Browsing by Author "Zhao, Bo"
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Item Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin(Elsevier, 2021-03) McGrath, Jamis; Tung, Chun-Yu; Liao, Xiayi; Belyantseva, Inna A.; Roy, Pallabi; Chakraborty, Oisorjo; Li, Jinan; Berbari, Nicolas F.; Faaborg-Andersen, Christian C.; Barzik, Melanie; Bird, Jonathan E.; Zhao, Bo; Balakrishnan, Lata; Friedman, Thomas B.; Perrin, Benjamin J.; Biology, School of ScienceStereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.Item ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points(JCB, 2022-02-17) Krey, Jocelyn F.; Liu, Chang; Belyantseva, Inna A.; Bateschell, Michael; Dumont, Rachel A.; Goldsmith, Jennifer; Chatterjee, Paroma; Morrill, Rachel S.; Fedorov, Lev M.; Foster, Sarah; Kim, Jinkyung; Nuttall, Alfred L.; Jones, Sherri M.; Choi, Dongseok; Friedman, Thomas B.; Ricci, Anthony J.; Zhao, Bo; Barr-Gillespie, Peter G.; Otolaryngology -- Head and Neck Surgery, School of MedicineThe stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell’s cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed–TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.Item Autophagy proteins are essential for aminoglycoside-induced hearing loss(Taylor & Francis, 2023) Li, Jinan; Liu, Chang; Müller, Ulrich; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of MedicineAminoglycosides (AGs) are widely used to treat severe infections. However, systemically administered AGs preferentially kill cochlear hair cells, resulting in irreversible hearing loss. Recently, we found that AGs bind to RIPOR2 and trigger its rapid translocation in cochlear hair cells. Reducing RIPOR2 expression entirely prevents AG-induced hair cell death and subsequent hearing loss in mice. Next using yeast two-hybrid screening, we found that RIPOR2 interacts with GABARAP, a key macroautophagy/autophagy pathway protein. Following AG treatment, RIPOR2 colocalizes with GABARAP and regulates the activation of autophagy. Remarkably, reducing the expression of GABARAP, or another key autophagy protein MAP1LC3B/LC3B, entirely prevents AG-induced hair cell death and subsequent hearing loss in mice. Furthermore, we found that AGs activate the autophagy pathway specific to mitochondria. Reducing the expression of PINK1 or PRKN/parkin, two key mitophagy proteins, protects hair cells against AG toxicity. Thus, our findings demonstrated that RIPOR2-mediated autophagic dysfunction is essential for AG-induced hearing loss and provided potential therapeutic strategies for preventing AG toxicity.Item Collapsin Response Mediator Protein 1 (CRMP1) Is Required for High-Frequency Hearing(Elsevier, 2022) Li, Jinan; Liu, Chang; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of MedicineCollapsin 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.Item GRXCR2 Regulates Taperin Localization Critical for Stereocilia Morphology and Hearing(Elsevier, 2018-10-30) Liu, Chang; Luo, Na; Tung, Chun-Yu; Perrin, Benjamin J.; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of MedicineMutations in human GRXCR2, which encodes a protein of undetermined function, cause hearing loss by unknown mechanisms. We found that mouse GRXCR2 localizes to the base of the stereocilia, which are actin-based mechanosensing organelles in cochlear hair cells that convert sound-induced vibrations into electrical signals. The stereocilia base also contains taperin, another protein of unknown function required for human hearing. We show that taperin and GRXCR2 form a complex and that taperin is diffused throughout the stereocilia length in Grxcr2-deficient hair cells. Stereocilia lacking GRXCR2 are longer than normal and disorganized due to the mislocalization of taperin, which could modulate the actin cytoskeleton in stereocilia. Remarkably, reducing taperin expression levels could rescue the morphological defects of stereocilia and restore the hearing of Grxcr2-deficient mice. Thus, our findings suggest that GRXCR2 is critical for the morphogenesis of stereocilia and auditory perception by restricting taperin to the stereocilia base.Item Large-scale annotated dataset for cochlear hair cell detection and classification(Springer Nature, 2024-04-23) Buswinka, Christopher J.; Rosenberg, David B.; Simikyan, Rubina G.; Osgood, Richard T.; Fernandez, Katharine; Nitta, Hidetomi; Hayashi, Yushi; Liberman, Leslie W.; Nguyen, Emily; Yildiz, Erdem; Kim, Jinkyung; Jarysta, Amandine; Renauld, Justine; Wesson, Ella; Wang, Haobing; Thapa, Punam; Bordiga, Pierrick; McMurtry, Noah; Llamas, Juan; Kitcher, Siân R.; López-Porras, Ana I.; Cui, Runjia; Behnammanesh, Ghazaleh; Bird, Jonathan E.; Ballesteros, Angela; Vélez-Ortega, A. Catalina; Edge, Albert S. B.; Deans, Michael R.; Gnedeva, Ksenia; Shrestha, Brikha R.; Manor, Uri; Zhao, Bo; Ricci, Anthony J.; Tarchini, Basile; Basch, Martín L.; Stepanyan, Ruben; Landegger, Lukas D.; Rutherford, Mark A.; Liberman, M. Charles; Walters, Bradley J.; Kros, Corné J.; Richardson, Guy P.; Cunningham, Lisa L.; Indzhykulian, Artur A.; Otolaryngology -- Head and Neck Surgery, School of MedicineOur sense of hearing is mediated by cochlear hair cells, of which there are two types organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains 5-15 thousand terminally differentiated hair cells, and their survival is essential for hearing as they do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. Machine learning can be used to automate the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, rat, guinea pig, pig, primate, and human cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 107,000 hair cells which have been identified and annotated as either