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Article |
Address correspondence to Bechara Kachar, Section on Structural Cell Biology, National Institute of Deafness and Other Communication Disorders, National Institutes of Health, Bldg. 50/Rm. 4249, 50 South Dr., Bethesda, MD 20892-8027. Tel.: (301) 402-1600. Fax: (301) 402-1765. email: Kacharb{at}nidcd.nih.gov
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Abstract |
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Key Words: hair cells; myosin XVa; myosin VIIa; espin; hearing
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Introduction |
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Recently, we exploited the preferential localization of the ß isoform of actin in the stereocilia to determine the locus of actin polymerization and to assess the degree of actin turnover in fully developed hair cells of the rat organ of Corti (Schneider et al., 2002). Transfecting cells with a DNA construct encoding ß actinGFP and imaging with confocal microscopy, we showed that the stereocilia actin filaments are continuously being turned over. Other recent discoveries using a genetic approach have also lead to an appreciation for unforeseen dynamic mechanisms within the stereocilia bundle. Genetic studies have shown that stereocilia formation and function depend on several novel actin-related proteins, including espin and several unconventional myosins, as mutations in their genes cause hereditary hearing loss by specifically disrupting stereocilia structure. Mutations in espin, an actin cross-linking protein, cause stereocilia to shorten and lose stiffness (Zheng et al., 2000); mutations in myosin XVa result in very short stereocilia; mutations in myosin VIIa lead to progressive disorganization of the stereocilia bundle; and mutations in myosin VI lead to stereocilia fusion (Steel and Kros, 2001). How stereocilia are formed and maintained while continuously functioning remains a fundamental question in inner ear research.
In this paper, we show that the actin paracrystal renewal process is organized as a "molecular conveyor belt" that mimics "treadmilling," a dynamic behavior of polarized polymers characterized by a net assembly at one end and disassembly at the other while maintaining steady-state length (Wanger et al., 1985). In the proposed stereocilia treadmill model, actin monomers are added at the tips and removed at the base of the stereocilia while the entire paracrystal moves basally. We show that espin is also incorporated at the tip of the stereocilia and moves rearwards at the same rate as actin. Most importantly, the treadmilling of the stereocilia paracrystal is highly regulated and the treadmill rates are scaled to the stereocilia length. We also characterize the localizations of myosins XVa, VIIa, and VI in relation to the actin paracrystal using light and electron microscopy immunolocalization techniques. We show that myosin XVa is localized at the barbed ends of the actin filaments of the paracrystal, whereas myosins VI and VIIa are localized alongside the paracrystal. We also show that the immunoreactivity levels for myosin XVa are proportional to the length of the stereocilia. The presence of these myosins at the tip and along the actin paracrystal suggests that they could be involved in paracrystal dynamics and stereocilia plasticity. Recognition of the dynamic nature of a structure previously regarded as the epitome of a stable cellular ensemble because of its ordered structure and mechanosensitivity is essential to understanding the development, repair, and maintenance of normal sensory function.
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Results |
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Characterization of the actin flux
To exclude the possibility that the observed actin incorporation and flux is a result of anomalous actin overexpression or a de novo nucleation of additional actin filaments at the actin paracrystal circumference, we analyzed the actin distribution by measuring pixel intensities in the fluorescence image. We used rhodamine/phalloidin to counterstain all the actin filaments and compared the fluorescence intensity profile measured along and across the stereocilia over the region where actin-GFP was incorporated. As shown in Fig. 2 A, such analysis shows that actin-GFP marks have a sharp leading edge, indicating that incorporation and flux is uniform for all filaments in the paracrystal. Also, there is no increase in rhodamine fluorescence across the region where actin-GFP is incorporated that would indicate formation of additional filaments laterally to the paracrystal (Fig. 2 A). Additional evidence illustrating the pattern of actin incorporation and flux was observed after serendipitous attenuation of ß actinGFP expression (Fig. 2 B). In this case, the incorporation appears as a band that has progressed halfway along the stereocilia. This band shows a sharp front facing the stereocilia base and a diffuse attenuated trailing edge facing the tip as incorporation of GFP-labeled actin is gradually reduced in favor of unlabeled actin. It also shows no change in the total actin fluorescence along the stereocilia, confirming that no anomalous actin filaments are added laterally to the actin paracrystal.
