Supervillin Is a Component of the Hair Cell\u27s Cuticular Plate and the Head Plates of Organ of Corti Supporting Cells

The organ of Corti has evolved a panoply of cells with extraordinary morphological specializations to harness, direct, and transduce mechanical energy into electrical signals. Among the cells with prominent apical specializations are hair cells and nearby supporting cells. At the apical surface of each hair cell is a mechanosensitive hair bundle of filamentous actin (F-actin)-based stereocilia, which insert rootlets into the F-actin meshwork of the underlying cuticular plate, a rigid organelle considered to hold the stereocilia in place. Little is known about the protein composition and development of the cuticular plate or the apicolateral specializations of organ of Corti supporting cells. We show that supervillin, an F-actin cross-linking protein, localizes to cuticular plates in hair cells of the mouse cochlea and vestibule and zebrafish sensory epithelia. Moreover, supervillin localizes near the apicolateral margins within the head plates of Deiters\u27 cells and outer pillar cells, and proximal to the apicolateral margins of inner phalangeal cells, adjacent to the junctions with neighboring hair cells. Overall, supervillin localization suggests this protein may shape the surface structure of the organ of Corti

Fascin 2b Is a Component of Stereocilia that Lengthens Actin-Based Protrusions

Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a Kd≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia

Predisposition to Cancer Caused by Genetic and Functional Defects of Mammalian Atad5

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5+/m) mice that were haploinsuffficient for Atad5. Atad5+/m mice displayed high levels of genomic instability in vivo, and Atad5+/m mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5+/m mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis

Primary cilia are present on endothelial cells of the hyaloid vasculature but are not required for the development of the blood-retinal barrier.

Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, ift57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with Shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown

Supervillin Is a Component of the Hair Cell’s Cuticular Plate and the Head Plates of Organ of Corti Supporting Cells

<div><p>The organ of Corti has evolved a panoply of cells with extraordinary morphological specializations to harness, direct, and transduce mechanical energy into electrical signals. Among the cells with prominent apical specializations are hair cells and nearby supporting cells. At the apical surface of each hair cell is a mechanosensitive hair bundle of filamentous actin (F-actin)-based stereocilia, which insert rootlets into the F-actin meshwork of the underlying cuticular plate, a rigid organelle considered to hold the stereocilia in place. Little is known about the protein composition and development of the cuticular plate or the apicolateral specializations of organ of Corti supporting cells. We show that supervillin, an F-actin cross-linking protein, localizes to cuticular plates in hair cells of the mouse cochlea and vestibule and zebrafish sensory epithelia. Moreover, supervillin localizes near the apicolateral margins within the head plates of Deiters’ cells and outer pillar cells, and proximal to the apicolateral margins of inner phalangeal cells, adjacent to the junctions with neighboring hair cells. Overall, supervillin localization suggests this protein may shape the surface structure of the organ of Corti.</p></div

Supervillin localizes to zebrafish hair cell CPs.

<p><b>(A,B)</b> Confocal micrographs of 4-dpf zebrafish anterior crista hair cells expressing GFP-fascin 2b (red) and labeled with anti-Svila (green). Arrowheads in <b>A</b> indicate hair cells out of focus or bent. Arrow indicates hair-bundle-localized GFP-fascin 2b. Asterisk indicates a CP. <b>(C,D,E)</b> Confocal micrographs of posterior macula hair cells from zebrafish expressing Acf7a-Citrine (red) and labeled with anti-Svila (green) <b>(C,E)</b> or phalloidin (green) <b>(D)</b>. Asterisks in <b>(C,D)</b> indicate CPs. Acf7a-Citrine encircles the CP, localizes to the CP base, out of the focal plane in <b>C-E</b>, and is found weakly throughout the CP. (<b>F</b>) Schematic of the zebrafish posterior macula tissue with the location of Svila immunolabeling in green and Acf7a-Citrine indicated in red. Scale bars, 2 μm.</p

The gene encoding supervillin is expressed in chicken hair cells.

<p><b>(A)</b> Detection of <i>SVIL</i> mRNA in chicken hair cells by RNA-seq. Depth of reads aligned to the chicken genome, with TopHat-predicted splice junctions (red) and exons of human <i>SVIL</i> aligned to the chicken genome (blue). <b>(B)</b> Major functional domains of supervillin: M, myosin II-binding region; A1-A3, actin-binding regions 1–3; G, gelsolin repeats; and VHP, villin headpiece. Purple line indicates region of mouse SVIL recognized by the H340 antibody (Oh et al., 2003), and the blue line indicates the region of zebrafish Svila recognized by novel antiserum. <b>(C)</b> Alignment of vertebrate supervillin protein sequences using Clustal/Jalview and default parameters. The regions of bovine supervillin shown to bind the myosin II heavy chain and F-actin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158349#pone.0158349.ref019" target="_blank">19</a>] are displayed.</p

Supervillin is expressed in the mouse and zebrafish ear.

