The Experts below are selected from a list of 4308 Experts worldwide ranked by ideXlab platform
Bechara Kachar - One of the best experts on this subject based on the ideXlab platform.
-
plastin 1 widens Stereocilia by transforming actin filament packing from hexagonal to liquid
Journal of Cell Biology, 2016Co-Authors: Jocelyn F. Krey, Rachel A. Dumont, Sarath Vijayakumar, Bechara Kachar, Evan S Krystofiak, Dongseok Choi, Francisco Rivero, Sherri M Jones, Peter G BarrgillespieAbstract: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.
-
alternative splice forms influence functions of whirlin in mechanosensory hair cell Stereocilia
Cell Reports, 2016Co-Authors: Seham Ebrahim, Bechara Kachar, Runjia Cui, Neil J Ingham, Morag A Lewis, Michael J Rogers, Johanna C Pass, Karen P SteelAbstract:WHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in Stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within Stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short Stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain Stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at Stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter Stereocilia, at the level of inter-Stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.
-
tectorins crosslink type ii collagen fibrils and connect the tectorial membrane to the spiral limbus
Journal of Structural Biology, 2016Co-Authors: Felipe T Salles, Leonardo R Andrade, Mhamed Grati, Uri Manor, Bechara KacharAbstract:Abstract All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and β tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell Stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and β-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4 ± 1.5 nm, width), resembling another collagen type protein, or chains of globular elements (15 ± 3.2 nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and β-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell Stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.
-
correlation of actin crosslinker and capper expression levels with Stereocilia growth phases
Molecular & Cellular Proteomics, 2014Co-Authors: Matthew R. Avenarius, Kateri J. Spinelli, Jung-bum Shin, Bechara Kachar, Katherine W Saylor, James M Pagana, Phillip A Wilmarth, Leonardo R Andrade, Megan R Lundeberg, Dongseok ChoiAbstract:During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell Stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when Stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at Stereocilia tips; it is abundant during growth phase II, when Stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when Stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea Stereocilia, quantitative mass spectrometry of Stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular Stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during Stereocilia development.
-
clic5 stabilizes membrane actin filament linkages at the base of hair cell Stereocilia in a molecular complex with radixin taperin and myosin vi
Cytoskeleton, 2014Co-Authors: Felipe T Salles, Bechara Kachar, Leona H Gagnon, Leonardo R Andrade, Mhamed Grati, Kenneth R Johnson, Soichi Tanda, Kathleen Lynn Plona, Mark BerrymanAbstract:Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell Stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of Stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of Stereocilia, were mislocalized in fused Stereocilia of jbg mice. TPRQ and RDX were dispersed even prior to Stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused Stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of Stereocilia.
Karen P Steel - One of the best experts on this subject based on the ideXlab platform.
-
alternative splice forms influence functions of whirlin in mechanosensory hair cell Stereocilia
Cell Reports, 2016Co-Authors: Seham Ebrahim, Bechara Kachar, Runjia Cui, Neil J Ingham, Morag A Lewis, Michael J Rogers, Johanna C Pass, Karen P SteelAbstract:WHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in Stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within Stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short Stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain Stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at Stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter Stereocilia, at the level of inter-Stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.
-
myosinviia interacts with twinfilin 2 at the tips of mechanosensory Stereocilia in the inner ear
PLOS ONE, 2009Co-Authors: Agnieszka K. Rzadzinska, Elisa M Nevalainen, Haydn M Prosser, Pekka Lappalainen, Karen P SteelAbstract:In vertebrates hearing is dependent upon the microvilli-like mechanosensory Stereocilia and their length gradation. The staircase-like organization of the Stereocilia bundle is dynamically maintained by variable actin turnover rates. Two unconventional myosins were previously implicated in Stereocilia length regulation but the mechanisms of their action remain unknown. MyosinXVa is expressed in Stereocilia tips at levels proportional to Stereocilia length and its absence produces staircase-like bundles of very short Stereocilia. MyosinVIIa localizes to the tips of the shorter Stereocilia within bundles, and when absent, the Stereocilia are abnormally long. We show here that myosinVIIa interacts with twinfilin-2, an actin binding protein, which inhibits actin polymerization at the barbed end of the filament, and that twinfilin localization in Stereocilia overlaps with myosinVIIa. Exogenous expression of myosinVIIa in fibroblasts results in a reduced number of filopodia and promotes accumulation of twinfilin-2 at the filopodia tips. We hypothesize that the newly described interaction between myosinVIIa and twinfilin-2 is responsible for the establishment and maintenance of slower rates of actin turnover in shorter Stereocilia, and that interplay between complexes of myosinVIIa/twinfilin-2 and myosinXVa/whirlin is responsible for Stereocilia length gradation within the bundle staircase.
