Hypervulnerability to Sound Exposure through Impaired Adaptive Proliferation of Peroxisomes.

A deficiency in pejvakin, a protein of unknown function, causes a strikingly heterogeneous form of human deafness. Pejvakin-deficient (Pjvk(-/-)) mice also exhibit variable auditory phenotypes. Correlation between their hearing thresholds and the number of pups per cage suggest a possible harmful effect of pup vocalizations. Direct sound or electrical stimulation show that the cochlear sensory hair cells and auditory pathway neurons of Pjvk(-/-) mice and patients are exceptionally vulnerable to sound. Subcellular analysis revealed that pejvakin is associated with peroxisomes and required for their oxidative-stress-induced proliferation. Pjvk(-/-) cochleas display features of marked oxidative stress and impaired antioxidant defenses, and peroxisomes in Pjvk(-/-) hair cells show structural abnormalities after the onset of hearing. Noise exposure rapidly upregulates Pjvk cochlear transcription in wild-type mice and triggers peroxisome proliferation in hair cells and primary auditory neurons. Our results reveal that the antioxidant activity of peroxisomes protects the auditory system against noise-induced damage.

Dispensability of Tubulin Acetylation for 15-protofilament Microtubule Formation in the Mammalian Cochlea.

The development of hearing in mammals requires the formation and maturation of a highly organized and specialized epithelium known as the organ of Corti. This epithelium contains two types of cells, the sensory cells, which are the true receptors of auditory information, and the surrounding supporting cells, which are composed of a highly developed cytoskeleton essential to the architecture of the mature organ of Corti. The supporting cells are the only mammalian cells reported to contain the unusual 15-protofilament microtubules. In this paper, we show that 15-protofilament microtubules appear between the second and fourth day after birth in the pillar cells of the organ of Corti in mice. We also show that contrary to what has been described in the nematode worm Caenorhabiditis. elegans, microtubule acetylation is not essential for the formation of 15-protofilament microtubules in mice but is required for fine-tuning of their diameter.Key words: Acetylation, cytoskeleton, microtubule, inner ear, supporting cells.

Proteostasis is essential during cochlear development for neuron survival and hair cell polarity.

Protein homeostasis is essential to cell function, and a compromised ability to reduce the load of misfolded and aggregated proteins is linked to numerous age-related diseases, including hearing loss. Here, we show that altered proteostasis consequent to Elongator complex deficiency also impacts the proper development of the cochlea and results in deafness. In the absence of the catalytic subunit Elp3, differentiating spiral ganglion neurons display large aggresome-like structures and undergo apoptosis before birth. The cochlear mechanosensory cells are able to survive proteostasis disruption but suffer defects in polarity and stereociliary bundle morphogenesis. We demonstrate that protein aggregates accumulate at the apical surface of hair cells, where they cause a local slowdown of microtubular trafficking, altering the distribution of intrinsic polarity proteins and affecting kinocilium position and length. Alleviation of protein misfolding using the chemical chaperone 4-phenylbutyric acid during embryonic development ameliorates hair cell polarity in Elp3-deficient animals. Our study highlights the importance of developmental proteostasis in the cochlea and unveils an unexpected link between proteome integrity and polarized organization of cellular components.

Visualization of Chromatin in the Yeast Nucleus and Nucleolus Using Hyperosmotic Shock.

Unlike in most eukaryotic cells, the genetic information of budding yeast in the exponential growth phase is only present in the form of decondensed chromatin, a configuration that does not allow its visualization in cell nuclei conventionally prepared for transmission electron microscopy. In this work, we studied the distribution of chromatin and its relationships to the nucleolus using different cytochemical and immunocytological approaches applied to yeast cells subjected to hyperosmotic shock. Our results show that osmotic shock induces the formation of heterochromatin patches in the nucleoplasm and intranucleolar regions of the yeast nucleus. In the nucleolus, we further revealed the presence of osmotic shock-resistant DNA in the fibrillar cords which, in places, take on a pinnate appearance reminiscent of ribosomal genes in active transcription as observed after molecular spreading ("Christmas trees"). We also identified chromatin-associated granules whose size, composition and behaviour after osmotic shock are reminiscent of that of mammalian perichromatin granules. Altogether, these data reveal that it is possible to visualize heterochromatin in yeast and suggest that the yeast nucleus displays a less-effective compartmentalized organization than that of mammals.

