Stereocilia Morphology Research Articles

BAIAP2L1 and BAIAP2L2 differently regulate hair cell stereocilia morphology.

Inner ear sensory hair cells are characterized by their apical F-actin-based cell protrusions named stereocilia. In each hair cell, several rows of stereocilia with different height are organized into a staircase-like pattern. The height of stereocilia is tightly regulated by two protein complexes, namely row-1 and row-2 tip complex, that localize at the tips of tallest-row and shorter-row stereocilia, respectively. Previously, we and others identified BAI1-associated protein 2-like 2 (BAIAP2L2) as a component of row-2 complex that play an important role in maintaining shorter-row stereocilia. In the present work we show that BAIAP2L1, an ortholog of BAIAP2L2, localizes at the tips of tallest-row stereocilia in a way dependent on known row-1 complex proteins EPS8 and MYO15A. Interestingly, unlike BAIAP2L2 whose stereocilia-tip localization requires calcium, the localization of BAIAP2L1 on the tips of tallest-row stereocilia is calcium-independent. Therefore, our data suggest that BAIAP2L1 and BAIAP2L2 localize at the tips of different stereociliary rows and might regulate the development and/or maintenance of stereocilia differently. However, loss of BAIAP2L1 does not affect the row-1 protein complex, and the auditory and balance function of Baiap2l1 knockout mice are largely normal. We hypothesize that other orthologous protein(s) such as BAIAP2 might compensate for the loss of BAIAP2L1 in the hair cells.

CIB2 function is distinct from Whirlin in the development of cochlear stereocilia staircase pattern.

Variations in genes coding for calcium and integrin binding protein 2 (CIB2) and whirlin cause deafness both in humans and mice. We previously reported that CIB2 binds to whirlin, and is essential for normal staircase architecture of auditory hair cells stereocilia. Here, we refine the interacting domains between these proteins and provide evidence that both proteins have distinct role in the development and organization of stereocilia bundles required for auditory transduction. Using a series of CIB2 and whirlin deletion constructs and nanoscale pulldown (NanoSPD) assays, we localized the regions of CIB2 that are critical for interaction with whirlin. AlphaFold 2 multimer, independently identified the same interacting regions between CIB2 and whirlin proteins, providing a detailed structural model of the interaction between the CIB2 EF2 domain and whirlin HHD2 domain. Next, we investigated genetic interaction between murine Cib2 and Whrn using genetic approaches. Hearing in mice double heterozygous for functionally null alleles (Cib2 KO/+ ;Whrn wi/+ ) was similar to age-matched wild type mice, indicating that partial deficiency for both Cib2 and Whrn does not impair hearing. Double homozygous mutant mice (Cib2 KO/KO ;Whrn wi/wi ) had profound hearing loss and cochlear stereocilia exhibited a predominant phenotype seen in single Whrn wi/wi mutants. Furthermore, over-expression of Whrn in Cib2 KO/KO mice did not rescue the stereocilia morphology. These data suggest that, CIB2 is multifunctional, with key independent functions in development and/or maintenance of stereocilia staircase pattern in auditory hair cells.

Deletion of Luzp2 Does Not Cause Hearing Loss in Mice.

Deafness is the prevailing sensory impairment among humans, impacting every aspect of one's existence. Half of congenital deafness cases are attributed to genetic factors. Studies have shown that Luzp2 is expressed in hair cells (HCs) and supporting cells of the inner ear, but its specific role in hearing remains unclear. To determine the importance of Luzp2 in auditory function, we generated mice deficient in Luzp2. Our results revealed that Luzp2 has predominant expression within the HCs and pillar cells. However, the loss of Luzp2 did not result in any changes in auditory threshold. HCs or synapse number and HC stereocilia morphology in Luzp2 knockout mice did not show any notable distinctions. This was the first study of the role of Luzp2 in hearing in mice, and our results provide important guidance for the screening of deafness genes.

Tmc regulates membrane viscosity in mammalian cochlear hair cells.

