Background
There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex.Principal Findings
We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis.Conclusions/Significance
These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein. 相似文献A change in ambient temperature is predicted to disrupt cellular homeostasis by affecting all cellular processes in an albeit non-uniform manner. Diffusion is generally less temperature-sensitive than enzymes, for example, and each enzyme has a characteristic individual temperature profile. The actual effects of temperature variation on cells are still poorly understood at the molecular level. Towards an improved understanding, we have performed a genome-wide RNA interference screen with S2R?+?cells. This Drosophila cell line proliferates over a temperature range comparable to that tolerated by the parental ectothermic organism. Based on effects on cell counts and cell cycle profile after knockdown at 27 and 17 °C, respectively, genes were identified with an apparent greater physiological significance at one or the other temperature. While 27 °C is close to the temperature optimum, the substantially lower 17 °C was chosen to identify genes important at low temperatures, which have received less attention compared to the heat shock response. Among a substantial number of screen hits, we validated a set successfully in cell culture and selected ballchen for further evaluation in the organism. This gene encodes the conserved metazoan VRK protein kinase that is crucial for the release of chromosomes from the nuclear envelope during mitosis. Our analyses in early embryos and larval wing imaginal discs confirmed a higher requirement for ballchen function at temperatures below the optimum. Overall, our experiments validate the genome-wide screen as a basis for future characterizations of genes with increased physiological significance at the lower end of the readily tolerated temperature range.
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