-
The deaf mouse mutant whirler suggests a role for whirlin in actin filament dynamics and Stereocilia development
Cell motility and the cytoskeleton, 2007Co-Authors: Mette M. Mogensen, Agnieszka K. Rzadzinska, Karen P SteelAbstract:Stereocilia, finger-like projections forming the hair bundle on the apical surface of sensory hair cells in the cochlea, are responsible for mechanosensation and ultimately the perception of sound. The actin cytoskeleton of the Stereocilia contains hundreds of tightly cross-linked parallel actin filaments in a paracrystalline array and it is vital for their function. Although several genes have been identified and associated with Stereocilia development, the molecular mechanisms responsible for Stereocilia growth, maintenance and organisation of the hair bundle have not been fully resolved. Here we provide further characterisation of the Stereocilia of the whirler mouse mutant. We found that a lack of whirlin protein in whirler mutants results in short Stereocilia with larger diameters without a corresponding increase in the number of actin filaments in inner hair cells. However, a decrease in the actin filament packing density was evident in the whirler mutant. The electron-density at the tip of each stereocilium was markedly patchy and irregular in the whirler mutants compared with a uniform band in controls. The outer hair cell Stereocilia of the whirler homozygote also showed an increase in diameter and variable heights within bundles. The number of outer hair cell Stereocilia was significantly reduced and the centre-to-centre spacing between the Stereocilia was greater than in the wildtype. Our findings suggest that whirlin plays an important role in actin filament packing and dynamics during postnatal stereocilium elongation.
-
elongation of hair cell Stereocilia is defective in the mouse mutant whirler
The Journal of Comparative Neurology, 2002Co-Authors: Ralph H Holme, Steve D M Brown, Brent W Kiernan, Karen P SteelAbstract:The recessive mouse mutant whirler (wi) shows no response to sound and exhibits circling and head-tossing behaviour, indicative of both auditory and vestibular dysfunction. The wi mutation maps genetically to mouse chromosome 4. We examined the organ of Corti of whirler mutants to explore the possibility that the wi mutation affects sensory hair cells. Scanning electron microscopy (SEM) reveals that the specialised microvilli (Stereocilia) that are projected by the sensory hair cells and are vital for sound transduction are abnormal in wi homozygotes. Specifically, wi homozygous inner hair cell (IHC) Stereocilia are approximately half the length of equivalent Stereocilia in heterozygous littermates. They are arranged normally into ranks, but the gradation in height and width of Stereocilia in adjacent ranks is less prominent in wi homozygotes. Analysis of IHC Stereocilia during the course of their development shows that, by embryonic day 18.5, mutant Stereocilia are already significantly shorter than those in controls. Mutant Stereocilia elongate at a normal rate, at least until postnatal day 1, but prematurely stop elongating between postnatal days 1 and 4. Stereocilia length then decreases. At postnatal day 15, outer hair cell (OHC) Stereocilia in wi homozygotes appear short and are arranged in a rounded, "U" shape rather than the normal "W" or "V" shape. Eventually, both IHCs and OHCs degenerate. We show that the whirler locus encodes a protein(s) required for the elongation and maintenance of IHC and OHC Stereocilia.
