Multiple PDZ Domain Protein Maintains Patterning of the Apical Cytoskeleton in Sensory Hair Cells

Mapping Intimacies ◽

10.1101/2021.02.20.432099 ◽

2021 ◽

Author(s):

Amandine Jarysta ◽

Basile Tarchini

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Critical Role ◽

Pdz Domain ◽

Hair Bundle ◽

Structural Defects ◽

Sensory Hair ◽

High Frequencies ◽

Sensory Hair Cells ◽

Sound Transduction

SUMMARYSound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here we report a novel and critical role for Multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis. We show that MPDZ is enriched at the hair cell apical membrane, and required there to maintain the proper segregation of apical blueprints proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia in Mpdz mutants, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.

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Multiple PDZ domain protein maintains patterning of the apical cytoskeleton in sensory hair cells

Development ◽

10.1242/dev.199549 ◽

2021 ◽

Author(s):

Amandine Jarysta ◽

Basile Tarchini

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Critical Role ◽

Pdz Domain ◽

Hair Bundle ◽

Structural Defects ◽

Sensory Hair ◽

High Frequencies ◽

Sensory Hair Cells ◽

Sound Transduction

Sound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of actin-based stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here we report a novel and critical role for Multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis. We show that MPDZ is enriched at the hair cell apical membrane along with MAGUK p55 subfamily member 5 (MPP5/PALS1) and the Crumbs protein CRB3. MPDZ is required there to maintain the proper segregation of apical blueprints proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia placement in Mpdz mutant hair cells, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.

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Comparing Sensory Organs to Define the Path for Hair Cell Regeneration

Annual Review of Cell and Developmental Biology ◽

10.1146/annurev-cellbio-100818-125503 ◽

2019 ◽

Vol 35 (1) ◽

pp. 567-589 ◽

Cited By ~ 5

Author(s):

Nicolas Denans ◽

Sungmin Baek ◽

Tatjana Piotrowski

Keyword(s):

Hair Cell ◽

Olfactory Epithelium ◽

Current Literature ◽

Hair Cells ◽

Sensory Cell ◽

Cell Regeneration ◽

Hair Cell Regeneration ◽

Sensory Hair ◽

Sensory Hair Cells ◽

Technical Advances

Deafness or hearing deficits are debilitating conditions. They are often caused by loss of sensory hair cells or defects in their function. In contrast to mammals, nonmammalian vertebrates robustly regenerate hair cells after injury. Studying the molecular and cellular basis of nonmammalian vertebrate hair cell regeneration provides valuable insights into developing cures for human deafness. In this review, we discuss the current literature on hair cell regeneration in the context of other models for sensory cell regeneration, such as the retina and the olfactory epithelium. This comparison reveals commonalities with, as well as differences between, the different regenerating systems, which begin to define a cellular and molecular blueprint of regeneration. In addition, we propose how new technical advances can address outstanding questions in the field.

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In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness

Science Translational Medicine ◽

10.1126/scitranslmed.aay9101 ◽

2020 ◽

Vol 12 (546) ◽

pp. eaay9101 ◽

Cited By ~ 11

Author(s):

Wei-Hsi Yeh ◽

Olga Shubina-Oleinik ◽

Jonathan M. Levy ◽

Bifeng Pan ◽

Gregory A. Newby ◽

...

Keyword(s):

Hair Cell ◽

Point Mutation ◽

Hair Cells ◽

Point Mutations ◽

Sensory Transduction ◽

Auditory Function ◽

Sensory Hair ◽

Base Editing ◽

Sensory Hair Cells

Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G; resulting in the substitution p.Y182C) in Tmc1 that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction. To repair the mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual adeno-associated viruses (AAVs) using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild-type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing 4 weeks after injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.

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Functional Expression of Exogenous Proteins in Mammalian Sensory Hair Cells Infected With Adenoviral Vectors

Journal of Neurophysiology ◽

10.1152/jn.1999.81.4.1881 ◽

1999 ◽

Vol 81 (4) ◽

pp. 1881-1888 ◽

Cited By ~ 54

Author(s):

Jeffrey R. Holt ◽

David C. Johns ◽

Sam Wang ◽

Zheng-Yi Chen ◽

Robert J. Dunn ◽

...

Keyword(s):

Gene Transfer ◽

Hair Cell ◽

Hair Cells ◽

Cell Function ◽

Functional Expression ◽

Adenoviral Vectors ◽

Inward Rectifier ◽

Sensory Hair ◽

Sensory Hair Cells ◽

Infected Cells

Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell’s exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for β-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 × 107pfu/ml were sufficient for expression in 50% of the ∼1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel–GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by −17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.

