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Using FlyAtlas to detect novel functions for well-known genes
Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S131–S138
show that whole-fly arrays are only capable of detecting large changes in widely expressed genes, and their use should thus be deprecated. This work was funded by the UK Biotechnology and Biological Sciences Research Council. Reference Chintapalli, V.R., Wang, J., Dow, J.A.T., 2007. Using FlyAtlas to identify better Drosophila models of human disease. Nature Genetics 39, 715720. doi:10.1016/j.cbpa.2008.04.331
A11.25 Reversible dissociation of V-ATPases: Fact or artefact? A. Albertmelcher, O. Vitavska, M. Huss, H. Wieczorek (University of Osnabrueck) Primary active proton transport by eukaryotic V-ATPases is regulated via the reversible disassembly of the V1VO holoenzyme into its peripheral, catalytic V1 complex and its membrane bound, proton translocating VO complex. This nutrient dependent phenomenon had been first detected in the midgut epithelium of non-feeding, moulting tobacco hornworms (Manduca sexta), and in glucose deficient yeast cells (Saccharomyces cerevisiae). Immunocytochemical investigation of nonfeeding starving instead of moulting tobacco hornworms revealed that subunits of the V1 complex are, as expected, found in the cytoplasm, but that an astonishingly high amount appeared to be membrane bound. To clarify this surprising finding we investigated the reversible disassembly under approximately physiological conditions in living yeast cells, taking advantage of the existence of yeast strains with V-ATPase subunits fused to green fluorescent protein. By using yeast cells with fluorescent subunits of the V-ATPase, we found that only theV1 subunit C is released after displacement of extracellular glucose with galactose, whereas the other V1 subunits remain at or near the membrane. Neither disassembly nor reassembly of subunit C depends on the actin cytoskeleton, whereas disassembly but not reassembly depends on functional microtubules. Results from overlay blots and pull-down assays support the assumption that subunit C interacts directly with microtubules without involvement of linker proteins.
S137
exchangers is not important for alkalinization. Na+/K+ ATPase has been localized in the apical membrane, but ouabain did not affect alkalinization in vivo or in vitro, ruling out the hypothesis that the pump could act as an ATP-driven Na+/H+ exchanger. Failure of luminal Zn2+ (10 μM) to inhibit alkalinization in vitro is evidence against H+ absorption via apical H+ channels. Very high intracellular pH (N8) and high transapical membrane voltages (N−100 mV) were observed during alkalinization. The high intracellular pH could generate significant levels of intracellular CO2− 3 , possibly fated for secretion by a CO2− 3 transporter driven by the large transapical voltage. Other workers have presented in vivo studies suggesting a role for carbonic anhydrase (CA), but CA was not found in the tissue and, in our hands, CA inhibitors did not affect alkalinization in vitro or in vivo. Future experiments should be directed to the identification of a chloride independent and DIDS-insensitive carbonate transporter in the apical membrane. Supported by NIH RO1 AI 063463. doi:10.1016/j.cbpa.2008.04.333
A11.27 Using FlyAtlas to detect novel functions for well-known genes S. Graham, J. Dow, S. Davies (University of Glasgow) Historically Drosophila melanogaster has been a key model organism in developmental and neurobiological studies and indeed may well-known genes have been discovered and characterised in such studies. However it can be argued that characterising genes in this manner may function out with development and neurobiology. The new online resource FlyAtlas has given scientists the opportunity to look at gene expression at a tissue specific manner and may provide a key to discovering ‘new’ functions for well-known genes. To test this hypothesis we looked at the neuronal gene Fasciclin 2 (fas2), which has been exhaustively characterised (over 500 papers), with neural functions ranging from axonal growth in development to synapse stabilization in the adult. Surprisingly FlyAtlas showed fas2 is predominately expressed in the Malpighian tubule (a renal, rather than neural, tissue), hinting at a previously unreported function in this tissue. Quantitative PCR validated the extremely high level of transcription reported by FlyAtlas; however, Fas2 protein is not detectable in tubule by immunocytochemistry. These results imply a novel function for a fas2mRNA transcript specifically in tubule. Recent reports of extensive non-canonical transcription of over 85% of the Drosophila genome (Manaket al., 2007), suggest that fas2 might be an example of a gene with both translated and untranslated transcripts.