-
role of myosin vi in the differentiation of cochlear hair cells
Developmental Biology, 1999Co-Authors: Tim Self, Tama Sobe, Neal G Copeland, Nancy A Jenkins, Karen B Avraham, Karen P SteelAbstract:Abstract The mouse mutant Snell's waltzer (sv) has an intragenic deletion of the Myo6 gene, which encodes the unconventional myosin molecule myosin VI (K. B. Avraham et al., 1995, Nat. Genet. 11, 369–375). Snell's waltzer mutants exhibit behavioural abnormalities suggestive of an inner ear defect, including lack of responsiveness to sound, hyperactivity, head tossing, and circling. We have investigated the effects of a lack of myosin VI on the development of the sensory hair cells of the cochlea in these mutants. In normal mice, the hair cells sprout microvilli on their upper surface, and some of these grow to form a crescent or V-shaped array of modified microvilli, the Stereocilia. In the mutants, early stages of Stereocilia development appear to proceed normally because at birth many Stereocilia bundles have a normal appearance, but in places there are signs of disorganisation of the bundles. Over the next few days, the Stereocilia become progressively more disorganised and fuse together. Practically all hair cells show fused Stereocilia by 3 days after birth, and there is extensive Stereocilia fusion by 7 days. By 20 days, giant Stereocilia are observed on top of the hair cells. At 1 and 3 days after birth, hair cells of mutants and controls take up the membrane dye FM1-43, suggesting that endocytosis occurs in mutant hair cells. One possible model for the fusion is that myosin VI may be involved in anchoring the apical hair cell membrane to the underlying actin-rich cuticular plate, and in the absence of normal myosin VI this apical membrane will tend to pull up between Stereocilia, leading to fusion.
Gregory I. Frolenkov - One of the best experts on this subject based on the ideXlab platform.
-
building and repairing the Stereocilia cytoskeleton in mammalian auditory hair cells
Hearing Research, 2019Co-Authors: Catalina A Velezortega, Gregory I. FrolenkovAbstract:Despite all recent achievements in identification of the molecules that are essential for the structure and mechanosensory function of Stereocilia bundles in the auditory hair cells of mammalian species, we still have only a rudimentary understanding of the mechanisms of Stereocilia formation, maintenance, and repair. Important molecular differences distinguishing mammalian auditory hair cells from hair cells of other types and species have been recently revealed. In addition, we are beginning to solve the puzzle of the apparent life-long stability of the Stereocilia bundles in these cells. New data link the stability of the cytoskeleton in the mammalian auditory Stereocilia with the normal activity of mechanotransduction channels. These data suggest new ideas on how a terminally-differentiated non-regenerating hair cell in the mammalian cochlea may repair and tune its Stereocilia bundle throughout the life span of the organism.
-
mechanotransduction current is essential for stability of the transducing Stereocilia in mammalian auditory hair cells
eLife, 2017Co-Authors: Catalina A Velezortega, Artur A. Indzhykulian, Mary J Freeman, Jonathan M Grossheim, Gregory I. FrolenkovAbstract:Our sense of hearing depends on cells known as hair cells that line the inner ear. Each hair cell has tiny projections called Stereocilia, which are arranged in a bundle with rows of increasing height like a staircase and are connected to each other by tiny filaments called tip-links. When sound waves hit the Stereocilia, the tension on the tip-links increases, which opens “mechanotransduction” channels on the shorter Stereocilia that allow calcium ions to flow into the cells. To ensure that the ears can detect even the softest sounds, the tip-links always have a small amount of tension which allows a small, but continuous flow of calcium ions into the cell. Scientists generally consider this continuous flow of calcium ions as a potentially harmful byproduct of sensitive hearing. Velez-Ortega et al. isolated inner ear tissues from young mice and rats and exposed them to drugs that either block the flow of calcium ions through the mechanotransduction channels or break the tip-links on Stereocilia. Surprisingly, these drugs made profound changes in the shape of individual Stereocilia and the staircase architecture of the Stereocilia bundle. When the drugs were rinsed out of the hair cells, the Stereocilia went back to their normal shape. Sequestering of free calcium ions inside the hair cells had a similar effect on the shape of Stereocilia. These findings show that the flow of calcium ions into the sterocilia via mechanotransduction channels controls the exquisite staircase-like architecture of the Stereocilia bundle. More research is needed to identify which structural proteins cause the Stereocilia shape changes and to work out exactly how calcium ions are involved.
-
the 133 kda n terminal domain enables myosin 15 to maintain mechanotransducing Stereocilia and is essential for hearing
eLife, 2015Co-Authors: Qing Fang, Gregory I. Frolenkov, Inna A Belyantseva, Thomas B Friedman, Artur A. Indzhykulian, Gavin P Riordan, Mirna Mustapha, David F Dolan, Sally A Camper, Jonathan E BirdAbstract:The precise assembly of inner ear hair cell Stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based Stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt Stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different Stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal Stereocilia bundles. However, a critical subset of Stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized Stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of Stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.
