Meisosomes, folded membrane microdomains between the apical extracellular matrix and epidermis

Apical extracellular matrices (aECMs) form a physical barrier to the environment. In Caenorhabditis elegans, the epidermal aECM, the cuticle, is composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Here, we show that in mutants lacking furrows, the normal intimate connection between the epidermis and the cuticle is lost, specifically at the lateral epidermis, where, in contrast to the dorsal and ventral epidermis, there are no hemidesmosomes. At the ultrastructural level, there is a profound alteration of structures that we term 'meisosomes,' in reference to eisosomes in yeast. We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, alternately filled with cuticle. We propose that just as hemidesmosomes connect the dorsal and ventral epidermis, above the muscles, to the cuticle, meisosomes connect the lateral epidermis to it. Moreover, furrow mutants present marked modifications of the biomechanical properties of their skin and exhibit a constitutive damage response in the epidermis. As meisosomes co-localise to macrodomains enriched in phosphatidylinositol (4,5) bisphosphate, they could conceivably act, like eisosomes, as signalling platforms, to relay tensile information from the aECM to the underlying epidermis, as part of an integrated stress response to damage

Altimetry for the future: Building on 25 years of progress

In 2018 we celebrated 25 years of development of radar altimetry, and the progress achieved by this methodology in the fields of global and coastal oceanography, hydrology, geodesy and cryospheric sciences. Many symbolic major events have celebrated these developments, e.g., in Venice, Italy, the 15th (2006) and 20th (2012) years of progress and more recently, in 2018, in Ponta Delgada, Portugal, 25 Years of Progress in Radar Altimetry. On this latter occasion it was decided to collect contributions of scientists, engineers and managers involved in the worldwide altimetry community to depict the state of altimetry and propose recommendations for the altimetry of the future. This paper summarizes contributions and recommendations that were collected and provides guidance for future mission design, research activities, and sustainable operational radar altimetry data exploitation. Recommendations provided are fundamental for optimizing further scientific and operational advances of oceanographic observations by altimetry, including requirements for spatial and temporal resolution of altimetric measurements, their accuracy and continuity. There are also new challenges and new openings mentioned in the paper that are particularly crucial for observations at higher latitudes, for coastal oceanography, for cryospheric studies and for hydrology. The paper starts with a general introduction followed by a section on Earth System Science including Ocean Dynamics, Sea Level, the Coastal Ocean, Hydrology, the Cryosphere and Polar Oceans and the ‘‘Green” Ocean, extending the frontier from biogeochemistry to marine ecology. Applications are described in a subsequent section, which covers Operational Oceanography, Weather, Hurricane Wave and Wind Forecasting, Climate projection. Instruments’ development and satellite missions’ evolutions are described in a fourth section. A fifth section covers the key observations that altimeters provide and their potential complements, from other Earth observation measurements to in situ data. Section 6 identifies the data and methods and provides some accuracy and resolution requirements for the wet tropospheric correction, the orbit and other geodetic requirements, the Mean Sea Surface, Geoid and Mean Dynamic Topography, Calibration and Validation, data accuracy, data access and handling (including the DUACS system). Section 7 brings a transversal view on scales, integration, artificial intelligence, and capacity building (education and training). Section 8 reviews the programmatic issues followed by a conclusion

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Altimetry for the future: building on 25 years of progress

In 2018 we celebrated 25 years of development of radar altimetry, and the progress achieved by this methodology in the fields of global and coastal oceanography, hydrology, geodesy and cryospheric sciences. Many symbolic major events have celebrated these developments, e.g., in Venice, Italy, the 15th (2006) and 20th (2012) years of progress and more recently, in 2018, in Ponta Delgada, Portugal, 25 Years of Progress in Radar Altimetry. On this latter occasion it was decided to collect contributions of scientists, engineers and managers involved in the worldwide altimetry community to depict the state of altimetry and propose recommendations for the altimetry of the future. This paper summarizes contributions and recommendations that were collected and provides guidance for future mission design, research activities, and sustainable operational radar altimetry data exploitation. Recommendations provided are fundamental for optimizing further scientific and operational advances of oceanographic observations by altimetry, including requirements for spatial and temporal resolution of altimetric measurements, their accuracy and continuity. There are also new challenges and new openings mentioned in the paper that are particularly crucial for observations at higher latitudes, for coastal oceanography, for cryospheric studies and for hydrology. The paper starts with a general introduction followed by a section on Earth System Science including Ocean Dynamics, Sea Level, the Coastal Ocean, Hydrology, the Cryosphere and Polar Oceans and the “Green” Ocean, extending the frontier from biogeochemistry to marine ecology. Applications are described in a subsequent section, which covers Operational Oceanography, Weather, Hurricane Wave and Wind Forecasting, Climate projection. Instruments’ development and satellite missions’ evolutions are described in a fourth section. A fifth section covers the key observations that altimeters provide and their potential complements, from other Earth observation measurements to in situ data. Section 6 identifies the data and methods and provides some accuracy and resolution requirements for the wet tropospheric correction, the orbit and other geodetic requirements, the Mean Sea Surface, Geoid and Mean Dynamic Topography, Calibration and Validation, data accuracy, data access and handling (including the DUACS system). Section 7 brings a transversal view on scales, integration, artificial intelligence, and capacity building (education and training). Section 8 reviews the programmatic issues followed by a conclusion