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Cytochalasin D shortens stereocilia as it blocks actin polymerization
To evaluate the contribution of actin polymerization to the incorporation and flux mechanisms and the maintenance of stereocilia length, we analyzed stereocilia exposed to the actin polymerization inhibitor cytochalasin D. We applied 1 µM cytochalasin D to 7-d-old cultures from all regions of the cochlea, fixed the preparations at 8, 16, 24, and 32 h, and analyzed the stereocilia lengths. We observed a progressive reduction in stereocilia lengths without significant changes in the characteristic structure of each stereocilium and staircase organization of the bundle (Fig. 3 A). We measured the length of stereocilia from the tallest rows in the bundles of inner and outer hair cells from the middle turns of organ of Corti and plotted the length as a function of time (Fig. 3 B). The estimated rate of shortening was 2.4 µm/24 h. Next, we assessed if actin-GFP marks that have been incorporated into the paracrystal continue to move after assembly is blocked during the shortening process. We repeated the cytochalasin D experiment with stereocilia labeled with ß actinGFP for 8 h before the drug application. We observed (n = 30 cells) that the actin-GFP marks remained at the tips and did not increase in length or progress down the stereocilia over the ensuing 16 h after the incubation with cytochalasin (Fig. 3 C).
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To test if the pointed tips are indeed correlated to the presence of intact tip links and if they could remodel after tip link loss, we treated cultures with a transient exposure to BAPTA, a calcium chelator known to disrupt tip links and mechano-transduction of hair cells (Assad et al., 1991). We analyzed changes of stereocilia tip shape both by light microscopy and by scanning EM (SEM). We observed that when the tip links are disrupted by BAPTA, the tips of the second and lower rows of stereocilia progressively attenuate their sharp pointed ends and assume a more rounded shape within 30 min after the BAPTA treatment (Fig. 4 E). To quantify this remodeling, we estimated the average length of the tip of the stereocilia (n = 30) to be 0.55 ± 0.03 µm in the control samples and decrease to 0.34 ± 0.02 µm after the BAPTA treatment (Fig. 4 E). This observation strongly suggests that the presence of the tip link may dynamically influence the shape of the stereocilia tip and its actin paracrystal core. Interestingly, close inspection of previously published images of BAPTA-treated stereocilia of chick hair cells show similar progressive rounding of the stereocilia tip (Kachar et al., 2000), suggesting that this phenomenon is common to all stereocilia.
Myosin XVa immunoreactivity at stereocilia tips is scaled to their lengths
Abnormally short stereocilia are observed in the shaker 2 and 2J mice, which carry mutations on the motor and FERM domains of myosin XVa, respectively (Anderson et al., 2000). Myosin XVa is an unconventional myosin suggested to have a role in the formation of stereocilia. To extend previous results on the localization of myosin XVa in hair cells and to gain insight into its role in stereocilia actin dynamics, we analyzed its precise localization using immunofluorescence. We observed intense and well-localized fluorescence signals at the stereocilia tips in whole-mount preparations of adult mouse, rat, and guinea pig organ of Corti and vestibular sensory epithelia using two affinity-purified antibodies. Virtually all stereocilia had detectable levels of myosin XVa labeling at their tips (Fig. 5 A). Moreover, although the primary localization of myosin XVa was at the tips of the stereocilia, occasional fluorescence puncta were also visualized along the stereocilia (Fig. 5 A). Detailed analysis of the fluorescence image revealed that the levels of myosin XVa fluorescence signal are scaled to the stereocilia length (Fig. 5). In both auditory and vestibular hair cells, the highest levels of fluorescence were observed in the tallest stereocilia of the bundle, whereas stereocilia of the second and lower rows showed considerably lower levels. We extended these immunofluorescence studies to embryonic and early postnatal stages of rat inner ear development to determine when the gradation in myosin XVa immunofluorescence signal arises. We detected myosin XVa signals at the tips of the earliest observable stereocilia on the surface of hair cells in embryonic day 18 cochlea. The gradation in myosin XVa fluorescence levels between different stereocilia rows was noticeable from the earliest emergence of nascent supernumerary stereocilia (Fig. 5 D).