doi:10.1016/j.cbpa.2008.04.332 doi:10.1016/j.cbpa.2008.04.334 A11.26 Revisiting the mechanism of strong alkalinization in the anterior midgut of larval yellow fever mosquitoes Aedes aegypti H. Onken (Wagner College); S. Moffett (Washington State University); D. Moffett (Washington State University)
It has been well established that basolateral V-ATPases drive strong alkalinization in the anterior midgut of larval A. aegypti, but transporters responsible for transapical base secretion and/or acid absorption are still unknown. Transapical base secretion via Cl−/HCO−3 exchangers and/or Na+/2–3 HCO−3 cotransporters has been proposed, but neither DIDS nor Cl− free luminal saline inhibited alkalinization in isolated, perfused anterior midguts. Neither luminal amiloride nor high luminal K+ affected alkalinization in vitro, indicating that H+ absorption via apical K+/2H+
A11.28 Archazolid: A novel inhibitor of the V-ATPase S. Bockelmann (University of Osnabrück); D. Menche (HZI Braunschweig); F. Sasse (HZI Braunschweig); H. Wieczorek (University of Osnabrück); M. Huss (University of Osnabrück)
V-ATPases present a family of heteromultimeric proteins, which translocate protons via eukaryotic plasma and endomembranes. By this process they energize a multitude of secondary transport processes. Therefore it is not astonishing that the V-ATPases are involved in serious diseases such as osteoporosis or cancer. The investigation of this enzyme as a potential drug target is one of the
show that whole-fly arrays are only capable of detecting large changes in widely expressed genes, and their use should thus be deprecated. This work was funded by the UK Biotechnology and Biological Sciences Research Council. Reference Chintapalli, V.R., Wang, J., Dow, J.A.T., 2007. Using FlyAtlas to identify better Drosophila models of human disease. Nature Genetics 39, 715720. doi:10.1016/j.cbpa.2008.04.331
A11.25 Reversible dissociation of V-ATPases: Fact or artefact? A. Albertmelcher, O. Vitavska, M. Huss, H. Wieczorek (University of Osnabrueck) Primary active proton transport by eukaryotic V-ATPases is regulated via the reversible disassembly of the V1VO holoenzyme into its peripheral, catalytic V1 complex and its membrane bound, proton translocating VO complex. This nutrient dependent phenomenon had been first detected in the midgut epithelium of non-feeding, moulting tobacco hornworms (Manduca sexta), and in glucose deficient yeast cells (Saccharomyces cerevisiae). Immunocytochemical investigation of nonfeeding starving instead of moulting tobacco hornworms revealed that subunits of the V1 complex are, as expected, found in the cytoplasm, but that an astonishingly high amount appeared to be membrane bound. To clarify this surprising finding we investigated the reversible disassembly under approximately physiological conditions in living yeast cells, taking advantage of the existence of yeast strains with V-ATPase subunits fused to green fluorescent protein. By using yeast cells with fluorescent subunits of the V-ATPase, we found that only theV1 subunit C is released after displacement of extracellular glucose with galactose, whereas the other V1 subunits remain at or near the membrane. Neither disassembly nor reassembly of subunit C depends on the actin cytoskeleton, whereas disassembly but not reassembly depends on functional microtubules. Results from overlay blots and pull-down assays support the assumption that subunit C interacts directly with microtubules without involvement of linker proteins.
S137
exchangers is not important for alkalinization. Na+/K+ ATPase has been localized in the apical membrane, but ouabain did not affect alkalinization in vivo or in vitro, ruling out the hypothesis that the pump could act as an ATP-driven Na+/H+ exchanger. Failure of luminal Zn2+ (10 μM) to inhibit alkalinization in vitro is evidence against H+ absorption via apical H+ channels. Very high intracellular pH (N8) and high transapical membrane voltages (N−100 mV) were observed during alkalinization. The high intracellular pH could generate significant levels of intracellular CO2− 3 , possibly fated for secretion by a CO2− 3 transporter driven by the large transapical voltage. Other workers have presented in vivo studies suggesting a role for carbonic anhydrase (CA), but CA was not found in the tissue and, in our hands, CA inhibitors did not affect alkalinization in vitro or in vivo. Future experiments should be directed to the identification of a chloride independent and DIDS-insensitive carbonate transporter in the apical membrane. Supported by NIH RO1 AI 063463. doi:10.1016/j.cbpa.2008.04.333
A11.27 Using FlyAtlas to detect novel functions for well-known genes S. Graham, J. Dow, S. Davies (University of Glasgow) Historically Drosophila melanogaster has been a key model organism in developmental and neurobiological studies and indeed may well-known genes have been discovered and characterised in such studies. However it can be argued that characterising genes in this manner may function out with development and neurobiology. The new online resource FlyAtlas has given scientists the opportunity to look at gene expression at a tissue specific manner and may provide a key to discovering ‘new’ functions for well-known genes. To test this hypothesis we looked at the neuronal gene Fasciclin 2 (fas2), which has been exhaustively characterised (over 500 papers), with neural functions ranging from axonal growth in development to synapse stabilization in the adult. Surprisingly FlyAtlas showed fas2 is predominately expressed in the Malpighian tubule (a renal, rather than neural, tissue), hinting at a previously unreported function in this tissue. Quantitative PCR validated the extremely high level of transcription reported by FlyAtlas; however, Fas2 protein is not detectable in tubule by immunocytochemistry. These results imply a novel function for a fas2mRNA transcript specifically in tubule. Recent reports of extensive non-canonical transcription of over 85% of the Drosophila genome (Manaket al., 2007), suggest that fas2 might be an example of a gene with both translated and untranslated transcripts.
doi:10.1016/j.cbpa.2008.04.332 doi:10.1016/j.cbpa.2008.04.334 A11.26 Revisiting the mechanism of strong alkalinization in the anterior midgut of larval yellow fever mosquitoes Aedes aegypti H. Onken (Wagner College); S. Moffett (Washington State University); D. Moffett (Washington State University)
It has been well established that basolateral V-ATPases drive strong alkalinization in the anterior midgut of larval A. aegypti, but transporters responsible for transapical base secretion and/or acid absorption are still unknown. Transapical base secretion via Cl−/HCO−3 exchangers and/or Na+/2–3 HCO−3 cotransporters has been proposed, but neither DIDS nor Cl− free luminal saline inhibited alkalinization in isolated, perfused anterior midguts. Neither luminal amiloride nor high luminal K+ affected alkalinization in vitro, indicating that H+ absorption via apical K+/2H+
A11.28 Archazolid: A novel inhibitor of the V-ATPase S. Bockelmann (University of Osnabrück); D. Menche (HZI Braunschweig); F. Sasse (HZI Braunschweig); H. Wieczorek (University of Osnabrück); M. Huss (University of Osnabrück)
V-ATPases present a family of heteromultimeric proteins, which translocate protons via eukaryotic plasma and endomembranes. By this process they energize a multitude of secondary transport processes. Therefore it is not astonishing that the V-ATPases are involved in serious diseases such as osteoporosis or cancer. The investigation of this enzyme as a potential drug target is one of the