The hair bundle, composed of rows of actin-filled stereocilia, is the sensory organelle of inner ear hair cells. There is a limited but growing body of data suggesting that stereocilia membrane properties modulate mechanically-gated (MET) ion channels located on the stereocilia. Transmembrane Channel Like proteins (TMCs) are considered part of the MET channel machinery, but also have membrane scramblase activity. To further investigate the role of membrane in modulating MET channel and test whether the presence of TMCs might account for the previously reported differences in stereocilia and soma membrane properties, we used a novel viscosity sensor BODIPY1c. The lifetime changes of BODIPY1c based on viscosity allowed precise spatial and dynamic monitoring of membrane properties within live hair cells. BODIPY1c can also enter hair cells through MET channels and fluorescently label the cytoplasmic membranes, thus allowing to identify hair cells with functional MET channels. Using synthesized liposomes of sizes ranging from 90-2000 nm with lipid composition matching that of stereocilia, we show that membrane curvature established with stereocilia morphology will not account for the observed difference between stereocilia and soma. We also show that the membrane viscosity of stereocilia and soma of mammalian cochlear hair cells vary during development. Membrane viscosity decreases and strongly correlates with the onset of MET. TMIE and TMC1 mutant mice, both of which lack TMC1 in stereocilia, have significantly higher membrane viscosity compared to litter mate controls at P10. In TMC1 mutants, the stereocilia membrane viscosity strongly correlates to the level at which the bundles are transducing. Inhibition of MET channel current in P10 rats for 30 mins with 1 mM curare, however, did not alter the membrane viscosity suggesting that the MET current is not directly driving the decrease in membrane viscosity.

Occlusion of two semicircular canals does not disrupt normal hearing in adult mice.

Vertigo is a debilitating disease affecting 15–20% of adults worldwide. Vestibular peripheral vertigo is the most common cause of vertigo, often due to Meniere's disease and benign paroxysmal positional vertigo. Although some vertigo symptoms can be controlled by conservative treatment and/or vestibular rehabilitation therapy, these treatments do not work for some patients. Semicircular canal occlusion surgery has proven to be very effective for these patients with intractable vertigo. However, its application is limited due to concern that the procedure will disrupt normal hearing. In this study, we investigated if occlusion of two semicircular canals would jeopardize auditory function by comparing auditory function and hair cell morphology between the surgical and contralateral ears before and after the surgery in a mouse model. By measuring the auditory brainstem response and distortion product otoacoustic emission 4 weeks post-surgery, we show that auditory function does not significantly change between the surgical and contralateral ears. In addition, confocal imaging has shown no hair cell loss in the cochlear and vestibular sensory epithelia, and scanning electron microscopy also indicates normal stereocilia morphology in the surgical ear. More importantly, the endocochlear potential measured from the surgical ear is not significantly different than that seen in the contralateral ear. Our study suggests that occlusion of two semicircular canals does not disrupt normal hearing in the mouse model, providing a basis to extend the procedure to patients, even those with normal hearing, benefitting more patients with intractable vertigo attacks.

BAIAP2L2 Inactivation Does Not Affect Stereocilia Development or Maintenance in Vestibular Hair Cells.

Hair cells are mechanosensitive cells in the inner ear, characterized by dozens to hundreds of actin-based stereocilia and one tubulin-based kinocilium on the apical surface of each cell. Two types of hair cells, namely cochlear hair cells and vestibular hair cells (VHCs), are responsible for the sensation of sound and balancing information, respectively. In each hair cell, the stereocilia are organized into rows of increasing heights with the mechano-electrical transduction (MET) channels localized at the tips of shorter-row stereocilia. A so-called “row 2 protein complex” also localizes at the tips of shorter-row mechanotransducing stereocilia, which plays important roles in the maintenance of mechanotransducing stereocilia. Recently, we and others identified BAIAP2L2 as a new component of row 2 complex. Baiap2l2 inactivation causes degeneration of the mechanotransducing stereocilia in cochlear hair cells, and leads to profound hearing loss in mice. In the present work, we examined the role of BAIAP2L2 in the VHC stereocilia. Confocal microscopy reveals that BAIAP2L2 immunoreactivity is localized at the tips of shorter-row stereocilia in VHCs. However, stereocilia development and maintenance are unaffected in Baiap2l2–/– VHCs. Meanwhile, MET function of VHCs as well as vestibular functions are also unaffected in Baiap2l2–/– mice. Further investigations show that the stereociliary tip localization of CAPZB2, another known row 2 complex component, is not affected in Baiap2l2–/– VHCs, consistent with the unaltered stereocilia morphology. Taken together, our present data show that BAIAP2L2 inactivation does not affect vestibular hair cell stereocilia.

