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Wei Xiong - One of the best experts on this subject based on the ideXlab platform.
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Deletion of Kncn Does Not Affect Kinocilium and Stereocilia Bundle Morphogenesis and Mechanotransduction in Cochlear Hair Cells
Frontiers in molecular neuroscience, 2018Co-Authors: Li Guo, Chenmeng Song, Wei XiongAbstract:Auditory hair cells possess stunning cilia structure that composes of a bundle of stereocilia for mechano-electrical transduction and a single Kinocilium for guiding the polarity of hair bundle towards maturation. However, the molecules underlying Kinocilium function have not yet been fully understood. Hence, the proteins involved in hair bundle development and function are of a large interest. From a fine microarray analysis, we found that kinocilin (Kncn) was enriched in hair cell specific expression profile. Consistently, it has been reported that KNCN was a protein mainly located in the Kinocilium of hair cells in the inner ear. However, the hypothesis that KNCN is a Kinocilium protein has not been validated in mice with Kncn gene perturbed. In this study, we generated Kncn knockout mouse lines by CRISPR/Cas9 technique and further examined the morphology and function of cochlear hair cells. Our results showed that there was no obvious hearing loss in the knockout mice, determined by audiometry. Histological study demonstrated that the inner ear and hair cell structure were intact. Especially, there was no deficit of mechanotransduction (MET) in cochlear outer hair cells (OHCs). In summary, our work suggests that KNCN is not essential for Kinocilium-oriented hair bundle function in cochlear hair cells.
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Image_1_Deletion of Kncn Does Not Affect Kinocilium and Stereocilia Bundle Morphogenesis and Mechanotransduction in Cochlear Hair Cells.TIFF
2018Co-Authors: Li Guo, Chenmeng Song, Wei XiongAbstract:Auditory hair cells possess stunning cilia structure that composes of a bundle of stereocilia for mechano-electrical transduction and a single Kinocilium for guiding the polarity of hair bundle towards maturation. However, the molecules underlying Kinocilium function have not yet been fully understood. Hence, the proteins involved in hair bundle development and function are of a large interest. From a fine microarray analysis, we found that kinocilin (Kncn) was enriched in hair cell specific expression profile. Consistently, it has been reported that KNCN was a protein mainly located in the Kinocilium of hair cells in the inner ear. However, the hypothesis that KNCN is a Kinocilium protein has not been validated in mice with Kncn gene perturbed. In this study, we generated Kncn knockout mouse lines by CRISPR/Cas9 technique and further examined the morphology and function of cochlear hair cells. Our results showed that there was no obvious hearing loss in the knockout mice, determined by audiometry. Histological study demonstrated that the inner ear and hair cell structure were intact. Especially, there was no deficit of mechanotransduction (MET) in cochlear outer hair cells (OHCs). In summary, our work suggests that KNCN is not essential for Kinocilium-oriented hair bundle function in cochlear hair cells.
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Table_1_Deletion of Kncn Does Not Affect Kinocilium and Stereocilia Bundle Morphogenesis and Mechanotransduction in Cochlear Hair Cells.DOCX
2018Co-Authors: Li Guo, Chenmeng Song, Wei XiongAbstract:Auditory hair cells possess stunning cilia structure that composes of a bundle of stereocilia for mechano-electrical transduction and a single Kinocilium for guiding the polarity of hair bundle towards maturation. However, the molecules underlying Kinocilium function have not yet been fully understood. Hence, the proteins involved in hair bundle development and function are of a large interest. From a fine microarray analysis, we found that kinocilin (Kncn) was enriched in hair cell specific expression profile. Consistently, it has been reported that KNCN was a protein mainly located in the Kinocilium of hair cells in the inner ear. However, the hypothesis that KNCN is a Kinocilium protein has not been validated in mice with Kncn gene perturbed. In this study, we generated Kncn knockout mouse lines by CRISPR/Cas9 technique and further examined the morphology and function of cochlear hair cells. Our results showed that there was no obvious hearing loss in the knockout mice, determined by audiometry. Histological study demonstrated that the inner ear and hair cell structure were intact. Especially, there was no deficit of mechanotransduction (MET) in cochlear outer hair cells (OHCs). In summary, our work suggests that KNCN is not essential for Kinocilium-oriented hair bundle function in cochlear hair cells.
Wally Grant - One of the best experts on this subject based on the ideXlab platform.
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Biomechanical measurement of Kinocilium.
