scholarly journals Radixin deficiency causes deafness associated with progressive degeneration of cochlear stereocilia

2004 ◽  
Vol 166 (4) ◽  
pp. 559-570 ◽  
Author(s):  
Shin-ichiro Kitajiri ◽  
Kanehisa Fukumoto ◽  
Masaki Hata ◽  
Hiroyuki Sasaki ◽  
Tatsuya Katsuno ◽  
...  
Keyword(s):  
Hair Cells ◽  
Plasma Membranes ◽  
Wild Type ◽  
Cross Link ◽  
Erm Proteins ◽  
Hearing Ability ◽  
Adult Mice ◽  
Progressive Degeneration ◽  
Sensory Hair Cells

Ezrin/radixin/moesin (ERM) proteins cross-link actin filaments to plasma membranes to integrate the function of cortical layers, especially microvilli. We found that in cochlear and vestibular sensory hair cells of adult wild-type mice, radixin was specifically enriched in stereocilia, specially developed giant microvilli, and that radixin-deficient (Rdx−/−) adult mice exhibited deafness but no obvious vestibular dysfunction. Before the age of hearing onset (∼2 wk), in the cochlea and vestibule of Rdx−/− mice, stereocilia developed normally in which ezrin was concentrated. As these Rdx−/− mice grew, ezrin-based cochlear stereocilia progressively degenerated, causing deafness, whereas ezrin-based vestibular stereocilia were maintained normally in adult Rdx−/− mice. Thus, we concluded that radixin is indispensable for the hearing ability in mice through the maintenance of cochlear stereocilia, once developed. In Rdx−/− mice, ezrin appeared to compensate for radixin deficiency in terms of the development of cochlear stereocilia and the development/maintenance of vestibular stereocilia. These findings indicated the existence of complicate functional redundancy in situ among ERM proteins.

scholarly journals Cy3-ATP labeling of unfixed, permeabilized mouse hair cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Itallia V. Pacentine ◽  
Peter G. Barr-Gillespie
Keyword(s):  
Hair Cells ◽  
Actin Polymerization ◽  
Cytochalasin D ◽  
Myosin Isoforms ◽  
Substantial Fraction ◽  
Vestibular Hair Cells ◽  
Sensory Hair Cells ◽  
Latrunculin A ◽  
Actin Turnover

AbstractATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia of the mouse. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. In many cases, vanadate (Vi) traps nucleotides at the active site of myosin isoforms and presents nucleotide dissociation. Co-application with Vi enhanced the tip labeling, which is consistent with myosin isoforms being responsible. By contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~ 50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+stereocilia. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.

scholarly journals Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pankhuri Vyas ◽  
Megan Beers Wood ◽  
Yuanyuan Zhang ◽  
Adam C. Goldring ◽  
Fatima-Zahra Chakir ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Hair Cells ◽  
Outer Hair Cells ◽  
Synaptic Function ◽  
Auditory Brainstem Responses ◽  
Wild Type ◽  
Cochlear Hair Cells ◽  
Heterozygous Condition ◽  
Adult Mice ◽  
Efferent Neurons

AbstractNeurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.

scholarly journals Plastin 1 widens stereocilia by transforming actin filament packing from hexagonal to liquid

2016 ◽  
Vol 215 (4) ◽  
pp. 467-482 ◽  
Author(s):  
Jocelyn F. Krey ◽  
Evan S. Krystofiak ◽  
Rachel A. Dumont ◽  
Sarath Vijayakumar ◽  
Dongseok Choi ◽  
...  
Keyword(s):  
Inner Ear ◽  
Hair Cells ◽  
Double Mutant ◽  
Structure And Function ◽  
Wild Type ◽  
Auditory Function ◽  
Hexagonal Packing ◽  
Sensory Hair Cells ◽  
Actin Protrusions ◽  
And Function

With their essential role in inner ear function, stereocilia of sensory hair cells demonstrate the importance of cellular actin protrusions. Actin packing in stereocilia is mediated by cross-linkers of the plastin, fascin, and espin families. Although mice lacking espin (ESPN) have no vestibular or auditory function, we found that mice that either lacked plastin 1 (PLS1) or had nonfunctional fascin 2 (FSCN2) had reduced inner ear function, with double-mutant mice most strongly affected. Targeted mass spectrometry indicated that PLS1 was the most abundant cross-linker in vestibular stereocilia and the second most abundant protein overall; ESPN only accounted for ∼15% of the total cross-linkers in bundles. Mouse utricle stereocilia lacking PLS1 were shorter and thinner than wild-type stereocilia. Surprisingly, although wild-type stereocilia had random liquid packing of their actin filaments, stereocilia lacking PLS1 had orderly hexagonal packing. Although all three cross-linkers are required for stereocilia structure and function, PLS1 biases actin toward liquid packing, which allows stereocilia to grow to a greater diameter.