-
Regenerating Stereocilia links appear at the tips but not at the bottom of Stereocilia.
2013Co-Authors: Artur A. Indzhykulian, Ruben Stepanyan, Inna A Belyantseva, Thomas B Friedman, Zubair M Ahmed, Anastasiia Nelina, Kateri J. Spinelli, Peter G. Barr-gillespie, Gregory I. FrolenkovAbstract:(A–B) Conventional (secondary electron) SEM images of IHC Stereocilia before (A) and immediately after (B) link disruption. Dashed rectangles indicate the areas magnified in insets. Arrows point to the tip links. (C) Distribution of the links along the height of Stereocilia (0%, bottom; 100%, top) in the third (shortest) row at different stages of link recovery. (D) Backscatter SEM image of IHC bundle immuno-labeled with anti-PDCH15 antibody, HL5614 (10 nm gold particles seen as white dots). (E) The same as in (D), but primary antibody was omitted. (F) Percentage of immuno-gold particles observed on links of second and third row Stereocilia in IHCs at two different dilutions of HL5614. (G) Representative images of HL5614 labeling in third row Stereocilia immediately and 20 min after BAPTA treatment. (H) Cumulative distribution of PCDH15 immuno-gold particles on third row stereocilium in control and during link recovery. For each time point, 50–70 Stereocilia images were scaled to a common template (dashed line) and the location of every gold particle was shown by a semitransparent grey circle. (I) Distribution of PCDH15 immuno-gold particles along the height of third row Stereocilia. Data in panels (C), (F), and (I) are shown as mean ± SE. Age of the cells: P3–4 plus 2–3 days in vitro (P3–4+2–3 div).
-
Auditory mechanotransduction in the absence of functional myosin-XVa
The Journal of Physiology, 2006Co-Authors: Ruben Stepanyan, Inna A Belyantseva, Andrew J Griffith, Thomas B Friedman, Gregory I. FrolenkovAbstract:In hair cells of all vertebrates, a mechanosensory bundle is formed by Stereocilia with precisely graded heights. Unconventional myosin-XVa is critical for formation of this bundle because it transports whirlin and perhaps other molecular components responsible for programmed elongation of Stereocilia to the Stereocilia tips. A tip of a stereocilium is the site of Stereocilia growth and one of the proposed sites of mechano-electrical transduction. In adult shaker 2 mice, a mutation that disables the motor function of myosin-XVa results in profound deafness and abnormally short Stereocilia that lack Stereocilia links, an indispensable component of mechanotransduction machinery. Therefore, it was assumed that myosin-XVa is required for proper formation of the mechanotransduction apparatus. Here we show that in young postnatal shaker 2 mice, abnormally short Stereocilia bundles of auditory hair cells have numerous Stereocilia links and ‘wild type’ mechano-electrical transduction. We compared the mechanotransduction current in auditory hair cells of young normal-hearing littermates, myosin-XVa-deficient shaker 2 mice, and whirler mice that have similarly short Stereocilia but intact myosin-XVa at the Stereocilia tips. This comparison revealed that the absence of functional myosin-XVa does not disrupt adaptation of the mechanotransduction current during sustained bundle deflection. Thus, the hair cell mechanotransduction complex forms and functions independently from myosin-XVa-based hair bundle morphogenesis.
Inna A Belyantseva - One of the best experts on this subject based on the ideXlab platform.