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Putative pore-forming subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize to the site of mechanotransduction in zebrafish sensory hair cells

10.1101/393330 ◽

2018 ◽

Cited By ~ 1

Author(s):

Itallia V. Pacentine ◽

Teresa Nicolson

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Hair Cells ◽

Transmembrane Domain ◽

Cell Activity ◽

Sensory Hair ◽

Transmembrane Channel ◽

Extracellular Region ◽

Normal Site ◽

Sensory Hair Cells

AbstractMutations in transmembrane inner ear (TMIE) cause deafness in humans; previous studies suggest involvement in the mechano-electrical transduction (MET) complex in sensory hair cells, but TMIE’s precise role is unclear. In tmie zebrafish mutants, we observed that GFP-tagged Tmc1 and Tmc2b, which are putative subunits of the MET channel, fail to target to the hair bundle. In contrast, overexpression of Tmie strongly enhances the targeting of Tmc2b-GFP to stereocilia. To identify the motifs of Tmie underlying the regulation of the Tmcs, we systematically deleted or replaced peptide segments. We then assessed localization and functional rescue of each mutated/chimeric form of Tmie in tmie mutants. We determined that the first putative helix was dispensable and identified a novel critical region of Tmie, the extracellular region and transmembrane domain, which mediates both mechanosensitivity and Tmc2b-GFP expression in bundles. Collectively, our results suggest that Tmie’s role in sensory hair cells is to target and stabilize Tmc subunits to the site of MET.Author summaryHair cells mediate hearing and balance through the activity of a pore-forming channel in the cell membrane. The transmembrane inner ear (TMIE) protein is an essential component of the protein complex that gates this so-called mechanotransduction channel. While it is known that loss of TMIE results in deafness, the function of TMIE within the complex is unclear. Using zebrafish as a deafness model, Pacentine and Nicolson demonstrate that Tmie is required for the localization of other essential complex members, the transmembrane channel-like (Tmc) proteins, Tmc1/2b. They then evaluate twelve unique versions of Tmie, each containing mutations to different domains of Tmie. This analysis reveals that some mutations in Tmie cause dysfunctional gating of the channel as demonstrated through reduced hair cell activity, and that these same dysfunctional versions also display reduced Tmc expression at the normal site of the channel. These findings link hair cell activity with the levels of Tmc in the bundle, reinforcing the currently-debated notion that the Tmcs are the pore-forming subunits of the mechanotransduction channel. The authors conclude that Tmie, through distinct regions, is involved in both trafficking and stabilizing the Tmcs at the site of mechanotransduction.

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Identification of a 275-kD protein associated with the apical surfaces of sensory hair cells in the avian inner ear.

The Journal of Cell Biology ◽

10.1083/jcb.110.4.1055 ◽

1990 ◽

Vol 110 (4) ◽

pp. 1055-1066 ◽

Cited By ~ 42

Author(s):

G P Richardson ◽

S Bartolami ◽

I J Russell

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Hair Cells ◽

Proximal Region ◽

Basilar Papilla ◽

Relative Molecular Mass ◽

Apical Surface ◽

Sensory Hair ◽

Sensory Hair Cells ◽

Avian Inner Ear

Immunological techniques have been used to generate both polyclonal and monoclonal antibodies specific for the apical ends of sensory hair cells in the avian inner ear. The hair cell antigen recognized by these antibodies is soluble in nonionic detergent, behaves on sucrose gradients primarily as a 16S particle, and, after immunoprecipitation, migrates as a polypeptide with a relative molecular mass of 275 kD on 5% SDS gels under reducing conditions. The antigen can be detected with scanning immunoelectron microscopy on the apical surface of the cell and on the stereocilia bundle but not on the kinocilium. Double label studies indicate that the entire stereocilia bundle is stained in the lagena macula (a vestibular organ), whereas in the basilar papilla (an auditory organ) only the proximal region of the stereocilia bundle nearest to the apical surface is stained. The monoclonal anti-hair cell antibodies do not stain brain, tongue, lung, liver, heart, crop, gizzard, small intestine, skeletal muscle, feather, skin, or eye tissues but do specifically stain renal corpuscles in the kidney. Experiments using organotypic cultures of the embryonic lagena macula indicate that the antibodies cause a significant increase in the steady-state stiffness of the stereocilia bundle but do not inhibit mechanotransduction. The antibodies should provide a suitable marker and/or tool for the purification of the apical sensory membrane of the hair cell.

