Publications

Nicolas Grillet, PhD
Assistant Professor of Otolaryngology - Head & Neck Surgery (OHNS)

Publications

  • LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission. Nature communications Wang, P., Miller, K. K., He, E., Dhawan, S. S., Cunningham, C. L., Grillet, N. 2024; 15 (1): 7865

    Abstract

    Hair cell bundles consist of stereocilia arranged in rows of increasing heights, connected by tip links that transmit sound-induced forces to shorter stereocilia tips. Auditory mechanotransduction channel complexes, composed of proteins TMC1/2, TMIE, CIB2, and LHFPL5, are located at the tips of shorter stereocilia. While most components can interact with the tip link in vitro, their ability to maintain the channel complexes at the tip link in vivo is uncertain. Return, using mouse models, we show that an additional component, LOXHD1, is essential for keeping TMC1-pore forming subunits at the tip link but is dispensable for TMC2. Using SUB-immunogold-SEM, we showed that TMC1 localizes near the tip link but mislocalizes without LOXHD1. LOXHD1 selectively interacts with TMC1, CIB2, LHFPL5, and tip-link protein PCDH15. Our results demonstrate that TMC1-driven mature auditory channels require LOXHD1 to stay connected to the tip link and remain functional, while TMC2-driven developmental channels do not.

    View details for DOI 10.1038/s41467-024-51850-4

    View details for PubMedID 39256406

    View details for PubMedCentralID PMC11387651

  • SUB-immunogold-SEM reveals nanoscale distribution of submembranous epitopes. Nature communications Miller, K. K., Wang, P., Grillet, N. 2024; 15 (1): 7864

    Abstract

    Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identify four critical sample preparation factors contributing to the method's sensitivity. We validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane protein distribution. We evaluate the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale MYO15A-L rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM extends the application of SEM-based nanoscale protein localization to the detection of intracellular epitopes on the exposed surfaces of any cell.

    View details for DOI 10.1038/s41467-024-51849-x

    View details for PubMedID 39256352

    View details for PubMedCentralID PMC11387508

  • Single-cell transcriptomic atlas reveals increased regeneration in diseased human inner ear balance organs. Nature communications Wang, T., Ling, A. H., Billings, S. E., Hosseini, D. K., Vaisbuch, Y., Kim, G. S., Atkinson, P. J., Sayyid, Z. N., Aaron, K. A., Wagh, D., Pham, N., Scheibinger, M., Zhou, R., Ishiyama, A., Moore, L. S., Maria, P. S., Blevins, N. H., Jackler, R. K., Alyono, J. C., Kveton, J., Navaratnam, D., Heller, S., Lopez, I. A., Grillet, N., Jan, T. A., Cheng, A. G. 2024; 15 (1): 4833

    Abstract

    Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.

    View details for DOI 10.1038/s41467-024-48491-y

    View details for PubMedID 38844821

  • High-resolution immunofluorescence imaging of mouse cochlear hair bundles. STAR protocols Miller, K. K., Wang, P., Grillet, N. 2022; 3 (2): 101431

    Abstract

    High-resolution immunofluorescence imaging of cochlear hair bundles faces many challenges due to the hair bundle's small dimensions, fragile nature, and complex organization. Here, we describe an optimized protocol for hair-bundle protein immunostaining and localization. We detail the steps and solutions for extracting and fixing the mouse inner ear and for dissecting the organ of Corti. We further emphasize the optimal permeabilization, blocking, staining, and mounting conditions as well as the parameters for high-resolution microscopy imaging. For complete details on the use and execution of this protocol, please refer to Trouillet etal. (2021).

    View details for DOI 10.1016/j.xpro.2022.101431

    View details for PubMedID 35669049

  • Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1. Cell reports Deng, Q., Natesan, R., Cidre-Aranaz, F., Arif, S., Liu, Y., Rasool, R. U., Wang, P., Mitchell-Velasquez, E., Das, C. K., Vinca, E., Cramer, Z., Grohar, P. J., Chou, M., Kumar-Sinha, C., Weber, K., Eisinger-Mathason, T. S., Grillet, N., Grünewald, T., Asangani, I. A. 2022; 39 (11): 110971

    Abstract

    Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1::FLI1 transcription factor drives EwS by orchestrating an oncogenic transcription program through de novo enhancers. By integrative analysis of thousands of transcriptomes representing pan-cancer cell lines, primary cancers, metastasis, and normal tissues, we identify a 32-gene signature (ESS32 [Ewing Sarcoma Specific 32]) that stratifies EwS from pan-cancer. Among the ESS32, LOXHD1, encoding a stereociliary protein, is the most highly expressed gene through an alternative transcription start site. Deletion or silencing of EWSR1::FLI1 bound upstream de novo enhancer results in loss of the LOXHD1 short isoform, altering EWSR1::FLI1 and HIF1α pathway genes and resulting in decreased proliferation/invasion of EwS cells. These observations implicate LOXHD1 as a biomarker and a determinant of EwS metastasis and suggest new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.

    View details for DOI 10.1016/j.celrep.2022.110971

    View details for PubMedID 35705030

  • High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy. STAR protocols Grillet, N. 2022; 3 (1): 101213

    Abstract

    Scanning electron microscopy (SEM) allows cell surface imaging at a sub-nanometric resolution. However, the sample requires a specific preparation to sustain the high vacuum of the SEM and be electrically conductive. The sample preparation consists of dissection, fixation, dehydration, metal coating, and tissue mounting. Here we provide a comprehensive protocol to perform SEM on the mouse's inner ear, and image the hair bundles at high resolution. Hair bundles are the force-sensitive organelles located at the apical surface of hair cells. For complete details on the use and execution of this protocol, please refer to Trouillet etal. (2021).