Relationships between the structural and functional organization of the turtle cell nucleolus.

The nucleolus is a multifunctional structure of the eukaryotic cell nucleus. However, its primary role is ribosome formation. Although the factors and mechanisms involved in ribogenesis are well conserved in eukaryotes, two types of nucleoli have been observed under the electron microscope: a tricompartmentalized nucleolus in amniotes and a bicompartmentalized nucleolus in other species. A recent study has also revealed that turtles, although belonging to amniotes, displayed a nucleolus with bipartite organization, suggesting that this reptile group may have carried out a reversion phenomenon during evolution. In this study, we examine in great detail the functional organization of the turtle nucleolus. In liver and spleen cells cultured in vitro, we confirm that the turtle nucleolus is mainly formed by two components: a fibrillar zone surrounded by a granular zone. We further show that the fibrillar zone includes densely-contrasted strands, which are positive after silver-stained Nucleolar Organizer Region (Ag-NOR) staining and DNA labelling. We also reveal that the dense strands condensed into a very compact mass within the fibrillar zone after a treatment with actinomycin D or 5,6-dichlorobenzimidazole riboside. Finally, by using pulse-chase experiments with BrUTP, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we show that the topological and spatial dynamics of rRNA within the nucleolus extend from upstream binding factor (UBF)-positive sites in the fibrillar zone to the granular zone, without ever releasing the positive sites for the UBF. Together, these results seem to clearly indicate that the compartmentalization of the turtle nucleolus into two main components reflects a less orderly organization of ribosome formation.

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein.

BACKGROUND: Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. RESULTS: The epitope sequence was genetically inserted in the αB-αB" domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. CONCLUSIONS: The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

Varicella-Zoster Virus ORF9p Binding to Cellular Adaptor Protein Complex 1 Is Important for Viral Infectivity.

ORF9p (homologous to herpes simplex virus 1 [HSV-1] VP22) is a varicella-zoster virus (VZV) tegument protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular proteins interacting with ORF9p that might be important for this function. We found 31 ORF9p interaction partners, among which was AP1M1, the µ subunit of the adaptor protein complex 1 (AP-1). AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo proteins between endosomes and the trans-Golgi network via clathrin-coated vesicles. We confirmed that AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs was individually impaired and identified leucine 231 in ORF9p to be critical for the interaction with AP-1. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus. Leucine 231 is part of a dileucine motif conserved among alphaherpesviruses, and we showed that VP22 of Marek's disease virus and HSV-2 also interacts with AP-1. This indicates that the function of this interaction in secondary envelopment might be conserved as well.IMPORTANCE Herpesviruses are responsible for infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and strokes. The constant emergence of viral strains resistant to classical antivirals (mainly acyclovir and its derivatives) pleads for the identification of new targets for future antiviral treatments. Cellular adaptor protein (AP) complexes have been implicated in the correct addressing of herpesvirus glycoproteins in infected cells, and the discovery that a major constituent of the varicella-zoster virus tegument interacts with AP-1 reveals a previously unsuspected role of this tegument protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets.

Study of intact virus-like particles of human papillomavirus by capillary electrophoresis.

Virus-like particles of human papillomavirus (HPV-VLP), resulting from the self-assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil(®) and Cervarix(®), are based on HPV-VLP L1. Analytical techniques currently used to characterize HPV-VLP, such as SDS-PAGE, Western blot, ELISA, are time-consuming and semiquantitative. In this study, CE was evaluated for the analysis of intact HPV16-VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH, and detergent additives were investigated. Reproducible HPV-VLP analysis in CE was achieved using poly(ethylene oxide)-coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV-VLP detection in less than 10 min (migration times RSD: 1.6%). The identity of HPV-VLP peak was confirmed by comparison with a sample obtained from a wild-type baculovirus and with VLP-based vaccine, Gardasil(®) , after adjuvant dissolution. Finally, we applied the developed methodology to VLP-based vaccines, demonstrating that CE could be successfully used for vaccine quality control.

Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus.

BACKGROUND: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. METHODS: The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfer method. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. RESULTS: All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. CONCLUSIONS: Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. GENERAL SIGNIFICANCE: This finding suggests that Der p 1 may be valuable target against mites.

Spatio-temporal dynamics of ß-tubulin isotypes during the development of the sensory auditory organ in rat.

There are different ß-tubulin isoforms in microtubules of vertebrate tissues. However, their functional significance is still largely unknown. In the present study, we investigated the localization of five ß-tubulin isotypes (ß1-5) within the hearing organ during development in rat. By using confocal microscopy, we showed that with the exception of the ß3-tubulin isoform that was specific to nerve fibres, all the different ß-tubulin isoforms were mainly present in the supporting cells. Contrary to ß1-4-tubulins, we also found that the ß5-tubulin isoform appeared only at a key stage of the post-natal development in specific cell types (pillar cells and Deiters' cells). By using transmission electron microscopy, we revealed further that this developmental stage coincided with the formation of two separate bundles of microtubules from a unique one in these supporting cells. Together, these results suggest that the ß5-tubulin isoform might be involved in the generation of new microtubule bundles from a pre-existing one.

Interaction studies between human papillomavirus virus-like particles and laminin 332 by affinity capillary electrophoresis assisted by bio-layer interferometry.

Human papillomavirus (HPV) interacts, in vitro, with laminin 332 (LN332), a key component of the extracellular matrix. In this study, we performed bio-layer interferometry (BLI) and affinity capillary electrophoresis (ACE) to investigate the binding properties of this interaction. Virus-like particles (VLPs), composed of the HPV16 L1 major capsid protein, were used as HPV model and LN332 as the VLPs binding partner. Using BLI, we quantitatively determined the kinetics of the interaction, via the measurement of VLP binding and release from LN332 immobilized onto the surface of aminopropylsilane biosensors. We found an averaged kon of 1.74 x 104 M-1s-1 and an averaged koff of 1.50 x 10-4 s-1. Furthermore, an ACE method was developed to study the interaction under physiological conditions, where the interactants are moving freely in solution, without any fluorescence labeling. Specifically, a constant amount of HPV16-VLPs was preincubated with increasing LN332 concentrations and then the samples were injected in the capillary electrophoresis instrument. A shift in the migration time of the HPV16-VLP/LN332 complexes, carrying an increasing number of LN332 molecules bound per VLP, was observed. The mobility of the complexes was found to decrease with increasing LN332 concentrations in the sample. It was used to quantify stability constant. From BLI and ACE approaches, we reported an apparent equilibrium dissociation constant in the nanomolar range (8.89 nM and 17.7 nM, respectively) for the complex between HPV16-VLPs and LN332.

Differentiation of Boettcher's cells during postnatal development of rat cochlea.

Contrary to the highly specialized epithelial cells of the mammalian auditory organ, little is known about the surrounding cells and, in particular, Boettcher's cells (BC). Our morphological studies show that, in rats, these cells began their differentiation around postnatal day 8 (P8) reaching maturity around P20, when they are completely covered by Hensen's and Claudius' cells. Tight junctions were noted near the apex of BC, providing that they were in direct contact with the endolymphatic space, between approximately P8 and P16. We observed gap junctions between BC and adjacent cells before the end of the covering process suggesting the additional involvement of BC in potassium recycling into the endolymph. Adherens junctions were also seen between BC throughout their maturation. Importantly, we noticed cytoplasmic secretory granules and an accumulated material, probably a secretion, in the intercellular space, between P8 and P25. These results indicate that BC could basally take part in the secretion of the extracellular matrix of the basilar membrane. Finally, we show that the basolateral interdigitations of BC are longer and more tighlty grouped at maturity and harbour urea transporters as early as P18. Our observations thus support the view that BC perform several functions.