Cy3-ATP Labeling of Unfixed, Permeabilized Hair Cells

2021 ◽  
Author(s):  
Itallia V. Pacentine ◽  
Peter G. Barr-Gillespie
Keyword(s):  
Hair Cells ◽  
Actin Polymerization ◽  
Myosin Isoforms ◽  
Substantial Fraction ◽  
Vestibular Hair Cells ◽  
Sensory Hair Cells ◽  
Latrunculin A ◽  
Actin Turnover ◽  
Length Range

Abstract ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. Co-application with vanadate (VO43-) enhanced the tip labeling, which is consistent with myosin isoforms being responsible; by contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+ stereocilia of the same length range. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.

Hearing loss caused by progressive degeneration of cochlear hair cells in mice deficient for the Barhl1 homeobox gene

Development ◽  
2002 ◽  
Vol 129 (14) ◽  
pp. 3523-3532 ◽  
Author(s):  
Shengguo Li ◽  
Sandy M. Price ◽  
Hugh Cahill ◽  
David K. Ryugo ◽  
Michael M. Shen ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Hair Cell ◽  
Inner Ear ◽  
Hair Cells ◽  
Organ Of Corti ◽  
Cochlear Hair Cells ◽  
Sensory Hair ◽  
Progressive Degeneration ◽  
Age Related ◽  
Sensory Hair Cells

The cochlea of the mammalian inner ear contains three rows of outer hair cells and a single row of inner hair cells. These hair cell receptors reside in the organ of Corti and function to transduce mechanical stimuli into electrical signals that mediate hearing. To date, the molecular mechanisms underlying the maintenance of these delicate sensory hair cells are unknown. We report that targeted disruption of Barhl1, a mouse homolog of the Drosophila BarH homeobox genes, results in severe to profound hearing loss, providing a unique model for the study of age-related human deafness disorders. Barhl1 is expressed in all sensory hair cells during inner ear development, 2 days after the onset of hair cell generation. Loss of Barhl1 function in mice results in age-related progressive degeneration of both outer and inner hair cells in the organ of Corti, following two reciprocal longitudinal gradients. Our data together indicate an essential role for Barhl1 in the long-term maintenance of cochlear hair cells, but not in the determination or differentiation of these cells.

scholarly journals Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking

2021 ◽  
pp. mbc.E20-10-0657
Author(s):  
Aayushi Manchanda ◽  
Josephine A. Bonventre ◽  
Sean M. Bugel ◽  
Paroma Chatterjee ◽  
Robyn Tanguay ◽  
...  
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Gaba Receptors ◽  
Transmembrane Domain ◽  
Stop Codon ◽  
Wild Type ◽  
Zebrafish Model ◽  
Sensory Hair Cells ◽  
Early Developmental ◽  
Vestibular Pathways

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish. At the early developmental timepoint of 72 hours post-fertilization (hpf) low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after upregulation of mutant otoferlin. Analysis of membrane-reconstituted otoferlin measurements suggest a function for the transmembrane domain in liposome docking. We conclude that that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.

Ultrastructure of superficial neuromasts in the mottled sculpin, Cottus bairdi

1989 ◽  
Vol 47 ◽  
pp. 958-959
Author(s):  
W.R. Jones ◽  
S. Coombs ◽  
J. Janssen
Keyword(s):  
Hair Cells ◽  
Lateral Line System ◽  
Electron Microscopic ◽  
Line System ◽  
Dense Cores ◽  
Sensory Hair Cells ◽  
Cytoplasmic Vesicles ◽  
Cottus Bairdi ◽  
Mottled Sculpin ◽  
Upper Third

The lateral line system of the mottled sculpin, like that of most bony fish, has both canal (CNM) and superficial (SNM) sensory end organs, neuromasts, which are distributed on the head and trunk in discrete, readily identifiable groupings (Fig. 1). CNM and SNM differ grossly in location and in overall size and shape. The former are located in subdermal canals and are larger and asymmetric in shape, The latter are located directly on the surface of the skin and are much smaller and more symmetrical It has been suggested that the two may differ at a more fundamental level in such functionally related parameters as extent of myelination of innervating fibers and the absence of efferent innervation in SNM. The present study addresses the validity of these last two features as distinguishing criteria by examining the structure of those SNM populations indicated in Fig. 1 at both the light and electron microscopic levels.All of the populations of SNM examined conform in general to previously published descriptions, consisting of a neuroepithelium composed of sensory hair cells, support cells and mantle cells, Several significant differences from these accounts have, however, emerged. Firstly, the structural composition of the innervating fibers is heterogeneous with respect to the extent of myelination. All SNM groups, with the possible exception of the TRrs and CFLs, possess both myelinated and unmyelinated fibers within the neuroepithelium proper (Fig. 2), just as do CNM. The extent of myelina- tion is quite variable, with some fibers sheath terminating just before crossing the neuroepithelial basal lamina, some just after and a few retaining their myelination all the way to the base of the hair cells in the upper third of the neuroepithelium. Secondly, all SNMs possess fibers that may, on the basis of ultrastructural criteria, be identified as efferent. Such fibers contained numerous cytoplasmic vesicles, both clear and with dense cores. In regions where such fibers closely apposed hair cells, subsynaptic cisternae were observed in the hair cell (Fig. 3).

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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