Functional analysis of dynamin isoforms in Drosophila melanogaster.
Dynamin and dynamin-like proteins are required for endocytosis, synaptic vesicle recycling and membrane trafficking. From the shibire locus in Drosophila melanogaster, six different isoforms of dynamin are generated by alternative splicing. However, the roles of the individual isoforms in cellular processes are unknown. To investigate functional differences among the dynamin isoforms, transgenic lines were generated that individually expressed each of 3 different isoforms under UASGAL4 control. The expression of the isoforms was controlled by neural promoter (elav)-driven GAL4, or by a shibire-promoter driven GAL4 transgene. Reporter gene expression indicated that the shi promoter is active during embryogenesis, and in larvae, pupae, and adults in a pattern consistent with normal dynamin expression. To assay for the ability of dynamin isoforms to function in vivo, the isoforms expressed via these GAL4 drivers were tested for the ability to rescue shibire phenotypes. When expressed at very high levels all individual isoforms tested rescued the temperature-sensitive paralytic phenotype of shi(ts2) flies; however, this rescue was partial, suggesting that no single tested isoform is sufficient for synaptic vesicle recycling in vivo. When tested for ability to rescue lethality induced by heat-pulsing larvae during development, shi- promoter driven expression of individual isoforms conferred significant resistance to heat treatment during larval development. However, all 3 isoforms were unable to rescue the lethality of shi12-12B mutants which are severely hypomorphic (or null) for shibire function. Taken together, these observations suggest that individual shibire isoforms have specific molecular activities in vivo.
Staples
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RR
Department of Molecular and Cellular Biology, University of Arizona, Tucson 85721, USA.
Ramaswami
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M
D017398:Alternative Splicing; D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology; D005838:Genotype; D008870:Microtubules / Q000502:physiology; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D013572:Synaptic Vesicles / Q000502:physiology; D013696:Temperature; D019076:Transgenes
D029721:Drosophila Proteins; D020033:Protein Isoforms; D020558:GTP Phosphohydrolases; D034281:Dynamins; C439628:shi protein, Drosophila