Collapsin Response Mediator Protein 1 (CRMP1) Is Required for High-Frequency Hearing

Collapsin response mediator protein 1 (CRMP1), also known as dihydropyrimidinase-related protein 1, participates in cytoskeleton remodeling during axonal guidance and neuronal migration. In cochlear hair cells, the assembly and maintenance of the cytoskeleton is of great interest because it is crucial for the morphogenesis and maintenance of hair cells. Previous RNA sequencing analysis found that Crmp1 is highly expressed in cochlear hair cells. However, the expression profile and functions of CRMP1 in the inner ear remain unknown. In this study, the expression and localization of CRMP1 in hair cells was investigated using immunostaining, and was shown to be highly expressed in both outer and inner hair cells. Next, the stereocilia morphology of Crmp1-deficient mice was characterized. Abolishing CRMP1 did not affect the morphogenesis of hair cells. Interestingly, scanning electron microscopy detected hair cell loss at the basal cochlear region, an area responsible for high-frequency auditory perception, in Crmp1-deficient mice. Correspondingly, an auditory brainstem response test showed that mice lacking CRMP1 had progressive hearing loss at high frequencies. In summary, these data suggest that CRMP1 is required for high-frequency auditory perception.

Rab11a Regulates the Development of Cilia and Establishment of Planar Cell Polarity in Mammalian Vestibular Hair Cells.

Vestibular organs have unique planar cell polarity (Figure 1A), and their normal development and function are dependent on the regular polarity of cilia (Figure 1B) requires. Rab11a is a small G protein that participates in the transportation of intracellular and extracellular materials required for polarity formation; however, our understanding of the mechanisms of the actions of Rab11a in vestibular organs is limited. Here, we showed that the general shape of the utricle was abnormal in Rab11aCKO/CKO mice. These mice also showed abnormal morphology of the stereocilia bundles, which were reduced in both length and number, as well as disturbed tissue-level polarity. Rab11a affected the distribution of polarity proteins in the vestibular organs, indicating that the normal development of cilia requires Rab11a and intraflagellar transportation. Furthermore, small G protein migration works together with intraflagellar transportation in the normal development of cilia. FIGURE 1Morphological changes of stereocilia in the extrastriolar hair cells from Rab11a single or Rab11a/IFT88 double-mutant utricles. (A) Medial view of a mouse left inner ear with its five vestibular sensory organs (gray). Enlarged are the utricle showing their subdivisions, LPR (yellow line), and striola (blue). LES, lateral extrastriola; MES, medial extrastriola; LPR, line of polarity reversal. (B) Schematic view of vestibular hair cell. Kinocilium is marked with ace-tubulin. Basal body is marked with γ-tubulin. (C,C1,D,D1) Normal appearance of the stereocilia of extrastriolar hair cells of wild-type controls. (E,E1,F,F1) Altered morphology in Rab11aCKO/CKO animals. (G,G1,H,H1) The changes in the stereocilia morphology were more severe in Rab11aCKO/CKO/IFT 88CKO/+ mice. (I–L) Higher magnification of confocal images of hair cells. (M–P) Scanning electron microscopy images of hair cells from wild-type controls and Rab11a mutants. (I,M) Morphology of normal. hair cells of wild-type controls. (J,N) The number of stereocilia on a single hair cell was deceased in the Rab11a mutant. (K,O) Stereocilia were shorter in mutants compared to the wild-type controls. (L,P) The staircase-like hair bundle architecture of hair cells was lost in Rab11a mutant mice. (Q) The percentage of hair cells with abnormal development of static cilia bundles in the extrastriola region was counted as a percentage of the total (n = 5). The percentage of abnormal hair cells was higher in Rab11aCKO/CKO, IFT88CKO/+ mice compared to Rab11aCKO/CKO. The abnormal ratios of single and double knockout hair cells were 42.1 ± 5.7 and 71.5 ± 10.4, respectively. In (A–J), for all primary panels, hair cell stereociliary bundles were marked with phalloidin (green), the actin-rich cuticular plate of hair cells was labeled with β-spectrin (red), while the basal body of the hair cell was labeled with γ-tubulin (blue). Scale bars: 10 μm (C–H1), 5 μm (J–N). *P < 0.05.