Evidence for a partial epithelial-mesenchymal transition in postnatal stages of rat auditory organ morphogenesis.

The epithelial-mesenchymal transition (EMT) plays a crucial role in the differentiation of many tissues and organs. So far, an EMT was not detected in the development of the auditory organ. To determine whether an EMT may play a role in the morphogenesis of the auditory organ, we studied the spatial localization of several EMT markers, the cell-cell adhesion molecules and intermediate filament cytoskeletal proteins, in epithelium of the dorsal cochlea during development of the rat Corti organ from E18 (18th embryonic day) until P25 (25th postnatal day). We examined by confocal microscopy immunolabelings on cryosections of whole cochleae with antibodies anti-cytokeratins as well as with antibodies anti-vimentin, anti-E-cadherin and anti-ß-catenin. Our results showed a partial loss of E-cadherin and ß-catenin and a temporary appearance of vimentin in pillar cells and Deiters between P8 and P10. These observations suggest that a partial EMT might be involved in the remodelling of the Corti organ during the postnatal stages of development in rat.

Ultrastructure of sonic muscles of piranhas (Serrasalmidae).

Among piranhas, different species are able to produce sounds but not all of them use the same mechanism. In all species, the sound-producing muscle originates on the second vertebra, but the insertion differs. Pygopristis denticulata can produce two kinds of pulsed sounds emitted in trains. Its sound production mechanism is mainly based on a muscle bundle that inserts between the two first ribs. In Catoprion mento, the anterior part of the sonic muscle inserts directly on the swim bladder. The most derived species (Serrasalmus and Pygocentrus) make all harmonic tonal sounds. Their sonic muscles constitute a single functional unit transversally surrounding the swim bladder. This study aims to study the ultrastructure of sonic muscles in nine species from these four genera. Epaxial muscles were compared with sonic muscles, and the sonic muscles of the different species were compared between them. Results confirmed ultrastructure modifications in the sonic muscles in comparison to epaxial muscles. Fibers of the sonic muscle are thinner and possess a thicker subsarcolemmal ring housing mitochondria. In sonic muscles, myofibrils are also proportionally less abundant, and their sarcomeres are longer and thinner. Some of these differences allows to separate basal species (e.g., P. denticulata) from more derived species (genera Pygocentrus and Serrasalmus) and supposedly support the observed differences in the acoustic abilities.

Sox10 promotes the survival of cochlear progenitors during the establishment of the organ of Corti.

Transcription factors of the SoxE family are critical players that underlie various embryological processes. However, little is known about their function during inner ear development. Here, we show that Sox10 is initially expressed throughout the otic vesicle epithelium and becomes later restricted to supporting cells as cell differentiation proceeds in the organ of Corti. Morphological analyses of Sox10 mutant mice reveal a significant shortening of the cochlear duct likely resulting from the progressive depletion of cochlear progenitors. While Sox10 appears dispensable for the differentiation and patterning of the inner ear prosensory progenitors, our data support a critical role for this transcription factor in the promotion of their survival. We provide genetic evidences that Sox10, in a concentration-dependant manner, could play a role in the regulation of Jagged1, a gene known to be important for inner ear prosensory development. Together, our results demonstrate that Sox10 regulates the biology of early cochlear progenitors during inner ear development, but, in contrast to neural crest-derived cells, this transcription factor is dispensable for their differentiation. Evidence also suggests that this effect occurs via the activation of the Jagged1 gene.

Glial but not neuronal development in the cochleo-vestibular ganglion requires Sox10.