inner or outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair-cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to give other hearing research groups the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.Item Large-scale annotated dataset for cochlear hair cell detection and classification(bioRxiv, 2023-09-01) Buswinka, Christopher J.; Rosenberg, David B.; Simikyan, Rubina G.; Osgood, Richard T.; Fernandez, Katharine; Nitta, Hidetomi; Hayashi, Yushi; Liberman, Leslie W.; Nguyen, Emily; Yildiz, Erdem; Kim, Jinkyung; Jarysta, Amandine; Renauld, Justine; Wesson, Ella; Thapa, Punam; Bordiga, Pierrick; McMurtry, Noah; Llamas, Juan; Kitcher, Siân R.; López-Porras, Ana I.; Cui, Runjia; Behnammanesh, Ghazaleh; Bird, Jonathan E.; Ballesteros, Angela; Vélez-Ortega, A. Catalina; Edge, Albert S. B.; Deans, Michael R.; Gnedeva, Ksenia; Shrestha, Brikha R.; Manor, Uri; Zhao, Bo; Ricci, Anthony J.; Tarchini, Basile; Basch, Martin; Stepanyan, Ruben S.; Landegger, Lukas D.; Rutherford, Mark; Liberman, M. Charles; Walters, Bradley J.; Kros, Corné J.; Richardson, Guy P.; Cunningham, Lisa L.; Indzhykulian, Artur A.; Otolaryngology -- Head and Neck Surgery, School of MedicineOur sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 90'000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.Item The murine catecholamine methyltransferase mTOMT is essential for mechanotransduction by cochlear hair cells(eLife Sciences Publications, 2017-05-15) Cunningham, Christopher L.; Wu, Zizhen; Jafari, Aria; Zhao, Bo; Schrode, Kat; Harkins-Perry, Sarah; Lauer, Amanda; Müller, Ulrich; Otolaryngology -- Head and Neck Surgery, School of MedicineHair cells of the cochlea are mechanosensors for the perception of sound. Mutations in the LRTOMT gene, which encodes a protein with homology to the catecholamine methyltransferase COMT that is linked to schizophrenia, cause deafness. Here, we show that Tomt/Comt2, the murine ortholog of LRTOMT, has an unexpected function in the regulation of mechanotransduction by hair cells. The role of mTOMT in hair cells is independent of mTOMT methyltransferase function and mCOMT cannot substitute for mTOMT function. Instead, mTOMT binds to putative components of the mechanotransduction channel in hair cells and is essential for the transport of some of these components into the mechanically sensitive stereocilia of hair cells. Our studies thus suggest functional diversification between mCOMT and mTOMT, where mTOMT is critical for the assembly of the mechanotransduction machinery of hair cells. Defects in this process are likely mechanistically linked to deafness caused by mutations in LRTOMT/Tomt.Item Murine GRXCR1 Has a Different Function Than GRXCR2 in the Morphogenesis of Stereocilia(Frontiers Media, 2021-07-21) Liu, Chang; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of MedicineMutations in human glutaredoxin domain-containing cysteine-rich protein 1 (GRXCR1) and its paralog GRXCR2 have been linked to hearing loss in humans. Although both GRXCR1 and GRXCR2 are required for the morphogenesis of stereocilia in cochlear hair cells, a fundamental question that remains unclear is whether GRXCR1 and GRXCR2 have similar functions in hair cells. Previously, we found that GRXCR2 is critical for the stereocilia morphogenesis by regulating taperin localization at the base of stereocilia. Reducing taperin expression level rescues the morphological defects of stereocilia and hearing loss in Grxcr2-deficient mice. So far, functions of GRXCR1 in mammalian hair cells are still unclear. Grxcr1-deficient hair cells have very thin stereocilia with less F-actin content inside, which is different from Grxcr2-deficient hair cells. In contrast to GRXCR2, which is concentrated at the base of stereocilia, GRXCR1 is diffusely distributed throughout the stereocilia. Notably, GRXCR1 interacts with GRXCR2. In Grxcr1-deficient hair cells, the expression level of GRXCR2 and taperin is reduced. Remarkably, different from that in Grxcr2-deficient mice, reducing taperin expression level does not rescue the morphological defects of stereocilia or hearing loss in Grxcr1-deficient mice. Thus, our findings suggest that GRXCR1 has different functions than GRXCR2 during the morphogenesis of stereocilia.Item N-Terminus of GRXCR2 Interacts With CLIC5 and Is Essential for Auditory Perception(Frontiers Media, 2021-05-05) Li, Jinan; Liu, Chang; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of MedicineStereocilia of cochlear hair cells are specialized mechanosensing organelles that convert sound-induced vibration to electrical signals. Glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2) is localized at the base of stereocilia and is necessary for stereocilia morphogenesis and auditory perception. However, the detailed functions of GRXCR2 in hair cells are still largely unknown. Here, we report that GRXCR2 interacts with chloride intracellular channel protein 5 (CLIC5) which is also localized at the base of stereocilia and required for normal hearing in human and mouse. Immunolocalization analyses suggest that GRXCR2 is not required for the localization of CLIC5 to the stereociliary base during development, or vice versa. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we deleted 60 amino acids near the N-terminus of GRXCR2 essential for its interaction with CLIC5. Interestingly, mice harboring this in-frame deletion in Grxcr2 exhibit moderate hearing loss at lower frequencies and severe hearing loss at higher frequencies although the morphogenesis of stereocilia is minimally affected. Thus, our findings reveal that the interaction between GRXCR2 and CLIC5 is crucial for normal hearing.