Essential Role of Sptan1 in Cochlear Hair Cell Morphology and Function Via Focal Adhesion Signaling

Hearing loss is the most common human sensory deficit. Hearing relies on stereocilia, inserted into the cuticular plate of hair cells (HCs), where they play an important role in the perception of sound and its transmission. Although numerous genes have been associated with hearing loss, the function of many hair cell genes has yet to be elucidated. Herein, we focused on nonerythroid spectrin αII (SPTAN1), abundant in the cuticular plate, surrounding the rootlets of stereocilia and along the plasma membrane. Interestingly, mice with HC-specific Sptan1 knockout exhibited rapid deafness, abnormal formation of stereocilia and cuticular plates, and loss of HCs from middle and apical turns of the cochlea during early postnatal stages. Additionally, Sptan1 deficiency led to the decreased spreading of House Ear Institute-Organ of Corti 1 cells, and induced abnormal formation of focal adhesions and integrin signaling in mouse HCs. Altogether, our findings highlight SPTAN1 as a critical molecule for HC stereocilia morphology and auditory function via regulation of focal adhesion signaling.

Annexin A4 Is Dispensable for Hair Cell Development and Function.

Annexin A4 (ANXA4) is a Ca2+-dependent phospholipid-binding protein that is specifically expressed in the cochlear and vestibular hair cells, but its function in the hair cells remains unknown. In the present study, we show that besides localizing on the plasma membrane, ANXA4 immunoreactivity is also localized at the tips of stereocilia in the hair cells. In order to investigate the role of ANXA4 in the hair cells, we established Anxa4 knockout mice using CRISPR/Cas9 technique. Unexpectedly, the development of both cochlear and vestibular hair cells is normal in Anxa4 knockout mice. Moreover, stereocilia morphology of Anxa4 knockout mice is normal, so is the mechano-electrical transduction (MET) function. Consistently, the auditory and vestibular functions are normal in the knockout mice. In conclusion, we show here that ANXA4 is dispensable for the development and function of hair cells, which might result from functional redundancy between ANXA4 and other annexin(s) in the hair cells.

Phase separation-mediated condensation of Whirlin-Myo15-Eps8 stereocilia tip complex

Stereocilia, the mechanosensory organelles on the apical surface of hair cells, are necessary to detect sound and carry out mechano-electrical transduction. An electron-dense matrix is located at the distal tips of stereocilia and plays crucial roles in the regulation of stereocilia morphology. Mutations of the components in this tip complex density (TCD) have been associated with profound deafness. However, the mechanism underlying the formation of the TCD is largely unknown. Here, we discover that the specific multivalent interactions among the Whirlin-myosin 15 (Myo15)-Eps8 complex lead to the formation of the TCD-like condensates through liquid-liquid phase separation. The reconstituted TCD-like condensates effectively promote actin bundling. A deafness-associated mutation of Myo15 interferes with the condensates formation and consequently impairs actin bundling. Therefore, our study not only suggests that the TCD in hair cell stereocilia may form via phase separation but it also provides important clues for the possible mechanism underlying hearing loss.

Mechanotransduction current is essential for stability of the transducing stereocilia in mammalian auditory hair cells.

Mechanotransducer channels at the tips of sensory stereocilia of inner ear hair cells are gated by the tension of 'tip links' interconnecting stereocilia. To ensure maximal sensitivity, tip links are tensioned at rest, resulting in a continuous influx of Ca2+ into the cell. Here, we show that this constitutive Ca2+ influx, usually considered as potentially deleterious for hair cells, is in fact essential for stereocilia stability. In the auditory hair cells of young postnatal mice and rats, a reduction in mechanotransducer current, via pharmacological channel blockers or disruption of tip links, leads to stereocilia shape changes and shortening. These effects occur only in stereocilia that harbor mechanotransducer channels, recover upon blocker washout or tip link regeneration and can be replicated by manipulations of extracellular Ca2+ or intracellular Ca2+ buffering. Thus, our data provide the first experimental evidence for the dynamic control of stereocilia morphology by the mechanotransduction current.

A short splice form of Xin-actin binding repeat containing 2 (XIRP2) lacking the Xin repeats is required for maintenance of stereocilia morphology and hearing function.

Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-linkers result in hearing loss, we investigated the role of XIRP2 in hearing function. In the inner ear, XIRP2 is specifically expressed in hair cells, colocalizing with actin-rich structures in bundles, the underlying cuticular plate, and the circumferential actin belt. Analysis using peptide mass spectrometry revealed that the bundle harbors a previously uncharacterized XIRP2 splice variant, suggesting XIRP2's role in the hair cell differs significantly from that reported in myocytes. To determine the role of XIRP2 in hearing, we applied clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology to induce targeted mutations into the mouse Xirp2 gene, resulting in the elimination of XIRP2 protein expression in the inner ear. Functional analysis of hearing in the resulting Xirp2-null mice revealed high-frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. We thus conclude that XIRP2 is required for long-term maintenance of hair cell stereocilia, and that its dysfunction causes hearing loss in the mouse.