CHARACTERIZATION OF MUSCARINIC BINDING SITES IN THE ADULT AND DEVELOPING RAT COCHLEA

International audienceThe maturation of the cholinergic innervation of the rat cochlea is associated with a transient increase in the muscarinic-receptor activated inositol phosphate synthesis. In order to investigate the mechanisms involved in this transient enhancement of the inositol phosphate response, the binding properties of the cochlear muscarinic receptors were studied during rat cochlear development. Incubating the membranes from 4-day-old, 12-day-old and adult cochleas with [3H]quinuclidinyl benzylate indicates that their respective, mean concentrations of cholinoceptors are 454 +/- 51 (+/- SEM), 39 +/- 2 and 42 +/- 3 fmol/mg of protein. The dissociation constants at equilibrium are 207 +/- 80, 42 +/- 7 and 28 +/- 3 pM for the binding sites of the 4-day-old, 12-day-old and adult cochleas, respectively. Pharmacological characterization of the binding, using selective antagonists, shows that M3 cholinoceptors are expressed in developing and adult cochleas. The data demonstrate that changes in muscarinic receptor affinity and number do not correlate with the previously observed peak of the inositol phosphate metabolism. The transient enhanced inositol phosphate response is therefore not due to changes in cholinoceptors, but probably due to alterations involving the intrinsic activity of the phospholipase C and/or the efficacy of coupling of the transduction system

Neuroprotective effect of riluzole in acute noise-induced hearing loss.

International audienceRiluzole has been reported to protect against the deleterious effect of cerebral ischemia by blocking glutamatergic neurotransmission. Here, we investigated whether acoustic trauma-induced cochlear excitotoxicity could be attenuated by riluzole. Cumulative intracochlear perfusion of riluzole completely abolished single-nerve fiber activity in the guinea pig cochlea and the compound action potential of the auditory nerve. Guinea pigs treated with riluzole (100 microM) showed significantly less hearing threshold shift than untreated guinea pigs, and presented no sign of dendritic damage in the cochlea observable by electron microscopy. When coapplied with glutamate, riluzole did not prevent glutamate-induced swelling of auditory nerve dendrites, suggesting that the protective effect of riluzole was mediated principally by inhibition of glutamate release from sensory inner hair cells

ACTIVATION OF MUSCARINIC CHOLINERGIC RECEPTORS STIMULATES INOSITOL PHOSPHATES SYNTHESIS IN THE DEVELOPING AVIAN COCHLEAR DUCT