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A surface preparation study on the effect of methyl mercury on the sensory hair cell population in the cochlea of the harp seal (Pagophilus groenlandicus Erxleben, 1777)

Canadian Journal of Zoology ◽

10.1139/z77-025 ◽

1977 ◽

Vol 55 (1) ◽

pp. 223-230 ◽

Cited By ~ 17

Author(s):

F. Ramprashad ◽

K. Ronald

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Methyl Mercury ◽

Sensory Cell ◽

Cell Damage ◽

Sensory Hair ◽

Low Level ◽

Sensory Hair Cell ◽

Sensory Hair Cells ◽

Daily Dose

Surface preparations of the organ of Corti of four harp seals were used to study the effect of prolonged ingestion of methyl mercury on the sensory cell population.A low level of damage to the sensory hair cells occurred throughout the length of the cochlea. Damage was confined to the three outer rows of sensory hair cells especially the third outermost row. At each location along the length of the cochlea, sensory hair cell damage in the seals on a daily dose of 25.0 mg/kg of methyl mercury exceeded the damage to the cochlea of the seals fed on a daily dose of 0.25 mg/kg of methyl mercury. Greatest damage in all the mercury-treated seals occurred in the middle coil of the cochlea. Seals on the higher mercury diet showed a 20–24% sensory cell damage at the upper middle coil, about 19–26 mm from the base, whereas only 4–5% damage was found within same region in the cochlea of the seals on the lower mercury diet.This lack of specificity and low level of damage to the sensory hair cells seems characteristic of mercury and is a direct contrast to other known ototoxic agents.

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Synaptic Interactions Between Crista Hair Cells in the Statocyst of the Squid Alloteuthis subulata

Journal of Neurophysiology ◽

10.1152/jn.1998.80.2.656 ◽

1998 ◽

Vol 80 (2) ◽

pp. 656-666 ◽

Cited By ~ 7

Author(s):

Abdesslam Chrachri ◽

Roddy Williamson

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Cell Types ◽

Afferent Neurons ◽

Sensory Hair ◽

Postsynaptic Potentials ◽

First Order ◽

Inhibitory Postsynaptic Potentials ◽

Synaptic Interactions ◽

Sensory Hair Cells

Chrachri, Abdesslam and Roddy Williamson. Synaptic interactions between crista hair cells in the statocyst of the squid Alloteuthis subulata. J. Neurophysiol. 80: 656–666, 1998. Intracellular injections of the fluorescent dye Lucifer yellow into the various cell types within the anterior transverse crista segment of the statocyst of squid revealed that the primary sensory hair cells and both large and small first-order afferent neurons have relatively simple morphologies, each cell having a single, unbranched axon that passes directly into the small crista nerve that innervates the anterior transverse crista. However, the small first-order neurons have short dendritic processes occurring in the region of the sensory hair cells. The secondary sensory hair cells have no centripetal axons, but some have long processes extending from their bases along the segment. Simultaneous intracellular recordings from pairs of the different cell types in the anterior transverse crista segment demonstrated that electrical coupling is widespread; secondary sensory hair cells are coupled electrically along a hair cell row, as are groups of primary sensory hair cells. Secondary sensory hair cell also are coupled to neighboring small first-order afferent neurons. However, this coupling is rectifying in that it only occurs from secondary sensory hair cells to first-order afferent neurons. Direct electrical stimulation of the small crista nerve to excite the efferent axons revealed efferent connections to both the primary sensory hair cells and the small first-order afferent neurons. These efferent responses were of three types: excitatory or inhibitory postsynaptic potentials and excitatory postsynaptic potentials followed by inhibitory postsynaptic potentials. The functional significance of the cell interactions within the crista epithelium of the statocyst of squid is discussed and comparisons drawn with the balance organs of other animals.

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Stereocilia Rootlets: Actin-Based Structures That Are Essential for Structural Stability of the Hair Bundle

International Journal of Molecular Sciences ◽

10.3390/ijms21010324 ◽

2020 ◽

Vol 21 (1) ◽

pp. 324 ◽

Cited By ~ 3

Author(s):

Itallia Pacentine ◽

Paroma Chatterjee ◽

Peter G. Barr-Gillespie

Keyword(s):

Inner Ear ◽

Hair Cells ◽

Actin Filaments ◽

Hair Bundle ◽

Sensory Hair ◽

Sensory Hair Cells ◽

Associated Proteins ◽

Unique Manner ◽

Electrical Impulses ◽

Mechanical Movement

Sensory hair cells of the inner ear rely on the hair bundle, a cluster of actin-filled stereocilia, to transduce auditory and vestibular stimuli into electrical impulses. Because they are long and thin projections, stereocilia are most prone to damage at the point where they insert into the hair cell’s soma. Moreover, this is the site of stereocilia pivoting, the mechanical movement that induces transduction, which additionally weakens this area mechanically. To bolster this fragile area, hair cells construct a dense core called the rootlet at the base of each stereocilium, which extends down into the actin meshwork of the cuticular plate and firmly anchors the stereocilium. Rootlets are constructed with tightly packed actin filaments that extend from stereocilia actin filaments which are wrapped with TRIOBP; in addition, many other proteins contribute to the rootlet and its associated structures. Rootlets allow stereocilia to sustain innumerable deflections over their lifetimes and exemplify the unique manner in which sensory hair cells exploit actin and its associated proteins to carry out the function of mechanotransduction.

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