IU Indianapolis ScholarWorks :: Browsing by Subject "Stereocilia"

Browsing by Subject "Stereocilia"

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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 Medicine
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.
Essential nucleotide- and protein-dependent functions of Actb/β-actin
(National Academy of Sciences, 2018-07-31) Patrinostro, Xiaobai; Roy, Pallabi; Lindsay, Angus; Chamberlain, Christopher M.; Sundby, Lauren J.; Starker, Colby G.; Voytas, Daniel F.; Ervasti, James M.; Perrin, Benjamin J.; Biochemistry and Molecular Biology, School of Medicine
The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.
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 Medicine
Mutations 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.
Hedgehog Signaling Regulates Apical Actin Morphology
(Office of the Vice Chancellor for Research, 2016-04-08) Anderson, Matthew; Hege, Melissa; Berbari, Nicolas; Perrin, Benjamin
Stereocilia are highly patterned actin based cell protrusions found on the apical surface of auditory hair cells. They are formed mainly from bundled filamentous actin and its associated actin cross-linking proteins. Interestingly, stereocilia develop around another cell appendage, the microtubule based kinocilium, which is the primary cilium for a hair cell. Primary cilia are found on most somatic cells and play a significant role in the regulation and proper transduction of the Hedgehog (Hh) pathway. In the current study, we are testing the hypothesis that Hh pathway activity can alter actin bundling and elongation. In support of this idea, ectopic activation or repression of Hh signaling changed the morphology of stereocilia in vivo. To further test our hypothesis, we used a CL4 porcine kidney epithelial cell culture system stably expressing the actin crosslinking protein ESPN fused to green fluorescent protein. These cells serve as an in vitro model of apical actin protrusions similar to mature stereocilia in vivo. We manipulated Hh signaling in these cells using both a genetic and a pharmacological approach. In the pharmacological approach, CL4 cells were treated with the hedgehog agonist (Purmophamine) and antagonist (Cyclopamine), at varying concentrations for 48 hours. Genetically, the Hh pathway was ectopically activated by overexpressing the transcription factor Gli1, Gli2, Gli3, and SmoA1 repressed by expressing Gli3R. Immunofluorescent (IF) and scanning electron microscopy (SEM) revealed that CL4 cells dramatically altered the apical actin structures under these conditions. In particular, activating Gli transcription decreased apical actin-based structures while antagonizing activity resulted in more actinbased protrusions. This data strongly supports the hypothesis that the Hh signaling pathway can regulate the actin cytoskeleton.
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 Medicine
Hair 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.
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 Medicine
Mutations 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.
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 Medicine
Stereocilia 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.
Reducing Taperin Expression Restores Hearing in Grxcr2 Mutant Mice
(Elsevier, 2022) Liu, Chang; Luo, Na; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
Recessive mutations in GRXCR2 cause deafness in both humans and mice. In Grxcr2 null hair cells, the sensory receptors for sound in the inner ear, stereocilia are disorganized. Reducing the expression of taperin, a protein that interacts with GRXCR2 at the base of stereocilia, corrects the morphological defects of stereocilia and restores hearing in Grxcr2 null mice. To further validate this finding, this study generated two novel taperin mutant mouse lines that exhibit progressive hearing loss. Then Grxcr2 null mice were crossed with one of these taperin mutant mice. The following morphological analysis revealed that reducing taperin expression indeed corrected stereocilia morphological abnormalities in Grxcr2 null mice. Functional analysis further confirmed that reducing taperin expression partially restored hearing in Grxcr2 null mice.
The role of ADF and cofilin in auditory sensory cell development
(2020-12) McGrath, Jamis; Perrin, Benjamin; Cummins, Theodore; Belecky-Adams, Teri; Balakrishnan, Lata; Zhao, Bo
Our ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.
Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures
(Elsevier, 2021-02-02) Miyoshi, Takushi; Zhang, Qianli; Miyake, Takafumi; Watanabe, Shin; Ohnishi, Hiroe; Chen, Jiji; Vishwasrao, Harshad D.; Chakraborty, Oisorjo; Belyantseva, Inna A.; Perrin, Benjamin J.; Shroff, Hari; Friedman, Thomas B.; Omori, Koichi; Watanabe, Naoki; Biology, School of Science
Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.

Browsing by Subject "Stereocilia"

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