Annie Cavalier

The Inner Mitochondrial Membrane Has Aquaporin-8 Water Channels and Is Highly Permeable to Water

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.

Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract

A remarkable amount, of water is transported in the gastrointestinal (GI) organs to fulfil the secretory and absorptive functions of the GI tract. However, the molecular basis of water movement in the GI epithelial barriers is still poorly known. Important clues about the mechanisms by which water is transported in the GI tract were provided by the recent identification of multiple aquaporin water channels expressed in GI tissues. Here we define the mRNA and protein expression and the cellular and subcellular distribution of aquaporin-8 (AQP8) in the rat GI tract. By semi-quantitative RT-PCR the AQP8 mRNA was detected in duodenum, proximal jejunum, proximal colon, rectum, pancreas and liver and, to a lesser extent, in stomach and distal colon. Immunohistochemistry using affinity-purified antibodies revealed AQP8 staining in the absorptive epithelial cells of duodenum, proximal jejunum, proximal colon and rectum where labeling was largely intracellular and confined to the subapical cytoplasm. Confirming previous results, AQP8 staining was seen at the apical pole of pancreatic acinar cells. Interestingly, both light and immunoelectron microscopy analyses showed AQP8 reactivity in liver where labeling was associated to hepatocyte intracellular vesicles and over the plasma membrane delimiting the bile canaliculi. A complex pattern was observed by immunoblotting with total membranes of the above GI organs incubated with affinity-purified anti-AQP8 antibodies which revealed multiple bands with molecular masses ranging between 28 and 45 kDa. This immunoblotting pattern was not modified after deglycosylation with N-glycosidase F except the 34-kDa band of liver that, as already reported, was partially down-shifted to 28 kDa. No bands were detected after preadsorption of the anti-AQP8 antibodies with the immunizing peptide. The cellular and subcellular distribution of AQP8 suggest physiological roles for this aquaporin in the absorption of water in the intestine and the secretion of bile and pancreatic juice in liver and pancreas, respectively. The large intracellular expression of AQP8 may indicate its recycling between the cytoplasmic compartment and the plasma membrane. The cytoplasmic localization observed may also relate to the involvement of AQP8 in processes of intracellular osmoregulation.

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.

Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae

Seven hysterectomy derived piglets were repeatedly challenged with Mycoplasma hyopneumoniae during the first week of life. Samples of trachea, bronchi and lung tissue collected 2-11 weeks post-inoculation (p.i.) were examined using light and electron microscopy. Autoradiography was used to study in more detail the site of M. hyopneumoniae multiplication. Gross lesions were observed in lung tissue and were characterized by hyperplasia of the epithelium and an increased mononuclear cell accumulation in perivascular and peribronchiolar areas. Mild lesions of the trachea and the bronchi, including epithelial hyperplasia and infiltration of the lamina propria by inflammatory cells, were noted. Electron microscopy showed that, 2-6 weeks p.i., changes in the mid-trachea and bronchi surface consisted of the loss of cilia. Mycoplasmas covered tufts of cilia remaining on the epithelial cell surface. Scanning and transmission electron micrographs showed that they were predominantly found closely associated with the top of cilia. No specialized terminal structure could be seen and no mycoplasma cells were identified lying free in the lumen nor in close contact with the plasma membrane of cells or microvilli. Some fine fibrils radiating from one mycoplasma to another or to cilia were seen at higher magnification by scanning electron microscopy. Six to eleven weeks p.i., a disrupted epithelial surface lacking cilia was observed. Cells were desquamated and shed into the lumen with cellular remains containing droplets of mucus. Autoradiography revealed that label corresponded to the observed mycoplasma distribution. At the top of cilia, a high density of labeling was visible in the zone of high mycoplasma concentration. Therefore, incorporation of the label in the mycoplasma is proof or their multiplication in the trachea. The intimate association between the mycoplasma and cilia may be an important factor in the pathogenesis of the disease caused by M. hyopneumoniae (swine enzootic pneumonia).

Supramolecular organization of the yeast F1Fo‐ATP synthase

Background information. The yeast mitochondrial F1Fo‐ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and Pi. For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology.

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

The 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.

Aquaglyceroporins, one channel for two molecules.

In the light of the recently published structure of GlpF and AQP1, we have analysed the nature of the residues which could be involved in the formation of the selectivity filter of aquaporins, glycerol facilitators and aquaglyceroporins. We demonstrate that the functional specificity for major intrinsic protein (MIP) channels can be explained on one side by analysing the polar environment of the residues that form the selective filter. On the other side, we show that the channel selectivity could be associated with the oligomeric state of the membrane protein. We conclude that a non-polar environment in the vicinity of the top of helix 5 could allow aquaglyceroporins and GlpF to exist as monomers within the hydrophobic environment of the membrane.

AQUAPORIN-RELATED PROTEINS IN THE FILTER CHAMBER OF HOMOPTERAN INSECTS

Abstract.In the Homopteran order of insects, the plant xylem feeders exhibit a highly differentiated part of their digestive tract known as the filter chamber. In this tissue, water crosses plasma membranes through a transepithelial osmotic gradient. In previous studies on the filter chamber of Cicadella viridis, we purified and characterized from the plasma membranes a 25 kDa protein that we demonstrated to be an aquaporin (or water channel, member of the major intrinsic protein family, a group of membrane channels for small solutes). We called this protein AQPcic for Cicadella aquaporin. In the present study, we used polyclonal antibody anti-AQPcic in Western blotting and immunocytochemical analysis of the intestinal tract of Cercopis sanguinolenta, Philaenus spumarius, Aphrophora alni (Cercopidae), Euscelidius variegatus, and Scaphoideus titanus (Jassidae). Western blotting experiments revealed that immunologically related AQPcic proteins are found in those species. The molecular weight of these proteins is 15–26 kDa. Immunocytochemical studies on ultrathin filter-chamber sections revealed that the anti-AQPcic antibody systematically labelled the membrane microvilli of epithelial cells. A good correlation thus exists between the physiology of these cells and the presence of aquaporin-related proteins in their membranes.

Structural and biochemical observations on specialized membranes of the “filter chamber”, a water‐shunting complex in sap‐sucking homopteran insects

Many homopteran insects feed on plant sap which contains solutes in very low concentration. Their digestive tract presents a complex called the “filter chamber” where the excess dietary water is believed to flow directly from the initial part of the midgut to the terminal part of the midgut and the proximal reions of the Malpighian tubules.

The use of in situ hybridization to study the transgene pathway following cellular transfection with cationic phosphonolipids

Gene therapy is a promising field of research and biotechnological development. Considering their safety and non-immunogenicity, cationic lipids are widely used for gene transfer in vitro and show promise for in vivo gene transfer applications. However, a better understanding of the mechanisms by which transfection occurs and the limiting steps in cellular transfer of foreign DNA are critical for significant improvements of gene transfer. In this work, we have traced the plasmid DNA into human hematopoietic cell line (K562) using the in situ hybridization method in order to define the main difficulties in transfection and to design new agents better adapted to cellular constraints. In this hematopoietic cell line, after showing the efficiency of our synthetic vectors and optimizing their formulation, we observed that only 5 h after transfection the nucleus to cytoplasm signal ratio was three to one, whereas at 24 h it was one to one. In parallel, the level of the reporter protein strongly increased between these times. Those results emphasize the rapidity of transfection and lead one to imagine chemical modifications adjusted to the environment.