlea and vestibule were then carefully microdissected, treated with 0.2% Triton X-100 in PBS for 15 min, and soaked in 1% BSA in PBS at RT. They were then incubated with rat anti-ezrin (M11), anti-radixin (R11), or anti-moesin (M22) mAb for 30 min at RT. Samples were washed three times with PBS, followed by a 30-min incubation with Cy3- (Jackson ImmunoResearch Laboratories, Inc.) or Alexa Fluor® 488conjugated secondary antibody (Molecular Probes, Inc.). After a wash with PBS, they were embedded in 95% glycerol-PBS containing 0.1% paraphenylendiamine and 1% n-propylgalate. Fluorescence images were obtained with a confocal microscope (model LSM 510 META; Carl Zeiss MicroImaging, Inc.) or with a DeltaVision optical sectioning microscope (version 2.10; Applied Precision, Inc.), equipped with an Axioplan2 (Plan Apochromat 63x/1.40 NA oil immersion objective; Carl Zeiss MicroImaging, Inc.) or IX70 (PlanApo 60x/1.40 NA oil immersion objective; Olympus) microscope, respectively.
Immunoblotting
The membranous labyrinths of 5-wk-old mice were dissected under a microscope. Whole organ of Corti and vestibules isolated from each mouse were sonicated in 100 µl SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 2% 2-mercaptoethanol, and 0.01% bromophenol blue), applied to SDS-PAGE, and immunoblotted by a blotting detection kit with biotinylated Ig and streptavidin-conjugated alkaline phosphatase (Amersham Biosciences).
Scanning EM
Temporal bones obtained from 1-, 6-, 14-, or 40-d-old mice were fixed using perilymphatic perfusion as described above with 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). They were then washed with phosphate buffer and post-fixed in 1% OsO4 for 2 h, after which they were once again treated with perilymphatic perfusion. The organ of Corti or crista ampullaris was microdissected, dehydrated, critical-point dried, sputter coated, and observed by scanning EM (model S-800 microscope; Hitachi Co.).
Ultrathin-section EM
Samples were processed as previously described, using 2% formaldehyde, 2.5% glutaraldehyde, and 0.1 M sodium cacodylate buffer (pH 7.4) as a fixative (Yonemura et al., 2002).
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Acknowledgments |
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This work was supported in part by a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science and Culture, Japan to Shoichiro Tsukita, and in part by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science and Culture, Japan, to Sachiko Tsukita.
Note added in proof. While this paper was being reviewed, Pataky et al. presented evidence that radixin is a constituent of stereocilia in hair cells (Pataky, F., R. Pironkova, and A.J. Hudspeth. 2004. Proc. Natl. Acad. Sci. USA. 101:26012606).
Submitted: 2 February 2004
Accepted: 14 June 2004
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