-
Human deafness-associated variants alter the dynamics of key molecules in hair cell Stereocilia F-actin cores
Human Genetics, 2021Co-Authors: Takushi Miyoshi, Inna A Belyantseva, Shin-ichiro Kitajiri, Byung Yoon Choi, Hiroki Miyajima, Shin-ya Nishio, Shin-ichi Usami, Bong Jik Kim, Koichi Omori, Hari ShroffAbstract:Stereocilia protrude up to 100 µm from the apical surface of vertebrate inner ear hair cells and are packed with cross-linked filamentous actin (F-actin). They function as mechanical switches to convert sound vibration into electrochemical neuronal signals transmitted to the brain. Several genes encode molecular components of Stereocilia including actin monomers, actin regulatory and bundling proteins, motor proteins and the proteins of the mechanotransduction complex. A stereocilium F-actin core is a dynamic system, which is continuously being remodeled while maintaining an outwardly stable architecture under the regulation of F-actin barbed-end cappers, severing proteins and crosslinkers. The F-actin cores of Stereocilia also provide a pathway for motor proteins to transport cargos including components of tip-link densities, scaffolding proteins and actin regulatory proteins. Deficiencies and mutations of Stereocilia components that disturb this “dynamic equilibrium” in Stereocilia can induce morphological changes and disrupt mechanotransduction causing sensorineural hearing loss, best studied in mouse and zebrafish models. Currently, at least 23 genes, associated with human syndromic and nonsyndromic hearing loss, encode proteins involved in the development and maintenance of Stereocilia F-actin cores. However, it is challenging to predict how variants associated with sensorineural hearing loss segregating in families affect protein function. Here, we review the functions of several molecular components of Stereocilia F-actin cores and provide new data from our experimental approach to directly evaluate the pathogenicity and functional impact of reported and novel variants of DIAPH1 in autosomal-dominant DFNA1 hearing loss using single-molecule fluorescence microscopy.
-
actin at Stereocilia tips is regulated by mechanotransduction and adf cofilin
Current Biology, 2021Co-Authors: Jamis Mcgrath, Inna A Belyantseva, Chunyu Tung, Xiayi Liao, Pallabi Roy, Oisorjo Chakraborty, Nicolas F Berbari, Christian C Faaborgandersen, Melanie Barzik, Jonathan E BirdAbstract:Stereocilia 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.
-
the 133 kda n terminal domain enables myosin 15 to maintain mechanotransducing Stereocilia and is essential for hearing
eLife, 2015Co-Authors: Qing Fang, Gregory I. Frolenkov, Inna A Belyantseva, Thomas B Friedman, Artur A. Indzhykulian, Gavin P Riordan, Mirna Mustapha, David F Dolan, Sally A Camper, Jonathan E BirdAbstract:The precise assembly of inner ear hair cell Stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based Stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt Stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different Stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal Stereocilia bundles. However, a critical subset of Stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized Stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of Stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.
-
Regenerating Stereocilia links appear at the tips but not at the bottom of Stereocilia.
2013Co-Authors: Artur A. Indzhykulian, Ruben Stepanyan, Inna A Belyantseva, Thomas B Friedman, Zubair M Ahmed, Anastasiia Nelina, Kateri J. Spinelli, Peter G. Barr-gillespie, Gregory I. FrolenkovAbstract:(A–B) Conventional (secondary electron) SEM images of IHC Stereocilia before (A) and immediately after (B) link disruption. Dashed rectangles indicate the areas magnified in insets. Arrows point to the tip links. (C) Distribution of the links along the height of Stereocilia (0%, bottom; 100%, top) in the third (shortest) row at different stages of link recovery. (D) Backscatter SEM image of IHC bundle immuno-labeled with anti-PDCH15 antibody, HL5614 (10 nm gold particles seen as white dots). (E) The same as in (D), but primary antibody was omitted. (F) Percentage of immuno-gold particles observed on links of second and third row Stereocilia in IHCs at two different dilutions of HL5614. (G) Representative images of HL5614 labeling in third row Stereocilia immediately and 20 min after BAPTA treatment. (H) Cumulative distribution of PCDH15 immuno-gold particles on third row stereocilium in control and during link recovery. For each time point, 50–70 Stereocilia images were scaled to a common template (dashed line) and the location of every gold particle was shown by a semitransparent grey circle. (I) Distribution of PCDH15 immuno-gold particles along the height of third row Stereocilia. Data in panels (C), (F), and (I) are shown as mean ± SE. Age of the cells: P3–4 plus 2–3 days in vitro (P3–4+2–3 div).
