Michael Rützler

Odor Coding in the Maxillary Palp of the Malaria Vector Mosquito Anopheles gambiae

BACKGROUND Many species of mosquitoes, including the major malaria vector Anopheles gambiae, utilize carbon dioxide (CO(2)) and 1-octen-3-ol as olfactory cues in host-seeking behaviors that underlie their vectorial capacity. However, the molecular and cellular basis of such olfactory responses remains largely unknown. RESULTS Here, we use molecular and physiological approaches coupled with systematic functional analyses to define the complete olfactory sensory map of the An. gambiae maxillary palp, an olfactory appendage that mediates the detection of these compounds. In doing so, we identify three olfactory receptor neurons (ORNs) that are organized in stereotyped triads within the maxillary-palp capitate-peg-sensillum population. One ORN is CO(2)-responsive and characterized by the coexpression of three receptors that confer CO(2) responses, whereas the other ORNs express characteristic odorant receptors (AgORs) that are responsible for their in vivo olfactory responses. CONCLUSIONS Our results describe a complete and highly concordant map of both the molecular and cellular olfactory components on the maxillary palp of the adult female An. gambiae mosquito. These results also facilitate the understanding of how An. gambiae mosquitoes sense olfactory cues that might be exploited to compromise their ability to transmit malaria.

Molecular characterization of the Aedes aegypti odorant receptor gene family

The olfactory‐driven blood‐feeding behaviour of female Aedes aegypti mosquitoes is the primary transmission mechanism by which the arboviruses causing dengue and yellow fevers affect over 40 million individuals worldwide. Bioinformatics analysis has been used to identify 131 putative odourant receptors from the A. aegypti genome that are likely to function in chemosensory perception in this mosquito. Comparison with the Anopheles gambiae olfactory subgenome demonstrates significant divergence of the odourant receptors that reflects a high degree of evolutionary activity potentially resulting from their critical roles during the mosquito life cycle. Expression analyses in the larval and adult olfactory chemosensory organs reveal that the ratio of odourant receptors to antennal glomeruli is not necessarily one to one in mosquitoes.

Molecular biology of insect olfaction:recent progress and conceptual models

Insects have an enormous impact on global public health as disease vectors and as agricultural enablers as well as pests and olfaction is an important sensory input to their behavior. As such it is of great value to understand the interplay of the molecular components of the olfactory system which, in addition to fostering a better understanding of insect neurobiology, may ultimately aid in devising novel intervention strategies to reduce disease transmission or crop damage. Since the first discovery of odorant receptors in vertebrates over a decade ago, much of our view on how the insect olfactory system might work has been derived from observations made in vertebrates and other invertebrates, such as lobsters or nematodes. Together with the advantages of a wide range of genetic tools, the identification of the first insect odorant receptors in Drosophila melanogaster in 1999 paved the way for rapid progress in unraveling the question of how olfactory signal transduction and processing occurs in the fruitfly. This review intends to summarize much of this progress and to point out some areas where advances can be expected in the near future.

Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin–Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein–protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t1/2 = 5.1 h and t1/2 = 4.4 h, respectively) compared to WT-AQP2 (t1/2 = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein–protein interactions.

Olfactory responses in a gustatory organ of the malaria vector mosquito Anopheles gambiae

The proboscis is an important head appendage in insects that has primarily been thought to process gustatory information during food intake. Indeed, in Drosophila and other insects in which they have been identified, most gustatory receptors are expressed in proboscis neurons. Our previous characterization of the expression of AgOR7, a highly conserved odorant receptor (OR) of the Afrotropical malaria vector mosquito Anopheles gambiae in the labellum at the tip of the proboscis was suggestive of a potential olfactory function in this mosquito appendage. To test this hypothesis, we used electrophysiological recording and neuronal tracing, and carried out a molecular characterization of candidate OR expression in the labellum of A. gambiae. These studies have uncovered a set of labial olfactory responses to a small spectrum of human-related odorants, such as isovaleric acid, butylamine, and several ketones and oxocarboxylic acids. Molecular analyses indicated that at least 24 conventional OR genes are expressed throughout the proboscis. Furthermore, to more fully examine AgOR expression within this tissue, we characterized the AgOR profile within a single labial olfactory sensillum. This study provides compelling data to support the hypothesis that a cryptic set of olfactory neurons that respond to a small set of odorants are present in the mouth parts of hematophagous mosquitoes. This result is consistent with an important role for the labellum in the close-range discrimination of bloodmeal hosts that directly impacts the ability of A. gambiae to transmit malaria and other diseases.

Vasopressin-independent targeting of aquaporin-2 by selective E-prostanoid receptor agonists alleviates nephrogenic diabetes insipidus

In the kidney, the actions of vasopressin on its type-2 receptor (V2R) induce increased water reabsorption alongside polyphosphorylation and membrane targeting of the water channel aquaporin-2 (AQP2). Loss-of-function mutations in the V2R cause X-linked nephrogenic diabetes insipidus. Treatment of this condition would require bypassing the V2R to increase AQP2 membrane targeting, but currently no specific pharmacological therapy is available. The present study examined specific E-prostanoid receptors for this purpose. In vitro, prostaglandin E2 (PGE2) and selective agonists for the E-prostanoid receptors EP2 (butaprost) or EP4 (CAY10580) all increased trafficking and ser-264 phosphorylation of AQP2 in Madin-Darby canine kidney cells. Only PGE2 and butaprost increased cAMP and ser-269 phosphorylation of AQP2. Ex vivo, PGE2, butaprost, or CAY10580 increased AQP2 phosphorylation in isolated cortical tubules, whereas PGE2 and butaprost selectively increased AQP2 membrane accumulation in kidney slices. In vivo, a V2R antagonist caused a severe urinary concentrating defect in rats, which was greatly alleviated by treatment with butaprost. In conclusion, EP2 and EP4 agonists increase AQP2 phosphorylation and trafficking, likely through different signaling pathways. Furthermore, EP2 selective agonists can partially compensate for a nonfunctional V2R, providing a rationale for new treatment strategies for hereditary nephrogenic diabetes insipidus.

