Hereditary hearing loss affects about 1 per 1000 children. Mutations in GJB2, which encodes the connexin 26 protein (Cx26) involved in cochlear homeostasis, are found in about 50% of patients with ...autosomal recessive non-syndromic hearing loss. Deciphering the trafficking pathway of cochlear Cx26 in situ should represent an advance in understanding the pathogenic significance of many of these mutations. Connexins trafficking and delivery to lipid raft-associated gap junction plaques usually requires successively microtubule and actin networks. Here we show that cochlear Cx26 exhibits an unusual trafficking pathway. We observed that Cx26 assembly occurs in non-lipid raft membrane domains and that junctional plaques are devoid of actin and associated zonula occludens proteins. Using cytoskeleton-disrupting drugs in organotypic culture, we found that cochlear Cx26 gap junction assembly requires microtubules but not actin filaments. Altogether, our data provide an unexpected insight into Cx26 trafficking pathway and gap junction assembly in the cochlea.
•Cx26 gap junctions are devoid of cortical actin and associated ZOs in the cochlea.•Cochlear Cx26 assembles into non-lipid raft gap junction plaques.•Cochlear Cx26 gap junction assembly requires microtubules but not the actin network.
The cerebral cortex contains layers of neurons sequentially generated by distinct lineage-related progenitors. At the onset of corticogenesis, the first-born progenitors are apical progenitors (APs), ...whose asymmetric division gives birth directly to neurons. Later, they switch to indirect neurogenesis by generating intermediate progenitors (IPs), which give rise to projection neurons of all cortical layers. While a direct lineage relationship between APs and IPs has been established, the molecular mechanism that controls their transition remains elusive. Here we show that interfering with codon translation speed triggers ER stress and the unfolded protein response (UPR), further impairing the generation of IPs and leading to microcephaly. Moreover, we demonstrate that a progressive downregulation of UPR in cortical progenitors acts as a physiological signal to amplify IPs and promotes indirect neurogenesis. Thus, our findings reveal a contribution of UPR to cell fate acquisition during mammalian brain development.
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•The UPR is a homeostat regulating the specification of cortical stem cells•Elp3 loss in cortical stem cells triggers UPR by decreasing codon translation rates•Gradual UPR suppression promotes the switch from direct to indirect neurogenesis
Laguesse, Creppe et al. demonstrate that the unfolded protein response (UPR) contributes to neurogenesis in the developing cerebral cortex. Depletion of the Elongator complex component Elp3 triggers the UPR through interference with codon translation speed. UPR activation impairs the balance between direct and indirect neurogenesis, leading to premature neuron generation.
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.
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•Pejvakin-deficient mice and humans are hypervulnerable to sound exposure•Oxidative stress induces a pejvakin-dependent proliferation of peroxisomes•Peroxisome proliferation contributes to the physiological response to sound exposure•Pjvk gene transfer can rescue auditory dysfunction in Pjvk−/− mice
Hypervulnerability to sound exposure in pejvakin-deficient mice results from impaired adaptive proliferation of peroxisomes in response to the oxidative stress. In wild-type mice, loud sounds elicit adaptive peroxisomal proliferation.
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
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.
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.
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.
The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.
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•Turtle cells have a nucleolus composed of a fibrillar and a granular zone.•Fibrillar zones present dark strands positive for NOR and DNA.•Ribosome formation in turtle nucleolus seems ...less structured.
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.
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.
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.
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.
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.
This finding suggests that Der p 1 may be valuable target against mites.
•We elicited the activation mechanism of proteases in D. pteronyssinus.•Der p 1 is the major activator of digestive mite cysteine and serine proteases.•Der p 1 (recombinant or natural) cleaves proDer p 1, 3, 6 and probably proDer p 9.•The propeptide inhibits the proteolytic activity of Der p 6.•As Der p 1, Der p 6 chymotrypsin is present in the mite gut.
Gut content examination and trophic markers (fatty acids, stable isotopes of C and N) were combined to delineate the diet of the dominant species of amphipods from Mediterranean Posidonia oceanica ...seagrass meadows and to highlight trophic diversity among this community. Our results indicate that, although all dominant species heavily relied on macroalgal epiphytes, considerable interspecific dietary differences existed. Carbon stable isotope ratios notably showed that some of the amphipod species favored grazing on epiphytes from leaves or litter fragments (Apherusa chiereghinii, Aora spinicornis, Gammarus aequicauda), while others such as Dexamine spiniventris preferred epiphytes from rhizomes. The remaining amphipods (Caprella acanthifera, Ampithoe helleri and Gammarella fucicola) readily consumed both groups. In addition, SIAR modeling suggested that most species had a mixed diet, and relied on several food items. Fatty acid analysis and gut contents revealed that contributions of microepiphytic diatoms and of benthic and suspended particulate organic matter to the diet of amphipods were anecdotal. None of the examined species seemed to graze on their seagrass host low 18:2(n‐6) and 18:3(n‐3) fatty acids contents, but Gammarus aequicauda partly relied on seagrass leaf detritus, as demonstrated by the lesser ¹³C‐depletion of their tissues. Overall, our findings suggest that amphipods, because of their importance in the transfer of organic matter from primary producers and detritus to higher rank consumers, are key items in P. oceanica‐associated food webs.
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-β-catenin. Our results showed a partial loss of E-cadherin and β-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.
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