International audienceWe previously reported that the inositol phosphates (IPs) synthesis is induced by muscarinic agonists in the rat cochlea and that this stimulation is maximal at postnatal day 12. This peak response is concomitant with the onset of the efferent synaptogenesis at the outer hair cell level. Whether the correlation between this neuronal plasticity and the enhanced IPs formation is unique to the rat or a general feature of the developing vertebrate cochlea is not known. To examine this question, we measured, in the presence of LiCI, the accumulation of (3H)-IPs induced by carbachol, in the developing chick cochlear duct during a period ranging from embryonic day (E) 8 to post-hatching day (P) 20. Carbachol (1 mM) causes a significant increase of IPs formation relative to basal values at all ages. This IPs accumulation is maximal at E8 (1854% of the basal level), then, rapidly decreases until P13 when it reaches a steady-state level of 294% of the basal level. Strikingly, this gradual decline in IPs formation is interrupted between El5 and El9, by a transient increase in IPs synthesis. This rise peaks at El6 with a stimulation value of 757% of the control level. This maximal stimulation is inhibited by atropine in a dose-dependent manner, as is the case at E9, suggesting the involvement of muscarinic receptors. Interestingly, the occurrence of the peak response is concomitant with the plastic events associated with the maturation of the efferent innervation of the cochlear duct. Thus, these results suggest that there may be a correlation between cochlear plasticity and enhanced IPs synthesis, which is not species-specific. The possible significance of the overall decrease in IPs formation, occurring during embryonic development , is discussed. The degradation of membrane phosphatidylinositol 4,5 biphosphate, by the enzyme phospholi-pase C, leads to the formation of diacylglycerol and inositol phosphates (IPs). Among these metabolites, diacylglycerol and inositol 1,4,5-trisphosphate are considered as second messengers. 4 The former directly activates the protein kinase C enzymes; the latter elicits a massive release of calcium from intracellular stores. This transduction system has been found to be driven by specific agonist-activated receptors such as muscarinic cholinergic receptors. 7 We previously reported that this transduction system is stimulated, in the rat cochlea, by muscarinic cholinergic agonists probably, via the activation of a M3 muscarinic receptor, s During the postnatal development of the mammalian cochlea, the muscarinic agonist-induced IPs formation is characterized by a peak around postnatal day 12. 3 This peak coincides with a time period during which plastic events lead to the setting up of the mature efferent innervation of the outer hair cells of the organ of Corti. I1 These efferent terminals are thought to be cholinergic. 6 Thus, it is conceivable that the IPs metabolism may play a role in cochlear neural plasticity. Whether this concomitance, between the increased IPs synthesis and the efferent synaptogenesis, is an overall developmental process in the vertebrate inner ear or is a specific feature of the rat cochlea remains to be investigated. To address this question, we studied the pattern of the phosphoinositide breakdown during the development of the chick basiler papilla. Although phylogenetically remote, the avian basilar papilla and the mammalian organ of Corti share some morphological homologies. They both possess, for instance, two types of hair cells lying on a basilar membrane and covered by a tector-ial membrane. The sensory hair cells are innervated, in both classes, by four different types of fibres, two of them belonging to the efferent systems and the other two to the efferent systems. II'16'23'25 Finally, physiological evidence, supporting the possibility of the presence of muscarinic receptors in the chick auditory organ, as this is the case in the rat cochlea, 3,8 i

Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response.

Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycoside neomycin. Similarly, ultrastructural analysis revealed structural alteration among hair cells within 15 minutes of neomycin exposure. Animals exposed to a low, 25-microM concentration of neomycin exhibited hair cells with swollen mitochondria, but little other damage. Animals treated with higher concentrations of neomycin (50-200 microM) had more severe and heterogeneous cellular changes, as well as fewer hair cells. Both necrotic-like and apoptotic-like cellular damage were observed. Quantitation of the types of alterations observed indicated that mitochondrial defects appear earlier and more predominantly than other structural alterations. In vivo monitoring demonstrated that mitochondrial potential decreased following neomycin treatment. These results indicate that perturbation of the mitochondrion is an early, central event in aminoglycoside-induced damage

The selective AMPA receptor antagonist GYKI 53784 blocks action potential generation and excitotoxicity in the guinea pig cochlea.

International audienceThe role of AMPA receptors in cochlear synaptic transmission and excitotoxicity was investigated by comparing the actions of a selective AMPA antagonist GYKI 53784 (LY303070) with additional AMPA/kainate antagonists, GYKI 52466 and DNQX, and the NMDA antagonist, D-AP5, in several electrophysiological, neurotoxicological and histochemical tests. GYKI 53784 had the same potency as DNQX and was 10 times more potent than GYKI 52466 in reducing auditory nerve activity. The NMDA antagonist D-AP5 had no effect on auditory nerve activity. When single-fiber activity was blocked with GYKI 53784, the effects of AMPA or kainate were also antagonized. GYKI 53784 completely blocked excitotoxicity (i.e. destruction of the afferent nerve endings) induced by AMPA and kainate. The histochemical detection of Co(2+) uptake was used to study Ca(2+) influx within the primary auditory nerve cells. Application of AMPA induced no significant Co(2+) uptake into the cells, suggesting that these receptors normally have a very low permeability to Ca(2+). Application of kainate induced significant Co(2+) uptake that was blocked by the AMPA receptor antagonist GYKI 53784 suggesting that kainate stimulated Ca(2+) entry through AMPA receptor channels. Results suggest that AMPA-preferring receptors are functionally located at the sensory cell-afferent synapse whereas NMDA and kainate receptors are not