Effect of Storage Media and Storage Time on Histological and Ultrastructural Changes in Cat Epididymal Cells

The aim of this study was to assess the histological and ultrastructural changes in cat epididymides (n = 22) stored at 4 °C in two different media [saline solution (SAL) or tris-egg yolk (TEY)]. Our hypothesis was that epididymides stored in TEY would have delayed epithelial cell autolysis. Four epididymides were fixed and processed immediately, and the remaining 18 epididymides were stored at 4 °C in SAL or TEY for 24, 48 or 72 h. In histological sections, the nuclear features and stereocilia morphology were scored from 0 to 3. Ultrastructurally, nuclear chromatin and stereocilia morphology were scored from 0 to 3. In addition, using transmission electron microscopy nuclear number, nuclear area, mitochondrial number and mitochondrial area were recorded. In the histological study, parameters changed with time and media (p < 0.01). A significant effect of time was observed (p < 0.01), and the morphological changes were greatest when the storage time increased. Morphological changes were higher in SAL compared with TEY (p < 0.01). In the ultrastructural study, nuclear chromatin and stereocilia morphology decreased with time and media as in the histological study (p < 0.01). In addition, nuclear number and nuclear area changed with time (p < 0.004; p < 0.001) but not with media. Conversely, mitochondrial number and mitochondrial area did not change with media or time (p > 0.05). In conclusion, these results show that TEY preserved epididymal epithelial cells better than SAL; this finding could help improve sperm quality of stored epididymides.

Genetics of hearing and deafness.

This article is a review of the genes and genetic disorders that affect hearing in humans and a few selected mouse models of deafness. Genetics is playing an increasingly critical role in the practice of medicine. This is not only in part to the importance that genetic knowledge has on traditional genetic diseases but also in part to the fact that genetic knowledge provides an understanding of the fundamental biological process of most diseases. The proteins coded by the genes related to hearing loss (HL) are involved in many functions in the ear, such as cochlear fluid homeostasis, ionic channels, stereocilia morphology and function, synaptic transmission, gene regulation, and others. Mouse models play a crucial role in understanding of the pathogenesis associated with these genes. Different types of familial HL have been recognized for years; however, in the last two decades, there has been tremendous progress in the discovery of gene mutations that cause deafness. Most of the cases of genetic deafness recognized today are monogenic disorders that can be broadly classified by the mode of inheritance (i.e., autosomal dominant, autosomal recessive, X-linked, and mitochondrial inheritance) and by the presence of associated phenotypic features (i.e., syndromic; and nonsyndromic). In terms of nonsyndromic HL, the chromosomal locations are currently known for ∼ 125 loci (54 for dominant and 71 for recessive deafness), 64 genes have been identified (24 for dominant and 40 for recessive deafness), and there are many more loci for syndromic deafness and X-linked and mitochondrial DNA disorders (http://hereditaryhearingloss.org). Thus, today's clinician must understand the science of medical genetics as this knowledge can lead to more effective disease diagnosis, counseling, treatment, and prevention.

Fucoidan promotes mechanosensory hair cell regeneration following amino glycoside-induced cell death

Objective Lateral line system of the zebrafish is a useful model for study of hair cell toxicity and regeneration. We found that low molecular weight fucoidan (LMWF) stimulated the regeneration of mechanosensory hair cells after neomycin-induced cell death in zebrafish lateral line. The aims of this study were to quantify the regenerative effects of LMWF and determine their relationship to the Notch and FGF signaling pathways. Methods Wild-type zebrafish and three different transgenic zebrafish lines ( Pou4f3::GFP, scm1::GFP, and ET20::GFP) were used. At 4.5–6 days post-fertilization, lateral line hair cells of larvae were eliminated using neomycin (500 μM). Larvae were then treated with LMWF. Neuromasts were observed using confocal microscopy. Stereocilia morphology was observed using scanning electron microscopy, and the location and status of regeneration was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Results Hair cells damaged by neomycin treatment regenerated faster in wild-type and Pou4f3::GFP larvae treated with LMWF (50 μg/ml) than in untreated controls. LMWF also enhanced the regeneration of supporting cells in scm1::GFP and ET20::GFP larvae. Increased numbers of BrdU-labeled cells were found after LMWF treatment in neuromast regions corresponding to internal and peripheral supporting cells. The effect of LMWF was mimicked by the Notch signaling inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT), but the effects of LMWF and DAPT were not additive. Conclusion LMWF enhances the regeneration of hair cells damaged by neomycin. The mechanism may involve the Notch signaling pathway. LMWF shows promise as a therapeutic agent for hearing and balance disorders.