-
twinfilin 2 regulates actin filament lengths in cochlear Stereocilia
The Journal of Neuroscience, 2009Co-Authors: Anthony W Peng, Inna A Belyantseva, Thomas B Friedman, Patrick D Hsu, Stefan HellerAbstract:Inner ear sensory hair cells convert mechanical stimuli into electrical signals. This conversion happens in the exquisitely mechanosensitive hair bundle that protrudes from the cell's apical surface. In mammals, cochlear hair bundles are composed of 50–100 actin-filled Stereocilia, which are organized in three rows in a staircase manner. Stereocilia actin filaments are uniformly oriented with their barbed ends toward Stereocilia tips. During development, the actin core of each stereocilium undergoes elongation due to addition of actin monomers to the barbed ends of the filaments. Here we show that in the mouse cochlea the barbed end capping protein twinfilin 2 is present at the tips of middle and short rows of Stereocilia from postnatal day 5 (P5) onward, which correlates with a time period when these rows stop growing. The tall Stereocilia rows, which do not display twinfilin 2 at their tips, continue to elongate between P5 and P15. When we expressed twinfilin 2 in LLC/PK1-CL4 (CL4) cells, we observed a reduction of espin-induced microvilli length, pointing to a potent function of twinfilin 2 in suppressing the elongation of actin filaments. Overexpression of twinfilin 2 in cochlear inner hair cells resulted in a significant reduction of Stereocilia length. Our results suggest that twinfilin 2 plays a role in the regulation of Stereocilia elongation by restricting excessive elongation of the shorter row Stereocilia thereby maintaining the mature staircase architecture of cochlear hair bundles.
David P. Corey - One of the best experts on this subject based on the ideXlab platform.
-
pkhd1l1 is a coat protein of hair cell Stereocilia and is required for normal hearing
Nature Communications, 2019Co-Authors: Maryna V Ivanchenko, Artur A. Indzhykulian, Hoor Al Jandal, Marcelo Cicconet, David P. CoreyAbstract:The bundle of Stereocilia on inner ear hair cells responds to subnanometer deflections produced by sound or head movement. Stereocilia are interconnected by a variety of links and also carry an electron-dense surface coat. The coat may contribute to Stereocilia adhesion or protect from Stereocilia fusion, but its molecular identity remains unknown. From a database of hair-cell-enriched translated proteins, we identify Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), a large, mostly extracellular protein of 4249 amino acids with a single transmembrane domain. Using serial immunogold scanning electron microscopy, we show that PKHD1L1 is expressed at the tips of Stereocilia, especially in the high-frequency regions of the cochlea. PKHD1L1-deficient mice lack the surface coat at the upper but not lower regions of Stereocilia, and they develop progressive hearing loss. We conclude that PKHD1L1 is a component of the surface coat and is required for normal hearing in mice. There is little known about the function or molecular identity of the electron-dense Stereocilia coat, which is transiently present at the surface of Stereocilia. In this study authors screened a database of hair-cell-enriched translated proteins to identify the expression of Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), a large, mostly extracellular protein, and show that it forms the coat at the tips of Stereocilia and is required for normal hearing in mice
-
Heterodimeric capping protein is required for Stereocilia length and width regulation.
The Journal of cell biology, 2017Co-Authors: Matthew R. Avenarius, David P. Corey, Jocelyn F. Krey, Rachel A. Dumont, Clive P Morgan, Connor B. Benson, Sarath Vijayakumar, Christopher L. Cunningham, Deborah I. Scheffer, Ulrich MüllerAbstract:Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and Stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear’s Stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at Stereocilia tips as hair cell development progressed, similar to the CAPZB-interacting protein TWF2. We deleted Capzb specifically in hair cells using Atoh1-Cre, which eliminated auditory and vestibular function. Capzb-null Stereocilia initially developed normally but later shortened and disappeared; surprisingly, Stereocilia width decreased concomitantly with length. CAPZB2 expressed by in utero electroporation prevented normal elongation of vestibular Stereocilia and irregularly widened them. Together, these results suggest that capping protein participates in Stereocilia widening by preventing newly elongating actin filaments from depolymerizing.
-
length regulation of mechanosensitive Stereocilia depends on very slow actin dynamics and filament severing proteins
PMC, 2015Co-Authors: Praveena Narayanan, David P. Corey, Paul Chatterton, Akihiro Ikeda, Sakae Ikeda, James M Ervasti, Benjamin J PerrinAbstract:Auditory sensory hair cells depend on Stereocilia with precisely regulated lengths to detect sound. Since Stereocilia are primarily composed of crosslinked, parallel actin filaments, regulated actin dynamics are essential for controlling Stereocilia length. Here we assessed Stereocilia actin turnover by monitoring incorporation of inducibly expressed β-actin-GFP in adult mouse hair cells in vivo and by directly measuring β-actin-GFP turnover in explants. Stereocilia actin incorporation is remarkably slow and restricted to filament barbed ends in a small tip compartment, with minimal accumulation in the rest of the actin core. Shorter rows of Stereocilia, which have mechanically gated ion channels, show more variable actin turnover than the tallest Stereocilia, which lack channels. Finally, the proteins ADF and AIP1, which both mediate actin filament severing, contribute to Stereocilia length maintenance. Altogether, the data support a model whereby Stereocilia actin cores are largely static, with dynamic regulation at the tips to maintain a critical length.