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Myosins VI and VIIa immunoreactivity alongside the actin paracrystal
Myosins VI and VIIa are two other unconventional myosins highly expressed in hair cells (Hasson et al., 1997). To determine if these other myosins also localize to the tips of stereocilia we investigated their pattern of localization. We found that whereas myosin XVa is predominantly localized to the tips of stereocilia, myosins VI and VIIa are virtually excluded from this region but are consistently localized between the actin core and the lateral membrane (Fig. 7).
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Discussion |
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Actin depolymerization and paracrystal disassemble at the stereocilia base
Stereocilia are anchored at their base to the cuticular plate, a network of crisscrossed and branched actin filaments. At the base of the stereocilia, the actin paracrystal is tapered and the majority of filaments terminate in close apposition to the plasma membrane, leaving only the central filaments of the paracrystal to insert in the cuticular plate (Tilney et al., 1983). Our observation that incorporation and flux of actin-GFP occurs continuously and uniformly from the tips to the base, whereas the stereocilia and cuticular plate maintain steady-state structure, strongly suggests that the paracrystal is disassembled at the base and the actin filaments are depolymerizing at their pointed end. The observation that stereocilia shortening and rearward movement of the actin-GFP marks when polymerization is blocked by cytochalasin D is consistent with this interpretation.
How does actin flow from the tip to the base of the stereocilia?
The entire process of assembly, rearwards flux, and disassembly has several parallels to a process termed treadmilling. Treadmilling has been described as a system where a polarized polymer (or population of polymers) exists in a steady state characterized by net plus end assembly and minus end disassembly. This results in plus-to-minus end-directed subunit flux through polymer. In an actin filament treadmill model (Wanger et al., 1985), monomers are continuously added to the filament barbed (plus) end and removed from its pointed (minus) end while the filament maintains constant length in a dynamic steady state. We believe that paracrystal assembly and subunit flux in the stereocilia follow a treadmill model as shown in Fig. 8 A. We base this interpretation and our model on the following observations: (a) we show a flux of labeled actin from the barbed to the pointed end as the actin filament paracrystal maintains constant length; (b) actin overexpression does not drive an increase or decrease of the length of the actin paracrystal; (c) there is no evidence for the addition of actin filaments to the periphery of the actin paracrystal; (d) when the polymerization is inhibited with cytochalasin D, the transient actin-GFP marks do not grow but continue to move down as the stereocilia shorten; and (e) the rate of shortening or depolymerization in the cytochalasin-treated stereocilia is equivalent to the rate of actin polymerization/flux in the untreated stereocilia when the length is maintained constant. We interpret the actin flux measured in the stereocilia as the paracrystal treadmilling rate in the unique special case where the lengths of the stereocilia are held constant.
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Treadmilling versus retrograde flow
In some cases, treadmilling has been shown to coexist with actions of a molecular motor (like a myosin), which exerts translocation force on the polymer(s) as they treadmill. Motor presence may affect either or both polymer dynamics and polymer translocation. Rearward movement of actin cytoskeletal assemblies assisted by myosins has been termed "retrograde flow" (Forscher et al., 1987), which has been described in systems with a very dynamic cytoskeletal/membrane framework often undergoing movements of extension and retraction against a rigid substrate. We cannot exclude the possibility that the rearward movement of the actin paracrystal is somehow assisted by myosins in a retrograde flow type movement. However, it is not clear how this would occur in the particular case of the stereocilia. Myosin VIIa located alongside the stereocilia paracrystal would be in a position to produce force between the paracrystal and the membrane. However, because the stereocilia are not attached to a substrate laterally, a myosin bridging the paracrystal and the membrane could not exert an effective action on the paracrystal unless the membrane is maintained under tension. Therefore, it remains to be determined how much of the rearward movement of the paracrystal is driven by a treadmill-type process and how much is driven by the action of myosins in a retrograde flow process against a tensed encapsulating membrane.
Is actin filament elongation influenced by tension on the stereocilia membrane?