Dynamic changes in hair cell stereocilia and cochlear transduction after noise exposure

The structures of cochlear transduction include stereocilia at the apical surface of hair cells and their connection to the tectorial membrane. The transduction site is one of the loci for noise-induced cochlear damage. Although stereocilia are susceptible to noise, it has been found that in the inner ears of avians, this fragile structure is largely self-repairing and is associated with recovery of hearing sensitivity after noise exposure, as observed in the difference between the temporal threshold shift (TTS) and the permanent threshold shift (PTS). In the mammalian cochleae, however, threshold shifts measured in the auditory brainstem responses (ABR) did not parallel the chronological changes in the stereocilia on hair cells. It is unclear how the morphological recovery of the stereocilia on the mammalian hair cells is correlated with the changes in cochlear transduction that can be assessed by measuring receptor potential. In the present study, guinea pigs were exposed to a broadband noise of 110dBSPL for 2h. Auditory sensitivity was evaluated using ABR and cochlear transduction was assessed using cochlear microphonics (CM). Stereocilia morphology was quantified at different time points after the noise and compared with the control. The noise produced a TTS of 55.69±14.13dB in frequency-averaged ABR thresholds. The threshold shift was reduced to 9.58±11.75dBSPL 1month later with virtually no loss of hair cells. Damage to the stereocilia immediately after noise exposure was found to be associated with depression of CM amplitude. Virtually no abnormal stereocilia were observed 1month after the noise in association with a fully recovered CM.

β-Actin and γ-Actin Are Each Dispensable for Auditory Hair Cell Development But Required for Stereocilia Maintenance

Hair cell stereocilia structure depends on actin filaments composed of cytoplasmic β-actin and γ-actin isoforms. Mutations in either gene can lead to progressive hearing loss in humans. Since β-actin and γ-actin isoforms are 99% identical at the protein level, it is unclear whether each isoform has distinct cellular roles. Here, we compared the functions of β-actin and γ-actin in stereocilia formation and maintenance by generating mice conditionally knocked out for Actb or Actg1 in hair cells. We found that, although cytoplasmic actin is necessary, neither β-actin nor γ-actin is required for normal stereocilia development or auditory function in young animals. However, aging mice with β-actin– or γ-actin–deficient hair cells develop different patterns of progressive hearing loss and distinct pathogenic changes in stereocilia morphology, despite colocalization of the actin isoforms. These results demonstrate overlapping developmental roles but unique post-developmental functions for β-actin and γ-actin in maintaining hair cell stereocilia.

In vivo and in vitro effects of two novel gamma-actin (ACTG1) mutations that cause DFNA20/26 hearing impairment

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

Coupling adhesion to actin bundles in the inner ear. Novel functions for novel cadherins.

Genotype and phenotype define two poles of modern biology. The great challenge is to understand the connections that translate gene products into tissue architecture and function. Two recent studies (Boeda et al ., 2002; Siemens et al ., 2002) exemplify how these connections can be identified during the development of the inner ear. The sensory hair cells of the inner ear detect sound and postural change by sensing movement in the endolymph fluid of the cochlea and the semicircular canals, respectively. At the subcellular level, mechano‐sensing is mediated by organelles called stereocilia (SC)—fine, finger‐like protrusions that project from the apices of the hair cells. The morphology and organization of SC are strikingly precise: in mature hair cells, SC increase regularly in height from one side of the cell to the other and are attached to one another by several lateral connections, thereby integrating individual SC into functional units. Developmental mechanisms must therefore exist to coordinate the growth and lateral interactions between SC. The importance of SC morphogenesis becomes apparent in Usher syndrome type 1. Mouse models of this disease show deafness and vestibular dysfunction and have SC that grow haphazardly and lose lateral interactions with one another. Seven genetic loci are independently able to cause this syndrome when mutated (reviewed in Petit, 2001). The corresponding genes for four of these loci encode the unconventional motor protein myosin VIIa, the cytoplasmic protein harmonin, and two transmembrane proteins of the cadherin superfamily, cadherin 23 (CDH23) and protocadherin 15. How these apparently diverse gene products generate …