Methods in enzymology, 2013Co-Authors: Corrie Spoon, Wally GrantAbstract:Vestibular hair cell bundles in the inner ear each contain a single Kinocilium that has the classic 9+2 axoneme microtubule structure. Kinocilia transmit movement of the overlying otoconial membrane mass and cupula to the mechanotransducing portion of the hair cell bundle. Force deflection techniques were used to measure turtle utricle Kinocilium shaft and base rotational stiffness. Kinocilia were modeled as homogenous cylindrical rods and their deformation examined as both isotropic Euler–Bernoulli beams (bending only) and as transversely isotropic Timoshenko beams (combined shear and bending). The measurements fit the transversely isotropic model much better with flexural rigidity EI = 10,400 pN·μm2 (95% confidence interval: 7182 to 13,630) and shear rigidity kGA = 247 pN (180 to 314). Resulting in a shear modulus (G=1.9 kPa) that was four orders of magnitude less than Young’s modulus (E=14.1 MPa), indicating that significant shear deformation occurs within deflected kinocilia. The base rotational stiffness (κ) was measured following BAPTA treatment to break the kinocilial links that bind the Kinocilium to the bundle along its shaft, and κ was measured as 177±47 pN·μm/rad. These parameters are important for understanding how forces arising from head movement are transduced and encoded.
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Biomechanics of hair cell kinocilia: experimental measurement of Kinocilium shaft stiffness and base rotational stiffness with Euler-Bernoulli and Timoshenko beam analysis.
The Journal of experimental biology, 2011Co-Authors: Corrie Spoon, Wally GrantAbstract:Vestibular hair cell bundles in the inner ear contain a single Kinocilium composed of a 9+2 microtubule structure. Kinocilia play a crucial role in transmitting movement of the overlying mass, otoconial membrane or cupula to the mechanotransducing portion of the hair cell bundle. Little is known regarding the mechanical deformation properties of the Kinocilium. Using a force-deflection technique, we measured two important mechanical properties of kinocilia in the utricle of a turtle, Trachemys (Pseudemys) scripta elegans. First, we measured the stiffness of kinocilia with different heights. These kinocilia were assumed to be homogenous cylindrical rods and were modeled as both isotropic Euler-Bernoulli beams and transversely isotropic Timoshenko beams. Two mechanical properties of the kinocilia were derived from the beam analysis: flexural rigidity (EI) and shear rigidity (kGA). The Timoshenko model produced a better fit to the experimental data, predicting EI=10,400 pN μm(2) and kGA=247 pN. Assuming a homogenous rod, the shear modulus (G=1.9 kPa) was four orders of magnitude less than Young's modulus (E=14.1 MPa), indicating that significant shear deformation occurs within deflected kinocilia. When analyzed as an Euler-Bernoulli beam, which neglects translational shear, EI increased linearly with Kinocilium height, giving underestimates of EI for shorter kinocilia. Second, we measured the rotational stiffness of the Kinocilium insertion (κ) into the hair cell's apical surface. Following BAPTA treatment to break the kinocilial links, the kinocilia remained upright, and κ was measured as 177±47 pN μm rad(-1). The mechanical parameters we quantified are important for understanding how forces arising from head movement are transduced and encoded by hair cells.
Guy P. Richardson - One of the best experts on this subject based on the ideXlab platform.
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FGFR1-mediated protocadherin-15 loading mediates cargo specificity during intraflagellar transport in inner ear hair-cell kinocilia.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Akira Honda, Guy P. Richardson, Tomoko Kita, Shri Vidhya Seshadri, Kazuyo Misaki, Zamal Ahmed, John E. Ladbury, Shigenobu Yonemura, Raj K. LadherAbstract:The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the Kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell Kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.
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Asymmetric Distribution of Cadherin 23 and Protocadherin 15 in the Kinocilial Links of Avian Sensory Hair Cells
The Journal of comparative neurology, 2010Co-Authors: Richard J. Goodyear, Andy Forge, P. Kevin Legan, Guy P. RichardsonAbstract:Cadherin 23 and protocadherin 15 are components of tip links, fine filaments that interlink the stereocilia of hair cells and are believed to gate the hair cell's mechanotransducer channels. Tip links are aligned along the hair bundle's axis of mechanosensitivity, stretching obliquely from the top of one stereocilium to the side of an adjacent, taller stereocilium. In guinea pig auditory hair cells, tip links are polarized with cadherin 23 at the upper end and protocadherin 15 at the lower end, where the transducer channel is located. Double immunogold labeling of avian hair cells was used to study the distribution of these two proteins in kinocilial links, a link type that attaches the tallest stereocilia of the hair bundle to the Kinocilium. In the kinocilial links of vestibular hair bundles, cadherin 23 localizes to the stereocilium and protocadherin 15 to the Kinocilium. The two cadherins are therefore asymmetrically distributed within the kinocilial links but of a polarity that is, within those links that are aligned along the hair bundle's axis of sensitivity, reversed relative to that of tip links. Conventional transmission electron microscopy of hair bundles fixed in the presence of tannic acid reveals a distinct density in the 120–130 nm long kinocilial links that is located 35–40 nm from the kinociliary membrane. The location of this density is consistent with it being the site at which interactions occur in an in trans configuration between the opposing N-termini of homodimeric forms of cadherin 23 and protocadherin 15. J. Comp. Neurol. 518:4288–4297, 2010. © 2010 Wiley-Liss, Inc.
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Initial characterization of kinocilin, a protein of the hair cell Kinocilium.