-
multi isotope imaging mass spectrometry reveals slow protein turnover in hair cell Stereocilia
Nature, 2012Co-Authors: Duan Sun Zhang, Agnieszka K. Rzadzinska, Haydn M Prosser, James M Ervasti, Benjamin J Perrin, Valeria Piazza, Collin J Poczatek, Mei Wang, David P. CoreyAbstract:Multi-isotope imaging mass spectrometry is used to quantify protein turnover in animal Stereocilia, showing that rapid turnover occurs only in Stereocilia tips. In the auditory system, the mechanosensory hair cells of the inner ear convert sound-induced vibrations into electrical signals. The apical surface of a hair cell consists of Stereocilia with a core of actin filaments that function as mechanosensors. It has been suggested that these actin filaments are replaced within two to three days by a treadmilling process. Using the newly developed multi-isotope imaging mass spectrometry (MIMS) technique, Zhang et al. quantify protein turnover in hair-cell Stereocilia in vivo and find that turnover is slow throughout the Stereocilia, except for the tip region, and does not involve a treadmilling process. Hair cells of the inner ear are not normally replaced during an animal’s life, and must continually renew components of their various organelles1. Among these are the Stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in Stereocilia has previously been studied2 by transfecting neonatal rat hair cells in culture with a β-actin–GFP fusion, and evidence was found that actin is replaced, from the top down, in 2–3 days. Overexpression of the actin-binding protein espin causes elongation of Stereocilia within 12–24 hours, also suggesting rapid regulation of Stereocilia lengths3. Similarly, the mechanosensory ‘tip links’ are replaced in 5–10 hours after cleavage in chicken and mammalian hair cells4,5. In contrast, turnover in chick Stereocilia in vivo is much slower6. It might be that only certain components of Stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a 15N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice, in vivo and in vitro, the Stereocilia were remarkably stable, incorporating newly synthesized protein at <10% per day. Only Stereocilia tips had rapid turnover and no treadmilling was observed. Other methods confirmed this: in hair cells expressing β-actin–GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of β- or γ-actin with tamoxifen-inducible recombination, neither actin isoform left the Stereocilia, except at the tips. Thus, rapid turnover in Stereocilia occurs only at the tips and not by a treadmilling process.
-
sliding adhesion confers coherent motion to hair cell Stereocilia and parallel gating to transduction channels
The Journal of Neuroscience, 2010Co-Authors: Domenica K Karavitaki, David P. CoreyAbstract:When the tip of a hair bundle is deflected by a sensory stimulus, the Stereocilia pivot as a unit, producing a shearing displacement between adjacent tips. It is not clear how Stereocilia can stick together laterally but still shear. We used dissociated hair cells from the bullfrog saccule and high-speed video imaging to characterize this sliding adhesion. Movement of individual Stereocilia was proportional to height, indicating that Stereocilia pivot at their basal insertion points. All Stereocilia moved by approximately the same angular deflection, and the same motion was observed at 1, 20, and 700 Hz stimulus frequency. Motions were consistent with a geometric model that assumes the stiffness of lateral links holding Stereocilia together is >1000 times the pivot stiffness of Stereocilia and that these links can slide in the plane of the membrane—in essence, that Stereocilia shear without separation. The same motion was observed when bundles were moved perpendicular to the tip links, or when tip links, ankle links, and shaft connectors were cut, ruling out these links as the basis for sliding adhesion. Stereocilia rootlets are angled toward the center of the bundle, tending to push Stereocilia tips together for small deflections. However, Stereocilia remained cohesive for deflections of up to ±35°, ruling out rootlet prestressing as the basis for sliding adhesion. These observations suggest that horizontal top connectors mediate a sliding adhesion. They also indicate that all transduction channels of a hair cell are mechanically in parallel, an arrangement that may enhance amplification in the inner ear.