Except for a very narrow neck at the tapered base that allows the central actin filaments to anchor to the cuticular plate, the entire paracrystal actin core is contained within the encapsulating stereocilia membrane. Therefore, membrane tension or variations on the tension could have an effect on the paracrystal structure and dynamics. An indication that the stereocilia-encapsulating membrane is under positive tension is seen in EM images of stereocilia obtained by freeze etching (Kachar et al., 2000) or thin sections as shown in Fig. 5. These images show the encapsulating membrane with a smooth profile consistent with a tensed membrane and gradation of the actin filament lengths that precisely matches the membrane profile. In order for the actin filaments to display such ordered gradation in lengths, the actin elongation process must directly or indirectly be influenced by the compressive force normal to the membrane tension. Moreover, actin polymerization and elongation at the ends of each filament would exert a reciprocal force on the membrane and influence its shape (Mogilner and Oster, 1996). In such a model, actin polymerization would stall at a certain level of balance between the opposing forces produced by the membrane tension and the actin polymerization. This reciprocal relationship between the actin polymerization and the membrane tension can be adjusted toward a net gain or net loss and help define the length and shape of the stereocilia tip. Tip links can directly influence membrane tension by pulling from the extracellular side of the membrane. Our experiment with BAPTA is consistent with this possibility. The disruption of the tip link with BAPTA and subsequent retailoring of actin filaments and progressive rounding of the stereocilia tips supports the hypothesis that membrane tension can influence actin incorporation. However, we cannot exclude that other signaling mechanisms such as Ca2+ entry through the transduction channel could also be involved.
Regulation of stereocilia length
Our data clearly demonstrate that treadmill rates are scaled to the lengths of the stereocilia in the bundle and that this process is dynamically regulated. To maintain the steady-state length of the stereocilia paracrystal, the two processes, assembly and disassembly, must be precisely matched. Treatment with cytochalasin D uncouples the two processes and results in the progressive shortening of the stereocilia. Moreover, while the shortening proceeds, the relative heights of the stereocilia in the staircase bundle are preserved, indicating that the depolymerization rates are also scaled to the length of the stereocilia. Therefore, the rate of paracrystal disassembly appears to be independently regulated from actin polymerization.
We have no leads as to what controls disassembly at the base. However, we have made several observations that suggest a critical role for myosin XVa in stereocilia paracrystal elongation. Myosin XVa immunoreactivity is detected at the stereocilia tips from the earliest stages of stereocilia formation. Most strikingly, the immunoreactivity levels are proportional to the actin incorporation rates and are scaled to the length of the stereocilia within the bundle staircase. Myosin XVa immunoreactivity is concentrated in the confined region between the membrane and the barbed ends of the actin filaments of the paracrystal in the region where they insert or contact the tip density visualized by EM as an osmiophilic (electron-dense) structure at the tips of the stereocilia. It is not known what makes up the stereocilia tip density. However, the correlation of the shape and intensity of immunoreactivity for myosin XVa and the shape of electron density when comparing the tallest and the second rows of stereocilia as shown in Fig. 6 suggests that myosin XVa is part of this electron-dense structure or "tip complex." Myosin XVa could be working with other components of the tip complex, including capping and scaffold proteins, to regulate actin polymerization. The observation that the shaker 2J mice have very short stereocilia with rounded, symmetric tips lacking myosin XVa and the electron-dense structure is consistent with the potential role of myosin XVa in stereocilia paracrystal elongation.
Relationship of myosins VIIa and VI to a treadmilling stereocilia core
The side links between adjacent stereocilia, presumed to be important in determining the dynamic properties of the bundle, are highly ordered in the bundle's geometry. Recent biochemical data suggest that myosin VIIa can form associations with stereocilia lateral link and scaffold proteins (Boeda et al., 2002). We have confirmed the localization of myosin VIIa along the paracrystal surface in the confined space between the paracrystal and the stereocilia membrane. Previously, it was shown that myosins could move a cargo on the surface of the stereocilia paracrystal toward the barbed end of actin filaments (Shepherd et al., 1990). It is possible that myosin VIIa, localized alongside the actin paracrystal, could "walk" on the treadmill and dynamically maintain stereocilia links and possibly other stereocilia components in place as the actin paracrystal treadmills rearwards (Fig. 8 B). In contrast, myosin VI, which is a unique myosin because it moves toward the pointed end of actin filaments, was reported to be present around the rootlet of stereocilia (Steel and Kros, 2001). We have now shown that it is also present along the actin paracrystal. Little is known about how the actin paracrystal tapers and how the membrane constricts at the stereocilia base. Future studies are needed to further characterize the precise role of myosin VI and its possible role in either translocating components along the paracrystal or helping to shape and stabilize the tapered base of the stereocilia.