Aquaporin-9 Protein Is the Primary Route of Hepatocyte Glycerol Uptake for Glycerol Gluconeogenesis in Mice

Background: Aquaporin-9 is a glycerol-permeable channel expressed in hepatocytes. Results: Newly identified chemical inhibitors, as well as aquaporin-9 gene deletion, eliminate glycerol-dependent murine hepatocyte glucose output. Conclusion: Aquaporin-9 is the primary route of murine hepatocyte glycerol uptake for gluconeogenesis. Significance: Aquaporin-9 may be a drug target in diabetes treatment It has been hypothesized that aquaporin-9 (AQP9) is part of the unknown route of hepatocyte glycerol uptake. In a previous study, leptin receptor-deficient wild-type mice became diabetic and suffered from fasting hyperglycemia whereas isogenic AQP9−/− knock-out mice remained normoglycemic. The reason for this improvement in AQP9−/− mice was not established before. Here, we show increased glucose output (by 123% ± 36% S.E.) in primary hepatocyte culture when 0.5 mm extracellular glycerol was added. This increase depended on AQP9 because it was absent in AQP9−/− cells. Likewise, the increase was abolished by 25 μm HTS13286 (IC50 ∼ 2 μm), a novel AQP9 inhibitor, which we identified in a small molecule library screen. Similarly, AQP9 deletion or chemical inhibition eliminated glycerol-enhanced glucose output in perfused liver preparations. The following control experiments suggested inhibitor specificity to AQP9: (i) HTS13286 affected solute permeability in cell lines expressing AQP9, but not in cell lines expressing AQPs 3, 7, or 8. (ii) HTS13286 did not influence lactate- and pyruvate-dependent hepatocyte glucose output. (iii) HTS13286 did not affect glycerol kinase activity. Our experiments establish AQP9 as the primary route of hepatocyte glycerol uptake for gluconeogenesis and thereby explain the previously observed, alleviated diabetes in leptin receptor-deficient AQP9−/− mice.

Heteromeric Anopheline Odorant Receptors Exhibit Distinct Channel Properties

Background Insect odorant receptors (ORs) function as odorant-gated ion channels consisting of a conventional, odorant-binding OR and the Orco coreceptor. While Orco can function as a homomeric ion channel, the role(s) of the conventional OR in heteromeric OR complexes has largely focused only on odorant recognition. Results To investigate other roles of odorant-binding ORs, we have employed patch clamp electrophysiology to investigate the properties of the channel pore of several OR complexes formed by a range of different odorant-specific Anopheles gambiae ORs (AgOrs) each paired with AgOrco. These studies reveal significant differences in cation permeability and ruthenium red susceptibility among different AgOr complexes. Conclusions With observable differences in channel function, the data support a model in which the odorant-binding OR also affects the channel pore. The variable effect contributed by the conventional OR on the conductive properties of odorant-gated sensory channels adds additional complexity to insect olfactory signaling, with differences in odor coding beginning with ORs on the periphery of the olfactory system.

Systematic analysis of sporulation phenotypes in 624 non-lethal homozygous deletion strains of Saccharomyces cerevisiae

A new high throughput mutant screening procedure for the detection of sporulation mutants was developed and used to analyse a set of 624 non‐lethal homozygous deletion mutants created in the European joint research program EUROFAN. The screening procedure involved determination of LL‐ and DL‐dityrosine, sporulation‐specific compounds, which were shown to be robust markers of the extent and arrest stage of sporulation mutants. Secondary screens consisted of light microscopy to detect mature and immature spores and DAPI staining to monitor the progress of meiotic nuclear divisions. We discovered new phenotypic classes of mutants defective in spore wall synthesis that were not discovered by previous screens for sporulation mutants. The genes corresponding to the sporulation mutants fell in several functional classes, some of which were previously unknown to be involved in spore formation. Peroxisomes seem to play a role in spore wall synthesis. Mitochondria play a role in sporulation that is not simply restricted to supply of ATP from respiratory metabolism. The deletion mutants included in the set were functionally unknown at the start of EUROFAN; however, within the last few years the importance to sporulation of some of them was also reported by other authors. Taken together, about 8% of all single gene deletion mutants of non‐essential genes of Saccharomyces cerevisiae seem to display a clear and reproducible sporulation phenotype. Copyright

Galpha encoding gene family of the malaria vector mosquito Anopheles gambiae: expression analysis and immunolocalization of AGalphaq and AGalphao in female antennae.

To initiate a comprehensive investigation of chemosensory signal transduction downstream of odorant receptors, we identified and characterized the complete set of genes that encode G‐protein α subunits in the genome of the malaria vector mosquito An. gambiae. Data are provided on the tissue‐specific expression patterns of 10 corresponding aga‐transcripts in adult mosquitoes and pre‐imago developmental stages. Specific immunoreactivity in chemosensory hairs of female antennae provides evidence in support of the participation of a subset of AGαq isoforms in olfactory signal transduction in this mosquito. In contrast, AGαo is localized along the flagellar axon bundle but is absent from chemosensory sensilla, which suggests that this G‐protein α subunit does not participate in olfactory signal transduction. J. Comp. Neurol. 499:533–545, 2006.