Hearing research, 2005Co-Authors: Michel Leibovici, Richard J. Goodyear, Guy P. Richardson, Elisabeth Verpy, Ingrid Zwaenepoel, Stéphane Blanchard, Sophie Lainé, Christine PetitAbstract:A subtracted library prepared from vestibular sensory areas [Nat. Genet. 26 (2000) 51] was used to identify a 960bp murine transcript preferentially expressed in the inner ear and testis. The cDNA predicts a basic 124aa protein that does not share any significant sequence homology with known proteins. Immunofluorescence and immunoelectron microscopy revealed that the protein is located mainly in the Kinocilium of sensory cells in the inner ear. The protein was thus named kinocilin. In the mouse, kinocilin is first detected in the kinocilia of vestibular and auditory hair cells at embryonic days 14.5, and 18.5, respectively. In the mature vestibular hair cells, kinocilin is still present in the Kinocilium. As the auditory hair cells begin to lose the Kinocilium during postnatal development, kinocilin becomes distributed in an annular pattern at the apex of these cells, where it co-localizes with the tubulin belt [Hear. Res. 42 (1989) 1]. In mature auditory hair cells, kinocilin is also present at the level of the cuticular plate, at the base of each stereocilium. In addition, as the Kinocilium regresses from developing auditory hair cells, kinocilin begins to be expressed by the pillar cells and Deiters cells, that both contain prominent transcellular and apical bundles of microtubules. By contrast, kinocilin was not detected in the supporting cells in the vestibular end organs. The protein is also present in the manchette of the spermatids, a transient structure enriched in interconnected microtubules. We propose that kinocilin has a role in stabilizing dense microtubular networks or in vesicular trafficking.
Andre Landin Malt - One of the best experts on this subject based on the ideXlab platform.
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Wnts regulate planar cell polarity via heterotrimeric G protein and PI3K signaling.
The Journal of cell biology, 2020Co-Authors: Andre Landin Malt, Arielle K. Hogan, Connor D. Smith, Maxwell S. MadaniAbstract:In the mammalian cochlea, the planar cell polarity (PCP) pathway aligns hair cell orientation along the plane of the sensory epithelium. Concurrently, multiple cell intrinsic planar polarity (referred to as iPCP) modules mediate planar polarization of the hair cell apical cytoskeleton, including the Kinocilium and the V-shaped hair bundle essential for mechanotransduction. How PCP and iPCP are coordinated during development and the roles of Wnt ligands in this process remain unresolved. Here we show that genetic blockade of Wnt secretion in the cochlear epithelium resulted in a shortened cochlear duct and misoriented and misshapen hair bundles. Mechanistically, Wnts stimulate Gi activity by regulating the localization of Daple, a guanine nucleotide exchange factor (GEF) for Gαi. In turn, the Gβγ complex signals through phosphoinositide 3-kinase (PI3K) to regulate Kinocilium positioning and asymmetric localizations of a subset of core PCP proteins, thereby coordinating PCP and iPCP. Thus, our results identify a putative Wnt/heterotrimeric G protein/PI3K pathway for PCP regulation.
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Par3 is essential for the establishment of planar cell polarity of inner ear hair cells.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Andre Landin Malt, Zachary Dailey, Julia Holbrook-rasmussen, Yuqiong Zheng, Arielle K. HoganAbstract:In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, Kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate Kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3-GEF-Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.
Arielle K. Hogan - One of the best experts on this subject based on the ideXlab platform.
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Wnts regulate planar cell polarity via heterotrimeric G protein and PI3K signaling.
The Journal of cell biology, 2020Co-Authors: Andre Landin Malt, Arielle K. Hogan, Connor D. Smith, Maxwell S. MadaniAbstract:In the mammalian cochlea, the planar cell polarity (PCP) pathway aligns hair cell orientation along the plane of the sensory epithelium. Concurrently, multiple cell intrinsic planar polarity (referred to as iPCP) modules mediate planar polarization of the hair cell apical cytoskeleton, including the Kinocilium and the V-shaped hair bundle essential for mechanotransduction. How PCP and iPCP are coordinated during development and the roles of Wnt ligands in this process remain unresolved. Here we show that genetic blockade of Wnt secretion in the cochlear epithelium resulted in a shortened cochlear duct and misoriented and misshapen hair bundles. Mechanistically, Wnts stimulate Gi activity by regulating the localization of Daple, a guanine nucleotide exchange factor (GEF) for Gαi. In turn, the Gβγ complex signals through phosphoinositide 3-kinase (PI3K) to regulate Kinocilium positioning and asymmetric localizations of a subset of core PCP proteins, thereby coordinating PCP and iPCP. Thus, our results identify a putative Wnt/heterotrimeric G protein/PI3K pathway for PCP regulation.
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Par3 is essential for the establishment of planar cell polarity of inner ear hair cells.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Andre Landin Malt, Zachary Dailey, Julia Holbrook-rasmussen, Yuqiong Zheng, Arielle K. HoganAbstract:In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, Kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate Kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3-GEF-Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.