    View details for DOI 10.1016/j.xpro.2022.101213

    View details for PubMedID 35257116

  • Loxhd1 mutations cause mechanotransduction defects in cochlear hair cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Trouillet, A., Miller, K. K., George, S. S., Wang, P., Ali, N., Ricci, A., Grillet, N. 2021

    Abstract

    Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions (called stereocilia) connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase alpha-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type (WT) levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the MET phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphological defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.SIGNIFICANCE STATEMENT:Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multi-repeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing.

    View details for DOI 10.1523/JNEUROSCI.0975-20.2021

    View details for PubMedID 33707295

  • Dimensions of a Living Cochlear Hair Bundle Front Cell Dev Biol Miller, K. K., Atkinson, P., Mendoza, K., Ó Maoiléidigh, D., Grillet, N. 2021; 9: 742529

    Abstract

    The hair bundle is the mechanosensory organelle of hair cells that detects mechanical stimuli caused by sounds, head motions, and fluid flows. Each hair bundle is an assembly of cellular-protrusions called stereocilia, which differ in height to form a staircase. Stereocilia have different heights, widths, and separations in different species, sensory organs, positions within an organ, hair-cell types, and even within a single hair bundle. The dimensions of the stereociliary assembly dictate how the hair bundle responds to stimuli. These hair-bundle properties have been measured previously only to a limited degree. In particular, mammalian data are either incomplete, lack control for age or position within an organ, or have artifacts owing to fixation or dehydration. Here, we provide a complete set of measurements for postnatal day (P) 11 C57BL/6J mouse apical inner hair cells (IHCs) obtained from living tissue, tissue mildly-fixed for fluorescent imaging, or tissue strongly fixed and dehydrated for scanning electronic microscopy (SEM). We found that hair bundles mildly-fixed for fluorescence had the same dimensions as living hair bundles, whereas SEM-prepared hair bundles shrank uniformly in stereociliary heights, widths, and separations. By determining the shrinkage factors, we imputed live dimensions from SEM that were too small to observe optically. Accordingly, we created the first complete blueprint of a living IHC hair bundle. We show that SEM-prepared measurements strongly affect calculations of a bundle's mechanical properties - overestimating stereociliary deflection stiffness and underestimating the fluid coupling between stereocilia. The methods of measurement, the data, and the consequences we describe illustrate the high levels of accuracy and precision required to understand hair-bundle mechanotransduction.

    View details for DOI 10.3389/fcell.2021.742529

    View details for PubMedCentralID PMC8657763

  • Mechanosensory hair cells express two molecularly distinct mechanotransduction channels NATURE NEUROSCIENCE Wu, Z., Grillet, N., Zhao, B., Cunningham, C., Harkins-Perry, S., Coste, B., Ranade, S., Zebarjadi, N., Beurg, M., Fettiplace, R., Patapoutian, A., Muller, U. 2017; 20 (1): 24-33

    Abstract

    Auditory hair cells contain mechanotransduction channels that rapidly open in response to sound-induced vibrations. We report here that auditory hair cells contain two molecularly distinct mechanotransduction channels. One ion channel is activated by sound and is responsible for sensory transduction. This sensory transduction channel is expressed in hair cell stereocilia, and previous studies show that its activity is affected by mutations in the genes encoding the transmembrane proteins TMHS, TMIE, TMC1 and TMC2. We show here that the second ion channel is expressed at the apical surface of hair cells and that it contains the Piezo2 protein. The activity of the Piezo2-dependent channel is controlled by the intracellular Ca(2+) concentration and can be recorded following disruption of the sensory transduction machinery or more generally by disruption of the sensory epithelium. We thus conclude that hair cells express two molecularly and functionally distinct mechanotransduction channels with different subcellular distributions.

    View details for DOI 10.1038/nn.4449

    View details for Web of Science ID 000391085500009

    View details for PubMedID 27893727

    View details for PubMedCentralID PMC5191906

  • TMIE Is an Essential Component of the Mechanotransduction Machinery of Cochlear Hair Cells. Neuron Zhao, B., Wu, Z., Grillet, N., Yan, L., Xiong, W., Harkins-Perry, S., Müller, U. 2014; 84 (5): 954-67

    Abstract

    Hair cells are the mechanosensory cells of the inner ear. Mechanotransduction channels in hair cells are gated by tip links. The molecules that connect tip links to transduction channels are not known. Here we show that the transmembrane protein TMIE forms a ternary complex with the tip-link component PCDH15 and its binding partner TMHS/LHFPL5. Alternative splicing of the PCDH15 cytoplasmic domain regulates formation of this ternary complex. Transducer currents are abolished by a homozygous Tmie-null mutation, and subtle Tmie mutations that disrupt interactions between TMIE and tip links affect transduction, suggesting that TMIE is an essential component of the hair cell's mechanotransduction machinery that functionally couples the tip link to the transduction channel. The multisubunit composition of the transduction complex and the regulation of complex assembly by alternative splicing is likely critical for regulating channel properties in different hair cells and along the cochlea's tonotopic axis.

    View details for DOI 10.1016/j.neuron.2014.10.041

    View details for PubMedID 25467981

    View details for PubMedCentralID PMC4258123