The inner mitochondrial membrane has aquaporin-8 water channels and is highly permeable to water.

International audience; Mitochondria are remarkably plastic organelles constantly changing their shape to fulfil their various functional activities. Although the osmotic movement of water into and out of the mitochondrion is central for its morphology and activity, the molecular mechanisms and the pathways for water transport across the inner mitochondrial membrane (IMM), the main barrier for molecules moving into and out of the organelle, are completely unknown. Here, we show the presence of a member of the aquaporin family of water channels, AQP8, and demonstrate the strikingly high water permeability (Pf) characterizing the rat liver IMM. Immunoblotting, electron microscopy, and biophysical studies show that the largest mitochondria feature the highest AQP8 expression and IMM Pf. AQP8 was also found in the mitochondria of other organs, whereas no other known aquaporins were seen. The osmotic water transport of liver IMM was partially inhibited by the aquaporin blocker Hg2+, while the related activation energy remained low, suggesting the presence of a Hg2+-insensitive facilitated pathway in addition to AQP8. It is suggested that AQP8-mediated water transport may be particularly important for rapid expansions of mitochondrial volume such as those occurring during active oxidative phosphorylation and those following apoptotic signals

An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus.

International audienceThe genome of influenza A viruses (IAV) is split into eight viral RNAs (vRNAs) that are encapsidated as viral ribonucleoproteins. The existence of a segment-specific packaging mechanism is well established, but the molecular basis of this mechanism remains to be deciphered. Selective packaging could be mediated by direct interaction between the vRNA packaging regions, but such interactions have never been demonstrated in virions. Recently, we showed that the eight vRNAs of a human H3N2 IAV form a single interaction network in vitro that involves regions of the vRNAs known to contain packaging signals in the case of H1N1 IAV strains. Here, we show that the eight vRNAs of an avian H5N2 IAV also form a single network of interactions in vitro, but, interestingly, the interactions and the regions of the vRNAs they involve differ from those described for the human H3N2 virus. We identified the vRNA sequences involved in five of these interactions at the nucleotide level, and in two cases, we validated the existence of the interaction using compensatory mutations in the interacting sequences. Electron tomography also revealed significant differences in the interactions taking place between viral ribonucleoproteins in H5N2 and H3N2 virions, despite their canonical '7 + 1' arrangement

Visualizing Compaction of Polysomes in Bacteria.

International audienceDuring protein synthesis, many translating ribosomes are bound together with an mRNA molecule to form polysomes (or polyribosomes). While the spatial organization of bacterial polysomes has been well studied in vitro, little is known about how they cluster when cellular conditions are highly constrained. To better understand this, we used electron tomography, template matching, and three-dimensional modeling to analyze the supramolecular network of ribosomes after induction of translational pauses. In Escherichia coli, we overexpressed an mRNA carrying a polyproline motif known to induce pausing during translation. When working with a strain lacking transfer-messenger RNA, the principle actor in the "trans-translation" rescuing system, the cells survived the hijacking of the translation machinery but this resulted in a sharp modification of the ribosomal network. The results of our experiments demonstrate that single ribosomes are replaced with large amounts of compacted polysomes. These polysomes are highly organized, principally forming hairpins and dimers of hairpins that stack together. We propose that these spatial arrangements help maintain translation efficiency when the rescue systems are absent or overwhelmed

Crystal Structure of Orthorhombic {bis-[(pyridin-2-yl)methyl](3,5,5,5-tetrachloropentyl)amine-κ\u3csup\u3e3\u3c/sup\u3e\u3cem\u3eN,N\u27,N\u27\u27\u3c/em\u3e}chloridocopper(II) Perchlorate

In the title compound, [CuCl(C17H19Cl4N3)]ClO4, the CuII ion adopts a distorted square-planar geometry defined by one chloride ligand and the three nitro­gen atoms from the bis­[(pyridin-2-yl)meth­yl](3,5,5,5-tetra­chloro­pent­yl)amine ligand. The perchlorate counter-ion is disordered over three sets of sites with refined occupancies 0.0634 (17), 0.221 (16) and 0.145 (7). In addition, the hetero-scorpionate arm of the bis­[(pyridin-2-yl)meth­yl](3,5,5,5-tetra­chloro­pent­yl)amine ligand is disordered over two sets of sites with refined occupancies 0.839 (2) and 0.161 (2). In the crystal, weak Cu⋯Cl inter­actions between symmetry-related mol­ecules create a dimerization with a chloride occupying the apical position of the square-pyramidal geometry typical of many copper(II) chloride hetero-scorpionate complexes

A supramolecular assembly formed by influenza A virus genomic RNA segments

The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common ‘transition zone’ located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5′ region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals

Observation of Membrane Proteins In Situ: AQPcic, the Insect Aquaporin Example

Aquaporins are water-selective channels widely distributed in prokaryotes, plants, and animals. Looking for the presence of a water channel in the filter chamber (FC) of a homopteran insect (Cicadella viridis), we conducted an electron microscopic study. On thin sections, FC displays thin epithelia with developed basal membrane folds (BMFs). Freeze fracture performed on FC shows an amazing network of intramembrane particles. Epithelial cell membranes were purified and observed by negative staining for control purity. Membrane solubilisation followed by PAGE showed that a 25-kDa polypeptide (P25) is the major protein constituent. Using a specific antibody, we located P25 on thin sections on the microvilli and on BMFs of the epithelial cells. Immunogold localisation of P25 on negatively stained membranes and examination of Pt/C shadowed membranes demonstrated that P25 has an asymmetric insertion within the membrane. cDNA cloning and heterologous expression confirmed that P25 is an aquaporin; thus, we called it AQPcic. The native state of crystallisation of this aquaporin in the membrane appeared to be unique and favourable for a structural investigation by negative staining, cryo-electron microscopy, and image processing. We demonstrated that, in the native membrane, AQPcic is a homotetramer forming a regular two-dimensional array

Discrepancies between creatinine-based and cystatin C-based equations in estimating prevalence of stage 3 chronic kidney disease in an elderly population.

Background . The prevalence of stage 3 chronic kidney disease (CKD) is increasing, calculated using the modification of diet in renal disease (MDRD) study equation for estimating glomerular filtration rate (GFR). Cystatin C-based equations are also being used to estimate GFR. Using creatinine-based and cystatin C-based equations, the aim of our study was to measure the difference in prevalence of stage 3 CKD in a population. Methods . CKD screening is organized in the Province of Liege, Belgium. On a voluntary basis, people aged between 45 and 75 years are invited for screening. GFR is estimated using the MDRD study equation and by the three recent cystatin C-based equations proposed by Levey's group. The Levey 1 equation is based on cystatin C only and the Levey 2 equation on cystatin C corrected for age and sex. The Levey 3 equation combines cystatin C, creatinine, age and sex. Results . The population screened comprised 754 people. Cystatin C is highly correlated with creatinine (r = 0.6196, p<0.0001). Prevalence of stage 3 CKD when GFR is estimated by the MDRD equation study is 17.2 %, which is significantly and much higher than the prevalence obtained when cystatin C-based equations are used. Indeed, prevalence is 2 %, 3.3 % and 5.8 % with the Levey 1, 2 and 3 equations, respectively. Conclusions . The prevalence of stage 3 CKD varies strongly following the method used for estimating GFR, creatinine-based or cystatin C-based equations. Such discrepancies must be confirmed and explained in additional studies using GFR measured with a reference method