The cochleo-vestibular ganglion contains neural crest-derived glial cells and sensory neurons that are derived from the neurogenic otic placode. Little is known about the molecular mechanisms that regulate the tightly orchestrated development of this structure. Here, we report that Sox10, a high-mobility group DNA-binding domain transcription factor that is required for the proper development of neural crest cell derivatives, is specifically expressed in post-migratory neural crest cells in the cochleo-vestibular ganglion. Using Sox10-deficient mice, we demonstrate that this transcription factor is essential for the survival, but not the generation, of the post-migratory neural crest cells within the inner ear. In the absence of these neural crest-derived cells, we have investigated the survival of the otocyst-derived auditory neurons. Surprisingly, auditory neuron differentiation, sensory target innervation and survival are conserved despite the absence of glial cells. Moreover, brain-derived neurotrophic factor expression is increased in the hair cells of Sox10-deficient mice, a compensatory mechanism that may prevent spiral ganglion neuronal cell death. Taken together, these data suggest that in the absence of neural crest-derived glial cells, an increase trophic support from hair cells promotes the survival of spiral ganglion neurons in Sox10 mutant mice.

Ultrastructural detection of nucleic acids within heat shock-induced perichromatin granules of HeLa cells by cytochemical and immunocytological methods.

The perichromatin granules (PGs) are enigmatic structures of the cell nucleus. The major drawbacks for a biological study are their rare occurrence and their small size in normal conditions. As heat shock has been shown to increase their number, we applied a hyperthermal shock on HeLa cells to investigate the nucleic acid content of PGs by means of cytochemical and immunocytological approaches. These heat shock-induced PGs (hsiPGs) appeared as clusters organized in the form of honeycomb structures and were always associated with some blocks of condensed chromatin, such as the perinucleolar chromatin shell. A stalk connecting the hsiPG to the chromatin could be observed. For the detection of RNA, we applied an immunocytological method involving two anti-RNA antibodies and quantified the gold labelling obtained. The results clearly revealed that hsiPGs contained RNA. Regarding to the detection of DNA, we used three different methods followed by quantitative analyses. The results seemed to indicate that a small amount of DNA was present in hsiPGs. Together, these findings suggest that hsiPGs might be RNP structures associated with particular regions of DNA.

Localization of Nopp140 within mammalian cells during interphase and mitosis.

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.

Early identification of inner pillar cells during rat cochlear development.

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far from being elucidated. Here, we examine its spatio-temporal development in rats from embryonic day 16 (E16) to E19 by using cytochemical and immunocytochemical methods at the light- and electron-microscope levels. We demonstrate that the organ of Corti develops from a non-proliferating cell zone that is located in the junctional region between two edges of the dorsal epithelium of the cochlear duct. We also reveal that the first cells to develop in this zone are the inner pillar cells, a particular type of non-sensory supporting cell, which arise in the base of the cochlear duct at the boundary between the two ridges at E16. Cell differentiation in this prosensory region continues according to a base-to-apex gradient; the inner hair cells appear in the greater epithelial ridge at E17 and the outer hair cells in the lesser epithelial ridge at E18. At E19, the various cell types of the organ of Corti are in place. Finally, we show that unlike the development of all the supporting cell types of the organ of Corti, the development of inner pillar cells within the prosensory domain seems not to involve Notch1 activation. These results highlight the central role that the inner pillar cells probably play in the development of the organ of Corti.

Cochlear connexin 30 homomeric and heteromeric channels exhibit distinct assembly mechanisms.

Many of the mutations in GJB2 and GJB6, which encode connexins 26 and 30 (Cx26 and Cx30), impair the formation of membrane channels and cause autosomal syndromic and non-syndromic hearing loss. In cochlear non-sensory supporting cells, Cx26 and Cx30 form two types of homomeric and heteromeric gap junctions. The biogenesis processes of these channels occurring in situ remain largely unknown. Here we show that Cx30 homomeric and Cx26/Cx30 heteromeric gap junctions exhibit distinct assembly mechanisms in the cochlea. When expressed as homomeric channels, Cx30 preferentially interacts with ß-actin in the peripheral non-junctional membrane region, called perinexus, and strongly relies on the actin network for gap junction plaque assembly. In contrast, we found that Cx26/Cx30 heteromeric gap junction plaques are devoid of perinexus and associated actin network, and resist to actin-depolymerizating drug. This supports that Cx26/Cx30 oligomers could be directly delivered from the interior of the cell to the junctional plaque. Altogether, our data provide a novel insight in homomeric and heteromeric gap junction plaque assembly in the cochlea.