Role of actin paracrystal turnover in stereocilia repair
The dynamic regulation of the actin treadmill and the observation that the dwell time for actin subunits within a stereocilia will be roughly equivalent throughout a population of stereocilia with varying lengths could represent the basis for stereocilia repair after damage from overstimulation. A substantial loss of cross-bridges was observed in the stereocilia actin paracrystal after sound overstimulation and suggested that the recovery process would involve some type of reformation of the actin cross-bridges over a period of several days (Tilney et al., 1982). A subsequent overstimulation study reported much faster recovery times (Duncan and Saunders, 2000). Perhaps overstimulation could produce actin paracrystal disruption at different portions along the paracrystal including the base of the stereocilia where the actin paracrystal structure is much thinner. The renewal process we describe continuously replaces the entire actin paracrystal from the elongation locus at the stereocilia tip downwards. The time required to complete the repair would depend on the extent and location of disruption along the paracrystal structure. In summary, we have now introduced a view in which stereocilia components are continuously turned over and the ordered staircase structure is not completely predetermined during development but undergoes continuous remodeling that may facilitate recovery from injury.
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Materials and methods |
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Organotypic cultures of rat and mouse inner ear tissue
Organotypic cultures of rat and mouse organ of Corti and vestibular sensory epithelia were prepared according to Sobkowicz et al. (1993). Postnatal day 04 rat (or mouse) pups were anesthetized using CO2 and decapitated according to National Institutes of Health (NIH) guidelines, and their temporal bones were isolated and placed into L-15 media (GIBCO BRL). The dissected organ of Corti was divided into pieces for culturing. Subsequently, the vestibular system was dissected. Tissues were attached to a Cell Tak (BD Biosciences)coated coverslip in a culture dish. Cultures were maintained at 37°C and 5% CO2 in DME F-12 supplemented with 7% FBS containing 1.5 µg/ml ampicilin (GIBCO BRL). For the cytochalasin D experiments, cultures were incubated 8, 16, 24, and 32 h with DME F-12 media containing 1 µM cytochalasin D, from a 2-mM DMSO stock (Sigma-Aldrich). For the BAPTA (1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, 4 Na) experiments, cultures were gently washed with L-15 media without calcium and 5 mM BAPTA (Calbiochem) was applied. After 10-min incubation at 37°C, BAPTA was washed out with DME F-12 media and cultures were placed back into the incubator for 2 h.
Cell transfection and immunofluorescence
Cultured auditory and vestibular hair cells were transfected with 1 µm of gold carrier on which plasmid DNA coding for ß actinGFP (BD Biosciences) and small espin-GFP (a gift from J. Bartles, Northwestern University, Chicago, IL; Chen et al., 1999) were precipitated. A Helios Gene Gun (Bio-Rad Laboratories) was used to shoot 1-µm gold particles coated with DNA. Cultures were fixed with 4% PFA in PBS for 1 h, permeabilized for 30 min with 0.5% Triton X-100 in PBS, and counterstained with rhodamine/phalloidin (Molecular Probes) for 30 min. BAPTA- and cytochalasin Dtreated samples were prepared as described above and actin filaments were counterstained with Alexa Fluor 488/phalloidin (Molecular Probes). Immunolocalization of myosins was performed on cultures and freshly dissected tissues fixed in 4% PFA in PBS at RT as described previously (Liang et al., 1999). Fluorescence images were obtained with a confocal microscope (model LSM 510 [Carl Zeiss MicroImaging, Inc.] or model Ultraview [PerkinElmer]). The relative amounts of myosin XVa expressed at the tips of stereocilia was quantified by measuring the integrated pixel intensity of the fluorescence image in a 250-nm diameter circular area of interest placed on the tips of stereocilia of different rows using the NIH image program.
Tissue preparation for EM
Unfixed rat and mouse organs of Corti were finely dissected and rapidly frozen, freeze-substituted in 1.5% uranyl acetate in absolute methanol at -90°C, infiltrated with Lowicryl HM-20 resin at -45°C, thin sectioned, and immuno-gold labeled as described previously (Dumont et al., 2001). Thin sections and freeze-etching replicas were viewed and photographed with an electron microscope (model 922; Leo). SEM of BAPTA-treated cultures and shaker 2J mice tissues (The Jackson Laboratory) were prepared as described previously (Anderson et al., 2000).
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Acknowledgments |
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Submitted: 13 October 2003
Accepted: 10 February 2004
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References |
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