<p><b>(A)</b> RT-PCR detection of <i>svila</i> and <i>svilc</i> mRNAs in zebrafish hair cells using two primer pairs to detect <i>svila</i> mRNA [RT plus (lanes 1,3), RT minus (lanes 2,4)] and two primer pairs to detect <i>svilc</i> mRNA [RT plus (lane 5,6), RT minus (lane 7,8)]. <i>Svilb</i> [RT plus (lane 9), RT minus (lane 10)] and <i>svild</i> [RT plus (lane 11), RT minus (lane 12)] mRNAs were detected in zebrafish maculae. <b>(B)</b> RT-PCR of <i>Svil</i> mRNA from mouse hair cells [RT plus (lane 1), RT minus (lane 2)]. <b>(C-H)</b> RNA <i>in situ</i> hybridization. Whole mount 4-dpf zebrafish treated with probes antisense to <i>svila</i> <b>(C)</b> and <i>svilc</i> <b>(F)</b> mRNAs. Both genes are expressed in the otocyst (arrowheads). Controls are displayed. Magnified otocysts show <i>svila</i> <b>(D)</b> and <i>svilc</i> <b>(G)</b> are expressed in the anterior macula (AM). Sense-probed controls <b>(E,H)</b>. White dashed lines denote the otic vesicles. Yellow boxes show positions of AM hair cells. PC indicates the region of the posterior cristae (out of focus).</p

Supervillin localizes to mouse hair cell CPs and cochlear supporting cell head plates.

<p><b>(A-E)</b> Confocal micrographs of mouse vestibular hair cells labeled with anti-SVIL (green) and phalloidin (red) at different developmental stages. (<b>A</b>) A top-down view of several hair cells from a mouse at P1. Supervillin labels the CPs (asterisk) but not the fonticulus (arrowhead). <b>(B,C)</b> Views of hair cells from a 6-month-old mouse. Supervillin labels the CP but not the fonticulus (arrowhead) or stereocilia (arrows). (<b>D,E</b>) Type I (<b>D</b>) and type II (<b>E</b>) vestibular hair cells from a P3 mouse co-labeled with anti-tubulin (blue), which marks somatic microtubules underlying the CPs (arrowheads). Arrow in (<b>E</b>) indicates a kinocilium from a neighboring hair cell. A region of interest (ROI, indicated by the purple line) was selected to span the hair bundle (top portion of the line), CP (middle portion of the line), and underlying microtubules (bottom portion of the line). (<b>F</b>) Fluorescence intensity profile using the ROI from <b>(E)</b>. The hair bundle (top portion of the purple ROI line) corresponds to the left region of the plot, showing robust F-actin-associated signal (red), while SVIL- (green) and tubulin-associated (blue) signals are minimal. The middle region of the plot corresponds to the CP (middle portion of the ROI line) and shows overlapping SVIL- and F-actin-associated signals; however, in the right region of the plot, only tubulin-associated signal is seen below the CP (bottom portion of the ROI line). (<b>G-M</b>) Confocal micrographs of mouse cochlear hair cells labeled with anti-SVIL (green) and anti-actin (red). (<b>G-J</b>) Hair cells at the middle (<b>G,H</b>) and apical (<b>I,J</b>) cochlear turns of a P1 mouse. At the middle turn (<b>G,H</b>), SVIL localizes to the CPs (asterisk) and to the region of the hair cell-supporting cell junctions (arrow). At the apical turn (<b>I,J</b>), SVIL co-localizes with actin near the apical surface of the developing hair cells. (<b>K,L</b>) In the middle turn of the P3 mouse cochlea, SVIL localizes to CPs of outer hair cells (OHCs) and inner hair cells (IHCs) and to supporting cell apicolateral margins, including those of Deiters’ cells (DC1, DC2, DC3), outer pillar cells (OP), and inner phalangeal cells (IPC). (<b>M</b>) Side view of two IHCs from the middle turn of a P3 mouse cochlea co-labeled with an antibody to tubulin (blue) demonstrates that SVIL localizes between the hair bundle (arrow) and somatic microtubules (arrowhead), at the region of the cuticular plate (asterisk). (<b>N</b>) Magnification of two OHCs and the Deiters’ cell between them (arrow) from the basal turn of a P1 mouse cochlea labeled with anti-SVIL (green) and anti-β-catenin (red). (<b>O</b>) Magnification of the first two rows of OHCs from the basal turn of a P2 mouse cochlea labeled with anti-SVIL (green) and anti-ZO-1 (red). SVIL strongly localizes to the apicolateral margins of the OPs (arrowhead) and the DCs (arrow). (<b>P</b>) Z-stacks of confocal sections were converted into a 3D model using the Leica Software. A 3D reconstruction of an OHC (asterisk) from the second row flanked by two DCs (arrow) labeled with anti-SVIL (green) and anti-ZO-1 (green) is seen. (<b>Q</b>) Fluorescence intensity profile of the cell in (<b>O</b>) using the ROI indicated by the purple line demonstrates that the signal associated with SVIL (green) is concentrated in the supporting cells, sandwiched between ZO-1-rich bands (red). In graphs in <b>F</b> and <b>Q</b>, intensity scales are linear, but the units are arbitrary. Scale bars, 2 μm.</p

Fascin 2b is a component of stereocilia that lengthens actin-based protrusions.

Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a K(d)≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia